N.A. Hayes’s research while affiliated with University College London and other places

We have examined the role of extracellular chloride in the mast cell secretion process. The immunologically‐directed ligand, antibody to IgE (anti‐IgE) required extracellular chloride ions for optimum secretion from rat peritoneal mast cells. In contrast, replacement of extracellular chloride did not alter the mast cell secretory response to compound 48/80, calcium ionophore A23187 or substance P. Anti‐IgE‐stimulation of mast cells evoked a significant uptake of chloride ions compared to non‐stimulated cells. The magnitude of chloride uptake correlated with the magnitude of stimulated histamine secretion. Compound 48/80, substance P and A23187 did not alter the rate of chloride ion uptake, although these agents caused significant histamine secretion. The Na ⁺ /K ⁺ /2Cl ⁻ cotransport inhibitor, furosemide, reduced the rate of anti‐IgE‐stimulated chloride uptake at a relatively high concentration (700 μ M ). However, the more potent Na ⁺ /K ⁺ /2Cl ⁻ cotransport inhibitors, bumetanide (100 μ M ) and piretanide (100 μ M ) had no effect on the stimulated chloride uptake. Furosemide inhibited anti‐IgE‐induced histamine secretion, bumetanide potentiated the response and piretanide had no effect. This suggests that their respective action on histamine secretion are unrelated to inhibition of the Na ⁺ /K ⁺ /2Cl ⁻ carrier. The chloride channel blocker, 5‐nitro‐2‐((3‐phenylpropyl)‐amino)‐benzoic acid (NPPB), reduced both anti‐IgE‐stimulated chloride uptake and the corresponding histamine secretion in a dose‐dependent manner. The magnitude of the inhibitory action of the drug on these two cellular processes was comparable, implying that chloride channel activity is related to the mechanism of histamine secretion. It is concluded that chloride uptake has a role in the control of Fc ε RI‐mediated histamine secretion from rodent mast cells. British Journal of Pharmacology (1997) 122 , 1188–1194; doi: 10.1038/sj.bjp.0701418

It has been argued that the digital cutaneous microvasculature is the site of the anomaly which causes Raynaud's phenomenon (RP). Both endothelin-1 (ET-1), a potent vasoconstrictor peptide present in the digital cutaneous microvasculature. and calcitonin gene-related peptide (CGRP). a powerful vasodilator present in digital cutaneous perivascular nerves, have been implicated in the pathogenesis of RP. Circulating ET-1 levels are raised, and there is a diminution of CORP-containing perivascular nerves in linger skin in RP We undertook a pharmacological study to investigate the sensitivity of the digital cutaneous microvasculature to intradermal ET-l and CGRP. Differences were found in RP compared with normal digital skin, supporting the idea that the digital cutaneous microvasculature is actively involved in the pathogenesis of RP. In RP, the erythematous response to ET-1 was diminished at both 20 and 5°C (a low temperature at which RP classically occurs) providing pharmacological support for the morphological evidence that in RP there is a deficiency of CGRP-containing nerves in the distal digital skin.

The aim of this study was to investigate in human skin in vivo the role of nitric oxide in maintaining resting vascular tone, in the vasodilatation caused by local warming and by ultraviolet B light exposure, and in the response to exogenous calcitonin gene-related peptide (CGRP). Cutaneous blood flow was assessed by planimetry of the visible erythema or pallor and by laser Doppler flowmetry. Intradermal injection of the inhibitor of nitric oxide synthase, NG-nitro-L-arginine methyl ester (L-NAME; 25 nmol), into forearm skin produced a visible pallor and a reduction of blood flow at a controlled ambient temperature of 21 degrees C. The control, NG-nitro-D-arginine methyl ester (D-NAME; 25 nmol) or NG-monomethyl-L-arginine (L-NMMA; 25 nmol) did not cause pallor or reduce blood flow. L-NAME and L-NMMA caused dose- and time-dependent increases in pallor, and reductions in cutaneous blood flow in skin that had been locally warmed by immersion in water at 45 degrees C and in skin that had been exposed to ultraviolet B light. D-NAME and D-NMMA at comparable concentrations did not have the effects on skin blood flow observed with the L forms. L-NAME and L-NMMA both inhibited the increased blood flow in human skin caused by the intradermal injection of CGRP (12.5 or 25 pmol). The reduction of CGRP-induced increase of blood flow by L-NAME was reversed by L-arginine. Neither D-NAME nor D-NMMA inhibited the increase in blood flow caused by CGRP. Neither L-NAME nor L-NMMA inhibited the increase in blood flow in human skin caused by the intradermal injection of prostaglandin E2 (63 pmol). The data show that nitric oxide is involved in the maintenance of resting blood flow in human skin and also in the cutaneous vasodilator responses to local warming, ultraviolet B irradiation, or injection of CGRP.

Antigen‐stimulated histamine secretion from rat peritoneal mast cells was inhibited when extracellular chloride was replaced by either isethionate or gluconate anions, but the histamine release still remained quite substantial. Rat peritoneal mast cells take up ³⁶ Cl and the uptake reaches a steady state after 60 min incubation with the isotope. At steady state, the intracellular chloride level in the cells was calculated to be 29 ± 11.5 m m . The chloride uptake in mast cells was exponential with a rate constant of 0.036 min ⁻¹ in resting cells. When the cells were stimulated with antigen, and rate constant for chloride uptake increased to 0.90 min ⁻¹ : an increase of 25 fold. Under identical experimental conditions histamine release increased 3 fold. The rate of chloride uptake in either resting cells or in antigen‐stimulated cells was not changed when the extracellular medium was nominally calcium‐free but histamine release was almost completely inhibited in the absence of extracellular calcium. The putative chloride channel blocker DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid) 0.3 to 30 μ m , produced a concentration‐related inhibition of antigen‐stimulated histamine secretion but DIDS (30 μ m ) did not inhibit the antigen‐stimulated increase of chloride uptake. The cyclic AMP analogue, dibutyryl cyclic AMP (1 m m ) produced a delayed increase in chloride uptake in resting mast cells but neither dibutyryl cyclic AMP nor 8‐bromo cyclic AMP per se induced any histamine secretion. Ouabain (1 m m ) which inhibits the Na ⁺ /K ⁺ ATPase in rat peritoneal mast cells, failed to affect the uptake of chloride in resting mast cells. The Na/K/2Cl‐cotransport inhibitor, furosemide (0.7 m m ), slowed the unstimulated chloride uptake in resting mast cells and abolished the increased antigen‐induced chloride uptake when added together with antigen. In contrast, spontaneous and antigen‐induced histamine release were unaffected by the presence of furosemide. However, when furosemide was added to the cell suspension 5 min before stimulation, furosemide was without effect on the antigen‐induced chloride uptake. In addition to the chloride uptake mediated by chloride channels which may be related to the mechanism of histamine secretion, crosslinking of the high affinity membrane receptors for IgE is followed by a fast chloride uptake that is likely to occur through a furosemide‐sensitive Na/K/2Cl‐cotransporter.

We have attempted to reproduce the findings of Benveniste and co-workers, who reported in 1988 that degranulation of human basophil leukocytes is triggered by very dilute (10(2)-10(120)) antiserum against IgE. The results were contrary to conventional scientific theory and were not satisfactorily explained. Following as closely as possible the methods of the original study, we can find no evidence for any periodic or polynomial change of degranulation as a function of anti-IgE dilution. Our results contain a source of variation for which we cannot account, but no aspect of the data is consistent with the previously published claims.

The CGRP antagonist, CGRP8-37, antagonized the ability of CGRP to increase blood flow in human skin and cause erythema. The mechanism of the antagonist effect of CGRP8-37 on the CGRP-induced erythema was an increase by the antagonist of the rate of decay of the CGRP-induced erythema. Since CGRP8-37 activates rat peritoneal mast cells to release their granular contents, we conclude that the degradation of CGRP by protease released from skin mast cells by CGRP8-37. It was not possible to demonstrate any antagonistic effect of CGRP8-37 on the CGRP receptor mediating increased blood flow in human skin.

When injected into human skin, endothelin-1 produces intense vasoconstriction localized to the site of the injection, but this area of vasoconstriction is surrounded by vasodilatation which spreads several centimetres from the injection site. The vasodilatation induced by intradermal injection of endothelin-1 (63 pmol) into human skin is prevented by local anaesthetic. Pretreatment of human skin with capsaicin also inhibits this response. Pretreatment of subjects with the selective histamine H1-receptor antagonist cetirizine, 10 mg orally 4 h before intradermal injections, inhibited vasodilatation caused by the intradermal injection of histamine (750 pmol), endothelin-1 (63 pmol), and carbachol (750 pmol). Endothelin-1 (0.3-10 microM) and carbachol (1-30 microM) failed to induce histamine release from rat peritoneal mast cells. We conclude that the vasodilatation caused by intradermal injection of endothelin-1 into human skin is neurogenic and is probably mediated by neuropeptide-containing primary afferent neurones. Because neither carbachol nor endothelin-1 cause histamine release from mast cells, our data suggest that histamine release from mast cells at the effector end of the axon reflex is responsible for the carbachol- and endothelin-induced vasodilatation in human skin.

Cutaneous wheal and flare responses to increasing concentrations of calcitonin gene-related peptide, substance P, neurokinin A, vasoactive intestinal polypeptide (VIP), compound 48/80, and phosphate-buffered saline were measured in 10 patients with chronic idiopathic urticaria and 10 healthy controls. A significant increase in VIP-induced wheal, but not flare or cutaneous blood flow, was seen in urticarial patients compared with controls (p less than 0.001). No significant differences in responses to other tested compounds were found between these groups. These data point to an increased sensitivity of microvasculature to VIP in patients with chronic idiopathic urticaria.