Massimo Parlani

The antagonism induced by ruthenium red of the actions of capsaicin on the peripheral terminals of sensory neurons: further studies

Ruthenium Red, an inorganic dye which blocks transmembrane calcium (Ca) fluxes in neural tissues, reduced the capsaicin-induced release of substance P-like immunoreactivity from muscle strips of the guinea-pig urinary bladder in a concentration-dependent (30 nM - 3 microM) manner, and protected the sensory fibers from capsaicin-induced densensitization. A similar antagonism of the actions of capsaicin was observed in functional experiments (capsaicin-induced contraction of the isolated guinea-pig bladder or inhibition of twitches of the isolated rat vas deferens). In view of its established action on the depolarization-coupled entry of Ca into synaptosomes and the secretion of transmitter, we propose that Ruthenium Red could antagonize the action of capsaicin on the peripheral terminals of sensory nerves by a similar mechanism, thereby suppressing transmitter secretion and preventing the establishment of desensitization.

The effect of calcium free medium and nifedipine on the release of substance P-like immunoreactivity and contractions induced by capsaicin in the isolated guinea-pig and rat bladder

1. Capsaicin produced a prompt release of substance P-like immunoreactivity (SP-LI) from superfused mucosa-free muscle strips excised from the guinea-pig urinary bladder. A second application of capsaicin had no further effect, indicating desensitization. 2. Neither tetrodotoxin (1 microM) or nifedipine (10 microM) had any inhibitory effect on SP-LI release by capsaicin nor influenced the establishment of the desensitized state. Nifedipine produced per se some SP-LI release. 3. SP-LI release by capsaicin was abolished by incubation in a Calcium(Ca)-free medium containing EDTA (1.0 mM) which also afforded a partial protection toward desensitization. A lower EDTA concentration (0.1 mM) did not suppress SP-LI release by capsaicin but still inhibited desensitization. 4. When the concentration of CaCl2 in the medium was lowered to 1/10-1/100 of that present in normal Krebs solution, capsaicin still evoked a marked SP-LI release and desensitization occurred. In a nominally Ca free medium (maximal Ca concentration due to impurities was 6.7 microM) SP-LI release was still observed and desensitization was incomplete. 5. In a nominally Ca free medium, removal of Mg ions enhanced the SP-LI release induced by capsaicin and enhanced desensitization. 6. In functional studies, nifedipine greatly reduced or abolished the capsaicin- or SP-induced contraction of the rat or guinea-pig isolated bladder but did not prevent desensitization. Likewise, SP-LI depletion in the rat bladder following systemic capsaicin desensitization was not prevented by nifedipine pretreatment. On the other hand, the protective action of Ca free media (containing EDTA) was confirmed in organ bath studies (guinea-pig bladder). 7. These findings indicate that: (a) the requirements of extracellular calcium for activation of neuropeptide release from sensory nerves by capsaicin are very low; (b) both excitation of sensory fibers (SP-LI release) and desensitization are dependent upon the presence of extracellular calcium and (c) L-type voltage-sensitive Ca channels are not likely to be involved in the actions of capsaicin on sensory nerve terminals.

Regional differences in the effects of capsaicin and tachykinins on motor activity and vascular permeability of the rat lower urinary tract

Summary1. The effects of capsaicin, substance P (SP) and neurokinin A (NKA) on motor activity and vascular permeability was investigated in the rat lower urinary tract (bladder dome and neck, proximal urethra and ureters). 2. Capsaicin produced contractions of the rat bladder dome and neck and of the proximal urethra in vitro, which were unaffected by tetrodotoxin and abolished by ganglionectomy. SP and NKA were almost equipotent in producing a contraction of the rat isolated bladder dome or neck and urethra. However, the maximal response to NKA was about twice that of SP on the urethra and bladder neck. 3. Capsaicin did not affect motility of the unstimulated rat isolated ureter, while NKA or SP activated rhythmic contractions, NKA being about 850 times more potent than SP. Either capsaicin or field stimulation produced a transient inhibition of the NKA-activated rhythmic contractions of the rat isolated ureter which was prevented by capsaicin-desensitization. 4. The capsaicin-(1 μM) or field stimulation-induced inhibition of NKA-activated rhythmic contractions of the rat isolated ureter were unaffected by removal of pelvic ganglia but abolished by cold storage (72 h at 4°C). 5. Intravenous capsaicin induced an inflammatory response (Evans blue leakage) in the bladder, proximal urethra and ureters in vivo. Plasma extravasation was greater in the ureters, urethra and bladder neck than in the dome. SP, NKA and histamine produced a dose-dependent dye leakage in all segments of the rat urinary tract, the response being slightly greater in the bladder neck than in the dome. 6. The capsaicin-induced inflammatory response was abolished by systemic capsaicin-desensitization and reduced, to a variable extent, by pelvic ganglionectomy, in the various tissues examined. Topical application of tetrodotoxin on the bladder dome failed to affect the capsaicin-induced plasma extravasation in the urinary bladder. 7. These findings indicate that chemoceptive, capsaicin-sensitive nerves are present throughout the whole rat lower urinary tract and their activation determines a variety of visceromotor responses and an increase of vascular permeability. In various instances the response to capsaicin may be explained by the action of tachykinins but some effects may involve other sensory neuropeptides.

The effect of nifedipine on spontaneous, drug-induced and reflexly-activated contractions of the rat urinary bladder: Evidence for the participation of an intracellular calcium store to micturition contraction

1. The effect of nifedipine on spontaneous and stimulated motility of the rat urinary bladder has been investigated in vitro (isolated detrusor strips) and in vivo (micturition reflex). 2. Nifedipine inhibited tone and spontaneous activity of the isolated rat bladder, its effect being greater in indomethacin-treated preparations. Nifedipine suppressed the KCl induced phasic and tonic contraction and inhibited by 60-80% the carbachol- or ATP- induced contractions. Nifedipine reduced by about 70% amplitude of the nerve-mediated bladder contractions. 3. Exposure to Ca free medium containing EDTA suppressed tone and spontaneous activity of the rat bladder. In these conditions the response to KCl or ATP was rapidly abolished while a response to carbachol was still evident even after a long exposure to the Ca free medium. 4. In vivo, nifedipine affected reflex micturition e.g. increased volume threshold and slightly reduced amplitude of micturition contraction. In addition, nifedipine reduced voiding efficiency e.g. increased residual volume after micturition. These effects were evident following ligation of the ureters because in normal conditions nifedipine induced a marked diuresis which masked its effect on volume threshold. 5. These findings indicate that in the rat urinary bladder Ca from both intra- and extracellular pools is mobilized during spontaneous or stimulated contractions. Mobilization of an intracellular Ca pool by cholinomimetics or other neurotransmitter(s) may be responsible for the nifedipine-resistant component of the voiding contraction in vivo.

Antiproliferative and differentiating activities of a novel series of histone deacetylase inhibitors.

Histone deacetylases are promising molecular targets for the development of antitumor agents. A novel series of histone deacetylase inhibitors of the hydroxamic acid type were synthesized for structure-activity studies. Thirteen tricyclic dibenzo-diazepine, -oxazepine, and -thiazepine analogues were studied and shown to induce variable degrees of histone H3/H4 and tubulin acetylation in a cellular model of myeloid leukemia sensitive to all-trans retinoic acid (ATRA). Multiparametric correlations between acetylation of the three substrates, tumor cell growth inhibition, and ATRA-dependent cytodifferentiation were performed, providing information on the chemical functionalities governing these activities. For two analogues, antitumor activity in the animal was demonstrated.

The presence of mucosa reduces the contractile response of the guinea-pig urinary bladder to substance P

The contractile response to substance P in‐vitro is greater in strips of guinea‐pig bladder freed of mucosa than in normal strips, while the response to field stimulation, histamine or KCl is unaffected by the presence of mucosa. Substance P has no inhibitory effect on histamine‐induced contractions of the guinea‐pig bladder. These findings support the possibility that the presence of mucosa may reduce accessibility of substance P to the muscle layer.

Impairment of myocardial contractility by anticancer anthracyclines: role of secondary alcohol metabolites and evidence of reduced toxicity by a novel disaccharide analogue

The anticancer anthracycline doxorubicin (DOX) causes cardiotoxicity. Enzymatic reduction of a side chain carbonyl group converts DOX to a secondary alcohol metabolite that has been implicated in cardiotoxicity. We therefore monitored negative inotropism, assessed as inhibition of post‐rest contractions, in rat right ventricle strips exposed to DOX or to analogues forming fewer amounts of their alcohol metabolites (epirubicin, EPI, and the novel disaccharide anthracycline MEN 10755). Thirty μM EPI exhibited higher uptake than equimolar DOX, but formed comparable amounts of alcohol metabolite due to its resistance to carbonyl reduction. MEN 10755 exhibited also an impaired uptake, and consequently formed the lowest levels of alcohol metabolite. Accordingly, DOX and EPI inhibited post‐rest contractions by ∼40 – 50%, whereas MEN 10755 inhibited by ∼6%. One hundred μM EPI exhibited the same uptake as equimolar DOX, but formed ∼50% less alcohol metabolite. One hundred μM MEN 10755 still exhibited the lowest uptake, forming ∼60% less alcohol metabolite than EPI. Under these conditions DOX inhibited post‐rest contractions by 88%. EPI and MEN 10755 were ∼18% (P<0.05) or ∼80% (P<0.001) less inhibitory than DOX, respectively. The negative inotropism of 30 – 100 μM DOX, EPI, or MEN 10755 correlated with cellular levels of both alcohol metabolites (r=0.88, P<0.0001) and carbonyl anthracyclines (r=0.79, P<0.0001). Nonetheless, multiple comparisons showed that alcohol metabolites were ∼20 – 40 times more effective than carbonyl anthracyclines in inhibiting contractility. The negative inotropism of MEN 10755 was therefore increased by chemical procedures, like side chain valeryl esterification, that facilitated its uptake and conversion to alcohol metabolite but not its retention in a carbonyl form. These results demonstrate that secondary alcohol metabolites are important mediators of cardiotoxicity. A combination of reduced uptake and limited conversion to alcohol metabolite formation might therefore render MEN 10755 more cardiac tolerable than DOX and EPI.

Capsaicin-induced relaxation in the rat isolated external urethral sphincter: characterization of the vanilloid receptor and mediation by CGRP

1 The potential role of capsaicin‐sensitive nerves in the relaxation of the rat external urethral sphincter (REUS) was evaluated by demonstrating the existence of specific vanilloid (capsaicin) receptors and by investigating the sensory neurotransmitter(s) putatively involved in this relaxation. 2 Capsaicin (1 μm) relaxed REUS strips precontracted with noradrenaline (NA) (0.1 mm). This effect underwent desensitization and it was absent in preparations taken from adult capsaicin‐pretreated rats. 3 Capsaicin‐induced relaxation of NA‐precontracted REUS was mimicked by calcitonin gene‐related peptide (CGRP, 0.3–10 μm), but not by substance P (1 μm), vasoactive intestinal polypeptide (VIP, 1 μm), α‐β methylene ATP (10 μm), γ‐aminobutyric acid (GABA, 3 mm) or galanin (1 μm). A cross‐tachyphylaxis between capsaicin (1 μm) and CGRP (1 μm) was observed. Both capsaicin and CGRP‐induced relaxation were partially antagonized by the proposed CGRP antagonist, CGRP (8–37) (10 μm). 4 Electrical field stimulation (EFS, 2.5 Hz, 60 V, 1 ms, trains of 5 s every 5 min) of REUS evoked a contraction characterized by a largely adrenergic slowly developing tonic contraction with superimposed fast twitches due to the striated component of the strips. Both capsaicin (1 μm) and CGRP (0.01–1 μm) produced an almost complete inhibition of EFS‐induced tonic contraction. A cross‐tachyphylaxis between capsaicin and CGRP was observed. Furthermore, these inhibitory actions were unaffected by CGRP (8–37) (10 μm). 5 [3H]‐resiniferatoxin displayed specific, saturable binding to rat urethral membranes. Data were consistent with a single site with a Kd of 105 pm and a Bmax of 40 fmol mg−1 protein. This binding was inhibited by capsaicin with a Ki of 0.6 μm and it was reduced by approximately 80% in preparations taken from rats that had undergone surgical ablation of the major pelvic ganglion 4 days earlier. 6 In conclusion we have demonstrated the existence of vanilloid receptors on capsaicin‐sensitive nerves innervating the rat urethra mainly through the major pelvic ganglion. The activation of this set of nerves could lead to a local release of CGRP that in turn elicits a remarkable urethral relaxation. Such a mechanism could be of relevance in physiological conditions to facilitate urine expulsion during micturition and in pathological conditions to help removal of noxious stimuli following mechanical/chemical irritation of the lower urinary tract.

Mechanical Irritation Induces Neurogenic Inflammation in the Rat Urethra

A catheter was inserted into the urethral meatus of urethane-anaesthetized rats and rotated (30 rotations/minute) during a three minute period. One hour later, microvascular permeability in the distal urethra was evaluated by means of the Evans Blue leakage technique. Dye extravasation increased significantly (74 +/- 12 ng./mg. of wet tissue weight, p less than 0.05), as compared to control values (18 +/- 2 ng./mg.). The effect of catheterism was prevented by about 50% by systemic pretreatment with capsaicin performed in either adult or newborn rats, as well as by bilateral removal of pelvic ganglia. Furthermore, pretreatment with capsaicin of adult rats, combined to pelvic ganglionectomy, virtually abolished the inflammatory response produced by mechanical irritation of the urethra. These results indicate that: 1) the increase of vascular permeability produced by mechanical irritation is nerve-mediated, 2) capsaicin-sensitive afferents participate in the inflammatory process and 3) capsaicin-insensitive nerves, which pass through the pelvic ganglia, contribute to the overall response.

Alkyl piperidine and piperazine hydroxamic acids as HDAC inhibitors.

We report here the strategy used in our research group to find a new class of histone deacetylase (HDAC) inhibitors. A series of N-substituted 4-alkylpiperazine and 4-alkylpiperidine hydroxamic acids, corresponding to the basic structure of HDAC inhibitors (zinc binding moiety-linker-capping group) has been designed, prepared, and tested for HDAC inhibition. Linker length and aromatic capping group connection were systematically varied to find the optimal geometric parameters. A new series of submicromolar inhibitors was thus identified, which showed antiproliferative activity on HCT-116 colon carcinoma cells.