Gabriele Barbanti

Cystometric Evidence that Capsaicin-Sensitive Nerves Modulate the Afferent Branch of Micturition Reflex in Humans

Intravesical instillation of capsaicin (0.1 to 10 microM) in six patients (five with hypersensitive disorders of the lower urinary tract, one with benign prostatic hyperplasia) produced a concentration-related reduction of the first desire to void, bladder capacity and pressure threshold for micturition. At a threshold concentration of one microM, capsaicin also produced a warm to burning sensation referred to the suprapubic area during the collecting phase and to the urethra during micturition. All the patients with hypersensitive disorders of the lower urinary tract reported disappearance or marked attenuation of their symptoms for a few days after capsaicin application. In three other patients with hypersensitive disorders of the lower urinary tract, intravesical instillation of capsaicins vehicle (0.1% ethanol in saline) did not produce significant cystometric changes nor modify the symptomatology. These observations provide the first indication that capsaicin-sensitive structures (nerves?) may be present in the human urinary bladder as they have been shown to occur in various other species.

Tachykinin antagonists inhibit nerve-mediated contractions in the circular muscle of the human ileum: Involvement of neurokinin-2 receptors

The effects of some newly developed tachykinin antagonists that are selective for the neurokinin (NK)-1 (L 668,169) or the NK-2 (MEN 10,207, L 659,877 and R 396) tachykinin receptor on the cholinergic and noncholinergic contraction and on the nonadrenergic noncholinergic relaxation produced by electrical field stimulation (50 Hz) were investigated in mucosa-free circular strips of the human ileum. The strips were contracted by substance P and neurokinin A as well as by selective NK-2-receptor ligands, [beta Ala8]neurokinin A(4-10), and MDL 28,564, the latter peptide being capable of discriminating between NK-2-receptor subtypes. The selectivity of the antagonists for NK-1 or NK-2 receptors was confirmed in pharmacological experiments using substance P, neurokinin A, and [beta Ala8]neurokinin A(4-10) as stimulants. Among the NK-2-selective antagonists, MEN 10,207 displayed the highest affinity, followed by L 659,877 and R 396. The antagonists MEN 10,207 and L 659,877 inhibited the noncholinergic contraction to electrical stimulation in a concentration-dependent manner; L 668,169 and R 396 were poorly effective. Thus the potency of antagonists toward the noncholinergic response closely paralleled their rank order of potency at NK-2 receptors. The cholinergic contraction and nonadrenergic noncholinergic relaxation were not inhibited by the antagonists. Both substance P- and neurokinin A-like immunoreactivities were detected in extracts of the human ileum, and the identity of the corresponding peptides was confirmed by reverse-phase high-performance liquid chromatography. It was concluded that in addition to NK-1 receptors, the circular muscle of the human ileum also contains NK-2 receptors. Activation of the latter is chiefly responsible for the noncholinergic contraction to nerve stimulation.

NK2 tachykinin receptors and contraction of circular muscle of the human colon: characterization of the NK2 receptor subtype

The contractile effect of substance P, neurokinin A, receptor selective agonists for tachykinin receptors and NK2 tachykinin receptor antagonists was investigated in mucosa-free circular strips of the human isolated colon. Neurokinin A and substance P produced concentration-dependent contractions which approached 80-90% of the maximal response to carbachol. Neurokinin A was about 370 times more potent than substance P. The action of neurokinin A and substance P was not modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The NK2 receptor selective agonist, [beta-Ala8]neurokinin A-(4-10) closely mimicked the response to neurokinin A while NK1 and NK3 receptor selective agonists were active only at microM concentrations. The pseudopeptide, MDL 28,564, which is one of the most selective NK2 ligands available, behaved as a full agonist. Responses to [beta-Ala8]neurokinin A were antagonized by NK2 receptor selective antagonists, with the rank order of potency MEN 10,376 greater than L 659,877 much greater than R 396. These data indicate that NK2 tachykinin receptors play a dominant role in determining the contraction of the circular muscle of the human colon to peptides of this family. The NK2 receptor subtype responsible for this effect belongs to the same subtype (NK2A) previously identified in the rabbit pulmonary artery and guinea-pig bronchi.

Potent contractile activity of endothelin on the human isolated urinary bladder

Endothelin (1 nm‐0.3μm) produced a concentration‐related contraction of mucosa‐free muscle strips excised from the dome of the human urinary bladder. The response to endothelin was unaffected by either atropine (1 μm) or nifedipine (1 μm) at concentrations that abolished the response to carbachol and KCl, respectively. These findings indicate that mechanisms other than activation of dihydropyridine‐ and voltage‐sensitive calcium channels may be involved in the action of endothelin on smooth muscles.

Effect of NG-monomethyl L-arginine (L-NMMA) and NG-nitro L-arginine (L-NOARG) on non-adrenergic non-cholinergic relaxation in the circular muscle of the human ileum.

1 We have investigated the effect of the NO synthesis inhibitors, NG‐monomethyl l‐arginine (l‐NMMA) and NG‐nitro l‐arginine (l‐NOARG) on the non‐adrenergic non‐cholinergic (NANC) relaxation produced by electrical field stimulation in the circular muscle of the human ileum. 2 In the presence of atropine and guanethidine (1 and 3 μm, respectively), electrical field stimulation produced tetrodotoxin‐sensitive relaxation of the strips. l‐NMMA, starting from 100 μm, produced a concentration‐dependent inhibition of the evoked relaxations (2 Hz); maximal inhibition at 1 mm averaged about 35%. 3 The inhibitory effect of l‐NMMA was unchanged by previous incubation with d‐arginine while it was prevented by l‐arginine (l‐Arg). l‐NMMA did not affect isoprenaline‐induced relaxation. 4 l‐NOARG (1–30 μm) concentration‐dependently inhibited the evoked relaxations at 2 Hz, up to a maximum of 65% inhibition, although in some strips complete inhibition of the response was observed. The effect of l‐NOARG was reversed by l‐Arg. l‐NOARG did not affect isoprenaline‐induced relaxation. 5 These results suggest that NO may be involved in inhibitory NANC transmission in the circular muscle of the human ileum.

Human isolated small intestine: motor responses of the longitudinal muscle to field stimulation and exogenous neuropeptides

Summary(1) Longitudinal muscle strips from the human small intestine (jejunum/ileum) responded to electrical field stimulation (1–50 Hz) with frequency-related primary contractions which were largely atropine- (3 μM) sensitive. When the tone was raised by addition of galanin (0.3 – 1 μM), prostaglandin (PG) E2 (1–10 μM) or neurokinin A (NKA, 0.1 μM), a frequency-related relaxation was evident which was potentiated by atropine. All the responses to field stimulation were abolished by tetrodotoxin (1 μM), thus indicating their neural origin. (2) The atropine-sensitive primary contraction to field stimulation was virtually abolished by omega conotoxin fraction GVIA (CTX, 0.1–0.3 μM) while the relaxations were CTX-resistant. The field stimulation-induced relaxations, which were observed in the presence of atropine and guanethidine (3 μM), were also unaffected by apamin (0.1 μM). (3) NKA and substance P (SP) produced a concentration- (1 nM−1 μM for both peptides) related contraction, NKA being about 53 times more potent than SP. [Pro9]SP sulphone and [MePhe7]-NKB, selective agonists of the NK-1 and NK-3 receptor, respectively, were barely effective. On the other hand, [\Ala8]NKA(4–10), a selective NK-2 receptor agonist, had a potent contractile activity, similar to that of NKA. (4) Galanin (1 nM–1μM) produced an atropine- and tetrodotoxin-resistant concentration-related contraction of longitudinal muscle of human isolated small intestine. The response to galanin did not show any sign of fading and was particularly suitable to study the evoked relaxations. (5) Calcitonin gene-related peptide (CGRP) (10–100 nM) consistently inhibited the nerve-mediated contractions of strips from the ileum while the effect on the jejunum was less pronounced. Vasoactive intestinal polypeptide (VIP, 0.1–1 μM) inhibited nerve-mediated contractions both in the ileum and the jejunum. (6) These experiments indicate that both cholinergic excitatory and non-adrenergic non-cholinergic nerves affect motility of the longitudinal muscle of the human small intestine. Furthermore, several neuropeptides produce potent motor effects, the contractile response to tachykinins being apparently mediated by activation of NK-2 receptors.

Multiple sources of calcium for contraction of the human urinary bladder muscle

1 KCl, carbachol, neurokinin A and endothelin produced concentration‐dependent contractions of mucosa‐free muscle strips from the dome of the human urinary bladder. The maximal response to carbachol or neurokinin A exceeded that to KCl, while the maximal response to endothelin approached that to KCl. 2 Nifedipine (1 μm) abolished the response to KCl, reduced the response to carbachol or neurokinin A but had no effect on the response to endothelin. Bay K 8644 (1 μm) markedly potentiated the response to KCl but had little or no effect on the response produced by the other stimulants. 3 Superfusion of the strips with a nominally calcium (Ca)‐free medium containing EDTA (1 mm) for 30 min markedly reduced the response to carbachol, neurokinin A and endothelin, although a small response was still evident at high concentrations. Likewise, after a prolonged (60 min) superfusion of the strips with a high K (80 mm) Ca‐free medium plus EDTA (1 mm) these three agonists still produced a small contractile response. 4 The nifedipine (1 μm) resistant response to carbachol, neurokinin A or endothelin was markedly depressed by LaCl3 (1 mm). In contrast, the nifedipine‐(1 μm) resistant response to carbachol was not modified by NiCl2 (0.1 mm) or ω‐conotoxin (0.1 μm). 5 Caffeine produced divergent effects depending upon the temperature of incubation: a relaxation at 37°C and a concentration‐dependent (2.5–20 mm) contraction at 25°C. The latter was markedly inhibited by procaine (3 mm) but unaffected by nifedipine (1 μm). 6 After a prolonged (60 min) superfusion with a high K, Ca‐free medium containing EDTA the response to carbachol (100 μm) was abolished by previous exposure to procaine (3 mm). Conversely, the response to endothelin (1 μm) was unaffected by procaine. The response to endothelin in these experimental conditions was also resistant to LaCl3 (1 mm). 7 These findings indicate that multiple sources of Ca are mobilized for contraction of the human bladder muscle by different stimulants. Dihydropyridine‐ and voltage‐sensitive Ca channels provide the major if not the sole source of Ca for the response to KCl, play some role in the response to muscarinic (carbachol) or NK‐2 tachykinin receptor stimulation but are not involved in the response to endothelin. Carbachol, neurokinin A and endothelin all mobilize a Ca pool (either extracellular or located at membrane level) which is LaCl3‐sensitive but nifedipine‐resistant. Neither T‐ nor N‐type channels appear to be involved in the response to carbachol. In addition, these agents mobilize a tightly bound Ca pool independently from membrane depolarization. This latter pool is probably a procaine‐sensitive intracellular source of activator Ca mobilized by caffeine and carbachol. The failure of procaine to prevent the response to endothelin in high K, Ca‐free medium raises the possibility that this peptide mobilizes an intracellular source of activator Ca, distinct from the caffeine‐ and carbachol‐sensitive pool.

Human isolated ileum: motor responses of the circular muscle to electrical field stimulation and exogenous neuropeptides

Summary(1) Circularly-oriented muscle strips from the human ileum responded to electrical field stimulation (1–50 Hz) with frequency-related primary relaxation at low frequency and primary contractions at high frequencies of stimulation. Both responses were abolished or markedly reduced by tetrodotoxin (1 μM). (2) Atropine (3 μ M) or omega conotoxin (0.1 μM) reduced but dit not abolish contraction to electrical field stimulation and enhanced the relaxation. Omega conotoxin (0.1 μM) did not affect carbachol-induced contraction nor isoprenaline-induced relaxation. (3) Neurokinin A and substance P (1 nM-1 μM) produced a concentration-dependent contraction. The NK-1 receptor selective agonist, [Pro9]SP sulfone and the NK-2 receptor selective agonist [βAla8]NKA(4-10) prodneed a contraction superimposable to that of substance P and neurokinin A, respectively. On the other hand, [MePhe7]-neurokinin B, an NK-3 receptor selective agonist was ineffective up to 1 [M. The response to substance P or neurokinin A was unaffected by atropine (3 μM). (4) Galanin, up to 0.1 μM produced a weak and inconsistent contraction. (5) Vasoactive intestinal polypeptide (10 nM - 1 μM) produced a concentration-dependent relaxation while human alpha calcitonin gene-related peptide exerted a weak and inconsistent relaxant effect. (6) These findings indicate that both cholinergic excitatory and non-cholinergic inhibitory nerves affect the motility of the circular muscle of the human small intestine. Transmitter release from excitatory nerves seems largely mediated by activation of omega conotoxin-sensitive (N-type) calcium channels. Tachykinins exert a potent contractile effect independently of cholinergic nerves via NK-1 and NK-2 receptors.

Motor response of the human isolated small intestine and urinary bladder to porcine neuromedin U-8.

1 Porcine neuromedin U‐8 produced a concentration (0.3 nm−1 μm)‐dependent contraction of the longitudinal muscle of the human isolated ileum, which was unaffected by either atropine (1 μm) or tetrodotoxin (1 μm). 2 By contrast, neuromedin U‐8 had only a weak effect on the circular muscle of the human isolated ileum. 3 Neuromedin U‐8 also produced a concentration‐dependent contraction of mucosa‐free muscle strips from the dome of the human isolated urinary bladder, its action being unaffected by either atropine or tetrodotoxin. 4 These findings indicate that neuromedin U‐8 exerts a direct contractile effect on smooth muscle of the human intestinal and urinary tract.

Release of VIP- but not CGRP-like immunoreactivity by capsaicin from the human isolated small intestine

Exposure to capsaicin (1 microM) produced a prompt and sustained release of vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) from mucosa-free strips of human small intestine (jejunum and ileum). A second application of capsaicin, 60 min later, had no effect indicating complete desensitization, a specific feature of the action of capsaicin on sensory nerves. By contrast no release of calcitonin gene-related peptide (CGRP)-LI was produced upon the first or second application of capsaicin.