Paolo Santicioli

The nonstop transvesical cystometrogram in urethane-anesthetized rats: A simple procedure for quantitative studies on the various phases of urinary bladder voiding cycle

A new cystometric procedure that involves a nonstop transvesical (through a needle inserted into the bladder dome) infusion of saline in urethane-anesthetized rats is described. This permits the obtainment of a series of repetitive voiding cycles in both male and female rats. Recording at high-chart speed permits quantitative measurements of the various phases of the voiding cycle. Micturition was almost invariably associated with appearance, during a sustained increase of intravesical pressure, of a series of high-frequency oscillations, which were paralleled by a stream-like emission of the infused fluid. Micturition was not observed after topical tetrodotoxin, after bilateral transection of pelvic nerves, or in acute spinal rats. Intravenous hexamethonium produced a dose-related impairment of the voiding cycle, and, at high doses, suppressed micturition, and overflow incontinence ensued. The experimental procedure described herein appears to be suitable for physiopharmacological studies dealing with regulation of voiding cycle.

Distribution of calcitonin gene-related peptide-like immunoreactivity in various rat tissues: correlation with substance P and other tachykinins and sensitivity to capsaicin

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) has been measured in various tissues of control rats and rats pretreated with systemic capsaicin, s.c. (50 mg/kg as newborns or as adults, 125 mg/kg as adults) and compared with the tissue level of substance P- and tachykinin-like immunoreactivities (SP-LI and TK-LI). The rank order of CGRP-LI concentration in various tissues was as follows: trigeminal ganglion greater than urinary bladder greater than ureter much greater than distal duodenum much greater than proximal duodenum much greater than skin (snout) greater than thymus = right atrium = vas deferens. A complete depletion of CGRP-LI following capsaicin treatment of both adult and newborn animals was observed in urinary bladder, ureter, atrium, vas deferens and skin. Capsaicin pretreatment of both adult and newborn rats reduced CGRP-LI in the duodenum by about 50%. CGRP-LI in trigeminal ganglion was reduced only in newborn animals, while it was not affected in the thymus. The CGRP-LI/SO-LI ratio varied in these tissues between 33.2 (urinary bladder) and 0.9 (proximal duodenum). A significant correlation was found between CGRP-LI and SP-LI or TK-LI in tissues where immunoreactivities were depleted by capsaicin, as well as in the urinary bladder of individual animals. The correlation between CGRP-LI with SP-LI and TK-LI upon treatment with capsaicin indicates that neurons containing SP and TK as well as CGRP, and neurons containing CGRP only, are affected in a similar manner by capsaicin.

The effects of topical capsaicin on rat urinary bladder motility in vivo

The effects of topical capsaicin on urinary bladder motility were investigated following saline-induced distension of the bladder wall in urethane-anaesthetized rats and compared to the effects of topical substance P and acetylcholine. Capsaicin and substance P produced similar excitatory effects in both quiescent and rhythmically contracting bladders, i.e., a TTX resistant tonic contraction followed by a series of rhythmic, TTX sensitive, phasic contractions. Acetylcholine, in doses equieffective in producing TTX resistant contractions was less effective than capsaicin or substance P in triggering neurogenic rhythmic contractions of bladder muscle. Atropine pretreatment prevented the neurogenic component of the excitatory effect of both capsaicin and substance P. Repeated applications of capsaicin but not of substance P led to desensitization. Bladders of animals pretreated (4 days before) with a large dose of s.c. capsaicin developed insensitivity to topical capsaicin and a larger volume of saline was required to trigger neurogenic rhythmic contractions of the detrusor muscle. These results suggest that capsaicin acts by interfering with the mechanism(s) regulating the threshold for the micturition reflex to occur.

The effect of omega conotoxin GVIA, a peptide modulator of the N-type voltage sensitive calcium channels, on motor responses produced by activation of efferent and sensory nerves in mammalian smooth muscle.

Summary1.The effect of omega-conotoxin (CTX) GVIA, a peptide which blocks neuronal calcium channels, were investigated on nerve-mediated motor responses in a variety of isolated smooth muscle preparations from rats and guinea-pigs.2.In the rat or guinea-pig isolated vas deferens CTX (1 nM − 1 μM) produced a concentration and time-related inhibition of the response to field stimulation, while the responses to KCI, noradrenaline or adenosine triphosphate were unaffected. In the presence of CTX a series of tetrodotoxin-resistant contractions could be elicited by field stimulation by increasing pulse width and/or voltage.3.In the rat or guinea-pig isolated urinary bladder, CTX produced a concentration and time-dependent inhibition of twitch responses to field stimulation without affecting the response to exogenous acetylcholine. In the rat bladder the maximal effect did not exceed 25% inhibition while a much larger fraction of the response (about 70%) was inhibited in the guinea-pig bladder. The CTX-resistant response was abolished, in both tissues, by tetrodotoxin.4.The effects of CTX in the rat bladder were also studied with a whole range of frequencies of field stimulation (0.1–50 Hz). Maximal inhibition was observed toward contractions elicited at frequencies of 2–5 Hz. At low frequencies the inhibitory effects of CTX and atropine were almost additive while at high frequencies of stimulation a large component of the atropine-sensitive response was CTX-resistant.5.In the rat isolated proximal duodenum, field stimulation in the- presence of atropine and guanethidine produced a primary relaxation followed by a rebound contraction. Both responses were abolished by tetrodotoxin, indicating the activation of intramural nonadrenergic noncholinergic nerves. The primary relaxation was totally CTX resistant while the rebound contraction was slightly inhibited.6.The motor responses produced by capsaicin (1 μM) in the rat or guinea-pig bladder (contraction) and in the rat proximal duodenum (relaxation) were unaffected by CTX. Likewise, the release of substance P-like immunoreactivity from sensory nerves of the guinea-pig bladder muscle was unaffected by CTX.7.These findings indicate that CTX-sensitive calcium channels modulate transmitter release in autonomic nerve terminals of mammals, but noticeable species and organ related variations exist in sensitivity to this peptide, possibly reflecting the existence of a heterogenous population of voltage-sensitive calcium channels. CTX-sensitive calcium channels are apparently not involved in the excitatory action of capsaicin on sensory nerve terminals.

Hydrogen sulfide (H2S) stimulates capsaicin-sensitive primary afferent neurons in the rat urinary bladder

In the rat isolated urinary bladder, NaHS (30 μM–3 mM) and capsaicin (10 nM–3 μM) produced concentration‐dependent contractile responses (pEC50=3.5±0.02 and 7.1±0.02, respectively) undergoing dramatic tachyphylaxis. In preparations in which sensory nerves were rendered desensitized (defunctionalized) by high‐capsaicin (10 μM for 15 min) pretreatment, neither capsaicin itself nor NaHS produced any motor effect. NaHS‐induced contractile effects were totally prevented by the simultaneous incubation with tachykinin NK1 (GR 82334; 10 μM) and NK2 (nepadutant; 0.3 μM) receptor‐selective antagonists. Tetrodotoxin (1 μM) only partially reduced the response to NaHS. These results provide pharmacological evidence that H2S stimulates capsaicin‐sensitive primary afferent nerve terminals, from which tachykinins are released to produce the observed contraction by activating NK1 and NK2 receptors. While the molecular site of action of H2S remains to be investigated, our discovery may have important physiological significance since H2S concentrations capable of stimulating sensory nerves overlap those occurring in mammalian tissues under normal conditions.

Cyclophosphamide cystitis in rats: involvement of capsaicin-sensitive primary afferents

The involvement of capsaicin-sensitive primary afferent neurons in cyclophosphamide (CYP)-induced cystitis has been investigated in rats. CYP (150 mg/kg) was administered 48 h before testing in both vehicle- and capsaicin- (50 mg/kg s.c., 4 days before) treated rats. Some experiments were also performed 96 h after bilateral removal of pelvic ganglia to produce bladder denervation. CYP administration produced a marked detrusor hyperreflexia which was abolished by capsaicin pretreatment, demonstrating that it is mediated through stimulation of capsaicin-sensitive afferent neurons. CYP administration was followed by a marked increase in bladder weight and plasma protein extravasation (measured by the Evans blue leakage technique). The latter effect was largely prevented by ganglionectomy but was aggravated by capsaicin pretreatment. The effect of capsaicin was suppressed by ganglionectomy. Isolated strips of detrusor muscle from CYP-treated animals developed less tension in response to various stimuli as compared to strips from vehicle-treated animals; however, when contractile responses were expressed as percentage of an internal standard (carbachol-induced contraction) no difference was evident between the two groups. The bladder content of calcitonin gene-related peptide, used as a marker of the bladder afferent fibres that are capsaicin-sensitive in adult rats, was slightly reduced as compared to controls, but the difference can be accounted for by the increased bladder weight. We conclude that CYP-induced cystitis is not accompanied by a toxic effect on bladder nerves and that the decrease in bladder capacity is entirely mediated through stimulation of capsaicin-sensitive afferent fibres, presumably linked to the formation of the irritant metabolite of CYP, acrolein.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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)

Characterization of the capsaicin-sensitive component of cyclophosphamide-induced inflammation in the rat urinary bladder

1 Cyclophosphamide (CYP) (150 mg kg−1, i.p. 0.5–48 h before) caused a time‐dependent plasma protein extravasation in the rat urinary bladder with the maximal extravasation occuring at between 2 and 4 h after administration of the drug. 2 Prior capsaicin desensitization of capsaicin‐sensitive primary afferent neurones (CSPANs) (50 mg kg−l, s.c., 4 days before) resulted in approximately 50% inhibition of the magnitude of the extravasation response at the 2 h time‐point. 3 Intraperitoneal (i.p.) pretreatment with the tachykinin NK1 receptor antagonist, RP 67,580 (0.44 mg kg−1) or the bradykinin B2 receptor antagonist, Hoe 140 (0.13 mg kg−1) had significant inhibitory effects, giving responses of 56 ± 6% and 39 ± 4% of the control extravasation response to CYP treatment after 2 h. Pretreatment with the tachykinin NK2 receptor antagonist, SR 48,968 (0.3 mg kg−1, i.p.), the histamine H1 receptor blocker, chlorpheniramine (10 mg kg−1, i.p.), the 5‐HT receptor blocker, methysergide (6 mg kg−l, i.p.) or the cyclo‐oxygenase inhibitor indomethacin (5 mg kg−1, i.p.) had no significant effect upon the development of the extravasation response at this same time‐point. 4 In rat isolated urinary bladder strips, the active metabolite of CYP, acrolein (1–300 μm) produced a concentration‐dependent contraction that was significantly reduced by in vitro capsaicin desensitization (10 μm for 15 min) indicating direct stimulation of CSPANs. CYP was without appreciable effect. 5 The effect of acrolein in vitro was significantly reduced by pretreatment of the bladder with a combination of tachykinin NK1 and NK2 receptor antagonists, RP 67,580 (3 μm) and SR 48,968 (1 μm). The dose‐response curve to acrolein was also significantly inhibited by treatment with indomethacin (10 μm) and slightly affected by Hoe 140 (1 μm). 6 These findings demonstrate the contribution of CSPANs to the development of CYP‐induced cystitis. Plasma protein extravasation involves activation of tachykinin NK1 and bradykinin B2 receptors. Activation of CSPANs in the urinary bladder is likely to be due to the conversion of CYP into its active metabolite, acrolein, and not to a direct effect of CYP upon these nerve‐endings.

Prostanoids modulate reflex micturition by acting through capsaicin-sensitive afferents

Topical application of exogenous prostanoids (PGE2, TBX B2) on the serosal surface of the urinary bladder of urethane-anaesthetized rats activated reflex micturition. Likewise, intravesical instillation of PGE2 during the cystometrogram lowered the threshold for reflex micturition. Both effects were prevented by systemic capsaicin desensitization (50 mg/kg s.c., 4 days before). Indomethacin pretreatment and systemic capsaicin desensitization each increased the micturition threshold without affecting the amplitude of micturition contraction. However, the effect of the two treatments combined was not greater than the effect of either alone. These findings support the idea that endogenous prostanoids facilitate reflex micturition by stimulating or sensitizing, directly or indirectly, the subset of bladder mechanoreceptors which is capsaicin-sensitive in adult rats.