Cutaneous vasculitidis: histology and immunofluorescence.

Cutaneous vasculitis comprises a wide spectrum of diseases that involve predominantly the blood vessels and surrounding tissues of the skin. Few vasculitic syndromes have pathognomonic clinical, radiographic and/or laboratory findings; thus, confident and accurate diagnosis of vasculitis requires histological confirmation. Skin biopsy should be done, optimally within 24 to 48 hours after vasculitic lesions appear. Deep excision biopsy must be preferred. Direct immunofluorescence of lesional skin is helpful in the diagnosis of vasculitides in the light of a proper clinico-pathological setting and diagnostic in some peculiarly forms. Cutaneous histological patterns can be used to generate relevant clinical differential diagnoses, and, when coupled with patient's history, clinical and laboratory data, allow more precise and accurate diagnosis of vasculitic syndromes. This review will focus on histopathological and immunologic pattern of the more common cutaneous vasculitis syndromes, based on the 2012 Revised International CHCC.

Regulatory T cells in skin lesions and blood of patients with bullous pemphigoid.

BACKGROUND: Although regulatory T cells (Tregs) are affected in several autoimmune skin diseases, only two studies have been performed in patients with bullous pemphigoid (BP) with contrasting results. OBJECTIVE: To characterize Tregs and to determine the serum levels of regulatory cytokines in patients with BP. METHODS: In BP lesional skin, immunohistochemistry and confocal microscopy were performed for CD4(+) , CD25(+) , forkhead/winged helix transcription factor (FOXP3)(+) , transforming growth factor (TGF)-ß(+) and interleukin (IL)-10(+) cells. In addition, the number of CD4(+) CD25(++) FOXP3(+) Tregs in peripheral blood was assessed by flow cytometry, and the levels of TGF-ß and IL-10 were determined in serum samples by enzyme-linked immunosorbent assay before and after steroid therapy. Controls included patients with psoriasis, atopic dermatitis (AD) and healthy donors. RESULTS: The frequency of FOXP3(+) cells was significantly reduced in skin lesions from patients with BP (P < 0.001) compared with psoriasis and AD. Moreover, the number of IL-10(+) cells was lower in BP than in psoriasis (P < 0.001) and AD (P = 0.002), while no differences were observed in the number of TGF-ß(+) cells. CD4(+) CD25(++) FOXP3(+) Treg in the peripheral blood of patients with BP was significantly reduced compared with healthy controls (P < 0.001), and augmented significantly after steroid therapy (P = 0.001). Finally, TGF-ß and IL-10 serum levels were similar in patients with BP compared with healthy controls. However, after therapy, BP patients showed significantly higher IL-10 serum levels than before therapy (P = 0.01). CONCLUSIONS: These data suggest that the depletion of Tregs and of IL-10 in patients with BP may be an important factor in the pathogenesis of the disease.

Serum levels of the regulatory cytokines transforming growth factor-ß and interleukin-10 are reduced in patients with discoid lupus erythematosus.

T-lymphocytes are believed to play an important role in the pathogenesis of discoid lupus erythematosus (DLE). However, the reasons that lead to loss of tolerance and to development of autoimmunity in DLE remain unclear. In the present paper, we investigated serum levels of the regulatory cytokines transforming growth factor (TGF)-ß and interleukin (IL)-10 in 25 newly diagnosed patients with DLE, 15 with systemic lupus erythematosus (SLE), 10 with psoriasis, 10 with atopic dermatitis (AD) and 20 healthy controls (HC). TGF-ß serum levels were significantly lower in patients with DLE compared with patients with psoriasis and HC, while no differences were found between DLE, SLE and AD (medians: DLE: 28.49 ng/ml; psoriasis: 42.77 ng/ml; HC: 43.71 ng/ml; DLE vs. psoriasis: p < 0.05; DLE vs. HC: p < 0.05). IL-10 concentrations were reduced in DLE serum samples with respect to SLE, psoriasis, AD and HC (medians: DLE: 46.42 pg/ml; SLE: 127.64 pg/ml; psoriasis: 109.3 pg/ml; AD: 76.3 pg/ml; HC: 114.71 pg/ml; DLE vs. SLE: p < 0.05; DLE vs. psoriasis: p < 0.05; DLE vs. AD: p < 0.05; DLE vs. HC: p < 0.05). The downregulation of TGF-ß and IL-10 in DLE may lead to defective immune suppression and thus to the generation of the tissue injury that is found in lupus patients.

Characterization of regulatory T cells in patients with dermatomyositis.

The purpose of this study was to characterize regulatory T cells (T(reg)) in skin lesions and peripheral blood from patients with dermatomyositis (DM) and to determine the serum levels of regulatory cytokines in the disease. In skin biopsy specimens from patients with DM, immunohistochemistry was performed for CD4(+), CD25(+), forkhead/winged helix transcription factor (FoxP3)(+), transforming growth factor (TGF)-ß(+) and interleukin (IL)-10(+) cells. Additionally, we defined the number of T(reg) subpopulations in peripheral blood by flow cytometry using monoclonal antibodies against CD4, CD25, FoxP3, CD45RO, CD95, CCR4 and CLA. The levels of TGF-ß and IL-10 were also determined in serum samples from patients with DM by enzyme-linked immunosorbent assays. Controls included patients with cutaneous lupus erythematosus, psoriasis and atopic dermatitis (AD) as well as healthy donors. The frequency of FoxP3(+) cells was significantly reduced in skin lesions from patients with DM (p < 0.001) compared to psoriasis and AD. Moreover, the number of cells positive for TGF-ß was lower in DM than in psoriasis and AD, while IL-10(+) cells were significantly reduced only compared to psoriasis. The number of CD4(+)CD25(++)FoxP3(+) T(reg) in the peripheral blood of patients with DM was significantly reduced compared to healthy controls (p < 0.05), whereas other cell populations showed no significant differences. Finally, TGF-ß and IL-10 serum levels were significantly lower in patients with DM compared to healthy controls (p < 0.05). These data suggest that the depletion of T(reg) and their main effector cytokines in the skin and the serum of patients with DM may be an important factor in the pathogenesis of the disease.

Regulatory T cells in the skin lesions and blood of patients with systemic sclerosis and morphoea.

BACKGROUND: Systemic sclerosis (SSc) and morphoea are connective tissue diseases characterized by fibrosis of the skin. Although to date their pathogenesis has not been clearly defined, it is thought that autoimmunity may play a role in the development of the skin lesions observed in both these diseases. As regulatory T cells (Tregs) play a key role in the modulation of immune responses, it has recently been suggested that Treg impairment may lead to the development of autoimmune diseases. OBJECTIVES: To investigate the presence of Tregs and their immunomodulatory cytokines, transforming growth factor (TGF)-beta and interleukin (IL)-10, in patients with SSc and morphoea. PATIENTS/METHODS: Fifteen patients with SSc and 15 with morphoea were enrolled. Immunohistochemistry was applied to identify FoxP3+ (forkhead/winged helix transcription factor) Tregs, TGF-beta+ cells and IL-10+ cells in the skin, cytofluorometry to detect CD4+CD25+FoxP3+ Tregs in the blood, and enzyme-linked immunosorbent assays to analyse TGF-beta and IL-10 serum levels. RESULTS: Fewer FoxP3+ Tregs and TGF-beta+ and IL-10+ cells were found in the skin of patients with scleroderma than in controls. Similarly, there were reduced TGF-beta and IL-10 serum levels and fewer circulating CD4+CD25brightFoxP3+ cells in patients with SSc or morphoea, than in controls. CONCLUSIONS: The quantitative reduction of Tregs, together with that of TGF-beta and IL-10 serum levels, may be responsible for the loss of tolerance observed in both SSc and morphoea.

Serum levels of IL-17 and IL-22 are reduced by etanercept, but not by acitretin, in patients with psoriasis: a randomized-controlled trial.

INTRODUCTION: There are no controlled trials comparing etanercept and acitretin efficacy and therapeutic mechanisms in psoriasis. MATERIALS AND METHODS: In the present study, 30 patients were given etanercept 50 mg twice weekly and 30 patients acitretin 0.4 mg/kg per day, both for 12 weeks. Before and after treatment, psoriasis area and severity index was calculated, and serum levels of interleukin (IL)-17, IL-22, and IL-23 were investigated. RESULTS: After treatment, psoriasis area and severity index was significantly lower for both groups. However, etanercept-treated patients showed lower psoriasis area and severity index than acitretin-treated ones. Psoriasis patients showed higher IL-17 and IL-22 levels than controls, while no IL-23 was found in any serum. Furthermore, a correlation between IL-17 levels and psoriasis severity was found. Only etanercept was able to reduce IL-17 and IL-22 levels. CONCLUSIONS: Our findings suggest that etanercept is more effective than acitretin in the treatment of psoriasis and that it is able to affect Th17 system.

Regional differences of adenylate cyclase stimulation by calcitonin and calcitonin gene-related peptide in the human kidney.

Calcitonin (CT) gene-related peptide (CGRP)-like immunoreactivity was detected in both the cortex and medullo-papillary portion of human kidneys. The two forms of human CGRP as well as rat CGRP were capable of stimulating renal cortical adenylate cyclase activity in a concentration-related manner, with a half-maximally effective concentration (EC50) similar to that of human CT and approximately 100-1000 times higher than that of salmon CT. However, in the medullo-papillary portion, in which both salmon CT and human CT were inactive, the two forms of human and rat CGRP increased adenylate cyclase activity by 100%, with EC50 values ranging from 36 nmol/L to 1 mumol/L. In cortical membrane preparations the effect of CGRP was additive to that of salmon CT. We concluded that regional differences exist in the effect of CT and CGRP in human renal tissue and that in the medullo-papillary portion and possibly in the cortex, CGRP stimulates adenylate cyclase activity through a CT-independent mechanism.

Effect of the GABAB antagonist, phaclofen, on baclofen-induced inhibition of micturition reflex in urethane-anesthetized rats.

The effect of intrathecal or intracerebroventricular administration of the GABAB receptor agonist, baclofen, on rhythmic contractions induced by distension of the urinary bladder (micturition reflex) was evaluated in urethane-anesthetized rats. Baclofen inhibited bladder motility acting at central nervous system sites (spinal and supraspinal) with a comparable potency. The inhibitory effect of i.t. baclofen (0.1-10 nmol) was blocked by i.t. phaclofen (200 nmol) while i.c.v. phaclofen did not affect i.c.v. baclofen (0.1-1 nmol). The inhibition of the micturition reflex induced by bladder distension observed after i.t. administration of baclofen was unaffected by systemic capsaicin pretreatment (50 mg/kg s.c., four days before). On the other hand, i.t. baclofen suppressed, in a phaclofen-sensitive manner, the reflex bladder contraction evoked by chemical stimulation (topical capsaicin) of capsaicin-sensitive bladder afferents. Intrathecal baclofen did not affect the hexamethonium-resistant tonic contraction produced by topical application of capsaicin on to the urinary bladder, which is ascribable to local peptide release from sensory nerves. Bladder motility inhibition by i.t. or i.c.v. baclofen (1 nmol) was unchanged by previous administration of p-chlorophenylalanine, indicating that the serotonergic pathways do not play a role in its action. Baclofen (100 microM) suppressed the release of calcitonin gene-related peptide-like immunoreactivity evoked by electrical field stimulation from the dorsal half of the rat spinal cord. This response was also abolished by in vitro capsaicin desensitization or tetrodotoxin, indicating that baclofen suppresses transmitter release from central endings of capsaicin-sensitive primary afferents. The present findings indicate that baclofen acts at both spinal and supraspinal sites to inhibit, with different mechanisms, the micturition reflex.(ABSTRACT TRUNCATED AT 250 WORDS)

Low pH-induced release of calcitonin gene-related peptide from capsaicin-sensitive sensory nerves: mechanism of action and biological response.

Protons can release in a Ca(2+)-dependent manner, calcitonin gene-related peptide (CGRP)-like immunoreactivity from peripheral endings of capsaicin-sensitive afferents. Here we have studied the mechanism by which proton promotes CGRP-like immunoreactivity release and whether the neuropeptide released might exert a biological action. In muscle slices of guinea-pig urinary bladder high pH (pH 8 or 9) media neither enhanced CGRP-like immunoreactivity outflow nor affected the capsaicin-evoked CGRP-like immunoreactivity release. The CGRP-like immunoreactivity release evoked by superfusion with pH 5 medium was not affected by tetrodotoxin (0.3 microM) indomethacin (10 microM) or the protein kinase C inhibitor H-7 (30 microM). However, it was reduced by 35% in the presence of the voltage-sensitive Ca(2+)-channel antagonists nifedipine (1 microM) and omega-conotoxin (0.1 microM) and by 80% in presence of the capsaicin "antagonist" Ruthenium Red (10 microM). The CGRP-like immunoreactivity release by capsaicin (10 microM) was reduced by 80% in the presence of Ruthenium Red, and not affected by voltage-sensitive Ca(2+)-channel blockers, while that evoked by 80 mM K+ was decreased by 82% in the presence of nifedipine and omega-conotoxin. The Ca(2+)-channel agonist Bay K 8644 enhanced the high K(+)-evoked CGRP-like immunoreactivity release but not that induced by capsaicin or pH 5 medium. Exposure to pH 6 solution of one half of the neck of guinea-pig urinary bladder induced a slowly developing inhibition of electrically evoked contractions, that was absent in the half pre-exposed in vitro to a desensitizing dose of capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS)

Bradykinin-induced release of calcitonin gene-related peptide from capsaicin-sensitive nerves in guinea-pig atria: mechanism of action and calcium requirements.

The mechanism of neuropeptide secretion induced by bradykinin from capsaicin-sensitive afferents was studied in guinea-pig atria. Both the inotropic response induced by bradykinin (0.1 microM) in the electrically driven isolated guinea-pig left atria and the bradykinin (10 microM)-induced release of calcitonin gene-related peptide calcitonin gene-related peptide-like immunoreactivity from slices of guinea-pig atria were abolished in vitro by capsaicin pretreatment or in the presence of indomethacin. Bradykinin-induced calcitonin gene-related peptide-like immunoreactive release was unaffected by tetrodotoxin (0.3 microM), the protein kinase C inhibitor, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (30 microM), nefedipine (1 microM) or Ruthenium Red (10 microM). It was significantly reduced by 79% in a Ca2(+)-free medium and by 52% in the presence of 0.1 microM omega-conotoxin (fraction GVIA). It is proposed that bradykinin releases calcitonin gene-related peptide from capsaicin-sensitive afferents in guinea-pig atria, via prostanoid generation. This mode of activation of the "efferent" function of capsaicin-sensitive nerves appears to be distinct from those produced by capsaicin or electrical field stimulation as they have been characterized in previous works. In fact, the bradykinin activation of capsaicin-sensitive afferents is not affected by tetrodotoxin and Ruthenium Red, but is partially sensitive to the selective blocker of N-type Ca2(+)-channels, omega-conotoxin.

Calcitonin gene-related peptide in the rat kidney: occurrence, sensitivity to capsaicin, and stimulation of adenylate cyclase.

The occurrence, effects and sensitivity to capsaicin and stimulation of adenylate cyclase of calcitonin gene-related peptide (CGRP) in the rat kidney have been investigated. CGRP-like immunoreactivity was higher in the medulla than in the papilla and the cortex. Capsaicin pretreatment significantly reduced CGRP-like immunoreactivity in the medulla and papilla while a small reduction was found in the cortex. CGRP-immunoreactive nerve fibres were observed surrounding blood vessels and occasionally in the vicinity of renal tubules and between the collecting ducts in the papilla. Some CGRP-immunoreactive fibres were also seen in kidneys from capsaicin-pretreated rats. Infusion of capsaicin (1 microM) through the renal artery of isolated and perfused rat kidney increased the CGRP-like immunoreactivity outflow from the venous effluent. This effect exhibited desensitization at the second challenge with the drug. Infusion of either capsaicin (1 microM) or CGRP (1 microM) reduced the increase of perfusion pressure induced by norepinephrine in isolated perfused rat kidney. Plasma protein extravasation was studied in the various regions of the rat kidney following infusion of capsaicin. No significant change was observed in the medulla, papilla or cortex after capsaicin administration. Adenylate cyclase activity was studied in membrane preparations from cortex, medulla and papilla of rat kidney. Cortical and medullary adenylate cyclase was stimulated in a concentration-dependent manner by salmon calcitonin, rat calcitonin and rat CGRP. Salmon calcitonin in these two areas showed half-maximal effective concentration approximately 1000 times lower and maximal stimulation only slightly higher than those of rat calcitonin and rat CGRP. However, in the papilla, only rat CGRP was able to induce a 60% increase of enzyme activity (half-maximal effective concentration, 19 +/- 1.6 nM). It is concluded that CGRP contained in capsaicin-sensitive sensory nerve may exert a local function in discrete areas of the rat kidney.

Effects of capsaicin and 5-HT3 antagonists on 5-hydroxytryptamine-evoked release of calcitonin gene-related peptide in the guinea-pig heart.

1. The effect of 5-hydroxytryptamine (5-HT) on the release of calcitonin gene-related peptide (CGRP) was studied directly in the isolated perfused heart and indirectly in the isolated left atria of guinea-pig. 2. 5-HT injection into the guinea-pig isolated and perfused heart evoked a dose-dependent (1-100 microM) release of CGRP-like immunoreactivity (LI) that was abolished by in vitro pretreatment with capsaicin and was not affected by indomethacin. 3. Chlorophenyldiguanide (CPD, 100 microM), but not 8-hydroxy-dipropylaminotetralin (8-OH-DPAT, 100 microM), sumatriptan (100 microM) or 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, 100 microM) evoked a release of CGRP-LI. Ondansetron (10 microM) or ICS205-930 (20 microM) completely abolished the 5-HT (100 microM)-evoked CGRP-LI release. 4. In the isolated electrically driven left atria of the guinea-pig 5-HT (1-10 microM) and CPD (3-100 microM) produced a positive inotropic response, which was abolished by capsaicin pretreatment. 8-OH-DPAT (10 microM) and DOI (10 microM) were inactive. Ondansetron inhibited the response to 5-HT with a pA2 of 6.50 (CL 6.08-6.91). 5. It is concluded that 5-HT causes a release of CGRP in the whole heart and a positive inotropic response in the isolated atria of guinea-pig. Both these effects are sensitive to capsaicin pretreatment and to 5-HT3 antagonists.

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 microM), 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 capsiacin, low pH medium and KCL, respectively. 3. Prior application of capsiacin (10 microM for 30 min followed by 30 min of washout) to produce capasaicin desensitization in vitro abolished CGRP-LI release induced by the three stimuli. 4. Capsazepine (1-100 microM, 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 microM capsazepine, respectively. Low pH-induced release was inhibited by 78, 84, 88 and 93% with 3, 10, 30 and 100 microM capsazepine, respectively. KCl-induced release was significantly inhibited by 55 and 93% with 30 and 100 microM (but not with 10 microM) 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.

Possible involvement of neuropeptidergic sensory nerves in alopecia areata.

Alopecia areata (AA) is a dermatosis involving the sudden occurrence of bald patches on the scalp. Although the aetiology is unknown, experimental data indicate that cutaneous microcirculation plays an important role. The skin is richly innervated by neuropeptidergic sensory nerves that help regulate microvascular circulation. This study shows a reduction of cutaneous levels of substance P and calcitonin gene-related peptide (CGRP) but not of vasoactive intestinal polypeptide in scalp biopsies from patients with AA. Laser-Doppler flowmetry was used to study microcirculation of the scalp. Results indicate that patients with AA have lower basal blood flow and greater vasodilatation following intradermal CGRP injection than control subjects. A vascular hyper-reactivity to vasodilatatory substances such as neuropeptides, probably because of the lack of these substances, is hypothesized.

Capsaicin releases calcitonin gene-related peptide from the human iris and ciliary body in vitro.

Slices of human iris or ciliary body, obtained post-mortem (8-12 h after death, n = 5), were superfused in vitro with capsaicin (10 microM) and the immunoreactivity for substance P (SP-LI) or calcitonin gene-related peptide (CGRP-LI) was measured in the effluent. In the iris and in the ciliary body CGRP-LI was 3.71 +/- 0.74 pmol/g and 3.01 +/- 0.55 pmol/g and SP-LI was 6.68 +/- 0.75 pmol/g and 6.55 +/- 0.84 pmol/g, respectively. A first exposure to capsaicin increased the CGRP-LI outflow from the ciliary body (427 +/- 46 fmol/g/30 min), whereas a second challenge with the drug 30 min later, failed to significantly enhance the CGRP-LI outflow (21.8 +/- 15.6 fmol/g/30 min). Likewise, the capsaicin-evoked increase in CGRP-LI outflow from the iris slices (472 +/- 62 fmol/g/30 min) was no longer observed at the second drug administration (38.4 +/- 12.8 fmol/g/30 min). Capsaicin failed to increase the SP-LI outflow from either the iris or the ciliary body. Reverse phase HPLC analysis of CGRP-LI indicated that authentic CGRP was contained in the tissue and in the superfusate collected during exposure to capsaicin. The present results show that in the human iris and ciliary body, capsaicin releases CGRP possibly contained in terminals of sensory nerves.

Different pathways by which extracellular Ca2+ promotes calcitonin gene-related peptide release from central terminals of capsaicin-sensitive afferents of guinea pigs: effect of capsaicin, high K+ and low pH media.

Different modes by which Ca2+, entering the nerve terminal, promotes transmitter secretion as well as the ability of protons to release neuropeptides, have been shown in peripheral endings of capsaicin-sensitive afferents. We have studied these two aspects in the central endings of these neurons by measuring the release of calcitonin-gene related peptide-like immunoreactivity (CGRP-LI) from slices of the dorsal half of the guinea pig spinal cord. Although capsaicin (1 microM) released both CGRP-LI and substance P-like immunoreactivity (SP-LI), CGRP-LI was chosen as the sole suitable marker of peptides released from central terminals of capsaicin-sensitive afferents, since after in vitro desensitization to capsaicin (1 microM capsaicin for 20 min), high K+ (80 mM) failed to evoke CGRP-LI release, whereas SP-LI release was still observed. The capsaicin (1 microM)-evoked CGRP-LI release was entirely dependent on extracellular Ca2+. It was unaffected by 0.3 microM tetrodotoxin (TTX), slightly reduced by 0.1 microM omega-conotoxin (CTX) and blocked by 10 microM Ruthenium red (RR). The Ca(2+)-dependent K+ (80 mM)-evoked CGRP-LI release was unaffected by TTX, markedly reduced by CTX and only moderately inhibited by RR. Low pH (pH 5) produced a remarkable increase in CGRP-LI outflow that was abolished after exposure to capsaicin, reduced by about 50% in Ca(2+)-free medium and unaffected by TTX (0.3 microM). The Ca(2+)-dependent component of the proton-evoked CGRP-LI release was abolished in the presence of RR (10 microM) and slightly inhibited by CTX (0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

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 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.

The 'efferent' function of capsaicin-sensitive nerves: ruthenium red discriminates between different mechanisms of activation.

We have investigated the ability of Ruthenium Red, an inorganic dye with calcium entry blocking properties, to interfere with the 'efferent' function of capsaicin-sensitive sensory nerves. These nerves were activated in the guinea-pig isolated bronchus (atropine in the bath) or left atria (reserpine-pretreated animals, atropine in the bath) by electrical field stimulation or with capsaicin. Both stimuli produced a contraction of the bronchus and a positive inotropic response in the atria, responses which are mediated by endogenous neuropeptides (tachykinins in the bronchus, calcitonin gene-related peptide in the atria) released from sensory nerves. Ruthenium Red (10 microM for 20 min in both cases) selectively inhibited the responses produced by the administration of capsaicin, while leaving the responses to electrical field stimulation unaffected. Likewise, the bronchoconstrictor response to exogenous neurokinin A and the atrial positive inotropic response to calcitonin gene-related peptide were unaffected by Ruthenium Red. A prejunctional site of action of Ruthenium Red was confirmed in release experiments where the dye strongly inhibited the capsaicin-evoked outflow of calcitonin gene-related peptide, which is taken as a marker of activation in sensory nerves. Together with other observations, these findings support the concept that there are two independent mechanisms for activating the 'efferent' function of sensory nerves, one of which is activated by capsaicin and is Ruthenium Red-sensitive but omega-conotoxin-resistant, while the other is activated by propagated action potentials (field stimulation) and is omega-conotoxin-sensitive and Ruthenium Red-resistant.