Elena Del Bianco

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.

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)

Release of calcitonin gene-related peptide like-immunoreactivity induced by electrical field stimulation from rat spinal afferents is mediated by conotoxin-sensitive calcium channels

Electrical field stimulation (EFS, 10 V, 50 mA/cm2, 5-50 Hz, 1 ms pulse duration, 10 s train every 20 s for 5 min) produced a rapid and reproducible outflow of calcitonin gene-related peptide like-immunoreactivity (CGRP-LI) from superfused slices from the dorsal half of the rat spinal cord which is abolished by tetrodotoxin (TTX, 0.3 microM), in vitro capsaicin desensitization (10 microM for 30 min) and in Ca-free medium. The response was unaffected by ruthenium red (10 microM), indomethacin (10 microM) and nifedipine (1 microM) while it was abolished by omega-conotoxin (omega-CTX, 0.1 microM) and, in a naloxone-sensitive manner, by morphine (3 microM). Since CGRP release from capsaicin-sensitive afferents evoked by EFS in rat peripheral tissues is conotoxin-resistant, these findings provide direct evidence for a qualitative difference between central and peripheral endings of capsaicin-sensitive primary afferents in the mechanisms regulating transmitter release in the same species.

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.

Adenosine A1 receptors mediate the presynaptic inhibition of calcitonin gene-related peptide release by adenosine in the rat spinal cord

Electrical field stimulation evoked a reproducible outflow of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from the dorsal half of the rat spinal cord, an effect which was abolished by prior application of capsaicin, tetrodotoxin or removal of extracellular Ca2+. Adenosine (EC50 3.2 microM) and the selective adenosine A1 receptor agonist N6-cyclohexyladenosine (EC50 8.2 nM) inhibited evoked CGRP-LI outflow, while the selective adenosine A2 receptor agonist CGS-21680 was ineffective up to 10 microM. The action of adenosine was prevented by the adenosine A1 receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (30 microM), which did not affect CGRP-LI release on its own.

Release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from organs of the genitourinary tract in rats

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was detected in the rat urinary bladder, ureter and vas deferens and was depleted by systemic capsaicin desensitization. Exposure to capsaicin in vitro produced a prompt increase in CGRP-LI outflow in superfusates of these tissues, while a second application of the drug was ineffective indicating desensitization. These findings provide further evidence for a transmitter role of CGRP from peripheral endings of sensory nerves and the involvement of CGRP-LI in the specific motor response to capsaicin in the rat genitourinary tract.

Effect of capsazepine on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) induced by low pH, capsaicin and potassium in rat soleus muscle.

1 We have determined the effect of the competitive antagonist capsazepine at the capsaicin receptor on the release of calcitonin gene‐related peptide‐like immunoreactivity (CGRP‐LI) from rat isolated soleus muscle induced by capsaicin (1 μm), by superfusion with low pH medium (pH 5) or by KCl (80 mm). 2 Each one of the three stimuli tested produced a marked CGRP‐LI release. Total evoked release (fmol g−1) was 482 ± 69, 169 ± 20 and 253 ± 43 for capsaicin, low pH medium and KCl, respectively. 3 Prior application of capsaicin (10 μm for 30 min followed by 30 min of washout) to produce capsaicin desensitization in vitro abolished CGRP‐LI release induced by the three stimuli. 4 Capsazepine (1–100 μm, 45 min preincubation) inhibited the evoked CGRP‐LI release. Capsaicin‐induced release was significantly inhibited by 77, 92 and 96% with 10, 30 and 100 μm capsazepine, respectively. Low pH‐induced release was inhibited by 78, 84, 88 and 93% with 3, 10, 30 and 100 μm capsazepine, respectively. KCl‐induced release was significantly inhibited by 55 and 93% with 30 and 100 μm (but not with 10 μm) capsazepine, respectively. 5 These findings demonstrate that capsazepine prevents low pH‐ and capsaicin‐induced CGRP‐LI release from rat soleus muscle at concentrations which do not affect the release evoked by KCl. These findings imply a relationship between the action of low pH and activation of the capsaicin receptor. At high concentrations, capsazepine produces a nonspecific inhibitory effect on CGRP‐LI release from peripheral endings of the capsaicin‐sensitive primary afferent neurone.

Release of sensory neuropeptides from dural venous sinuses of guinea pig.

Substance P- and calcitonin gene-related peptide-like immunoreactivities (SP-LI and CGRP-LI, respectively) were measured in superfusates of either superior sagittal sinus and transverse sinuses and attached dura mater or dura mater alone of guinea pig. Exposure of cerebral venous sinuses to capsaicin (1 microM) evoked the release of both SP-LI and CGRP-LI, which was no longer observed upon second challenge with the drug. Neuropeptide release was induced by 80 mM K+ either at the first or second administration. Bradykinin (10 microM) increased the outflow of CGRP-LI, but not of SP-LI, from cerebral venous sinuses. In vitro capsaicin pretreatment (10 microM) or incubation with 10 microM indomethacin completely abolished the bradykinin-evoked CGRP-LI release. Capsaicin (1 microM) failed to evoke release from dura mater without major intracranial venous vessels. Sensory neuropeptide released from the cerebral venous sinuses may take part in certain symptoms, such as vasodilatation and inflammation accompanying the pain of the migraine attack. Bradykinin, putatively via prostanoid generation, may participate in this event.

Adenosine inhibits action potential-dependent release of calcitonin gene-related peptide- and substance P-like immunoreactivities from primary afferents in rat spinal cord

Electrical field stimulation (5 Hz) evoked a prompt outflow of calcitonin gene-related peptide- and substance P-like immunoreactivities (CGRP-LI and SP-LI, respectively) from superfused slices of the dorsal but not ventral half of the rat spinal cord. The evoked outflow was abolished by tetrodotoxin, calcium-free medium or previous exposure to capsaicin, indicating that it is produced through action potentials invading the central terminals of capsaicin-sensitive primary afferents. Adenosine as well as gamma-aminobutyric acid (GABA) or the GABAB receptor agonist (-)-baclofen produced a concentration-dependent inhibition of the evoked CGRP-LI outflow. Adenosine also inhibited the evoked SP-LI outflow. These findings demonstrate that inhibition of transmitter release from primary afferent neurons should be considered as a possible mechanism of the antinociceptive action of adenosine and adenosine analogs.

The effects of tacrolimus ointment on regulatory T lymphocytes in atopic dermatitis.

Only very recently studies were conducted in order to evaluate the impact of regulatory T (Treg) cells in the pathophysiology of atopic dermatitis (AD). Nine adult patients with moderate-to-severe AD in riacutization period of a chronic disease were given tacrolimus ointment, while seven hydrocortisone butyrate ointment, that served as controls. We performed lesional-skin biopsies before and after treatment, that were stained immunohistochemically with monoclonal antibodies to CD4, CD25, forkhead/winged helix transcription factor (FoxP3), interleukin (IL)-10 and transforming growth factor (TGF)-β. CD4+ cells were significantly reduced in post-treatment series. Tacrolimus treatment achieved a significant reduction of CD25+ cells. FoxP3+ cells were present in untreated AD lesions. Both treatments did not significantly modify the number of FoxP3+ cells. The number of IL-10+ cells increased in post-treatment series. Tacrolimus enhanced the production of TGF-β, while hydrocortisone did not.Restoration of TGF-β-producing Treg cells may represent another important pharmacodynamic effect of tacrolimus on AD.