Mechanisms of vasoactive intestinal peptide-mediated vasodilation in human skin.

Vasoactive intestinal peptide (VIP) is known to induce histamine release in human skin and to include a nitric oxide (NO)-dependent dilation in several other vascular beds. However, the relative contribution of histamine and NO to VIP-mediated vasodilation in human skin is unknown. Forty-three subjects volunteered to participate in two studies designed to examine the mechanism of VIP-mediated vasodilation in human skin. Study 1 examined the contribution of NO in the skin blood flow response to eight doses of VIP ranging from 25 to 800 pmol. In addition, study 1 examined a specific role for NO in VIP-mediated dilation. Study 2 examined the relative contribution of NO and histamine to VIP-mediated dilation via H1 and H2 histamine receptors. Infusions were administered to skin sites via intradermal microdialysis. Red blood cell flux was measured by using laser-Doppler flowmetry (LDF), and cutaneous vascular conductance (CVC; LDF/mean arterial pressure) was calculated and normalized to maximal vasodilation. VIP-mediated vasodilation includes a NO-dependent component at doses above 100 pmol, where NO synthase inhibition significantly attenuates CVC (P < 0.05). Inhibition of H1 receptors attenuates the rise in CVC to exogenous VIP (P < 0.05); however, combined H1-receptor inhibition and NO synthase inhibition further reduced VIP-mediated vasodilation compared with either H1 inhibition or NO synthase inhibition alone (P < 0.05). In contrast to H1-receptor inhibition, H2-receptor inhibition did not affect vasodilation to exogenous VIP. Thus, in human skin, VIP-mediated vasodilation includes a NO-dependent component that could not be explained by H1- and H2-receptor activation.

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title: Immunohistochemical localization of a vasodilatory polypeptide (VIP) in cerebrovascular nerves
abstract: Brain vessels are richly supplied with sympathetic (adrenergic) and parasympathetic (cholinergic) nerves, which have been visualized by a variety of techiquesS,7,10,13, 14. In the course of our ultrastructural studies 7,13 on the cerebrovascular innervation of rats and cats, we observed a third type of nerves which, by the appearance of the vesicles present in their axon terminals, did not conform with either of the two reported systems of autonomic nerves. These axons were characterized by the predominance of large vesicles, 85-200 nm in diameter, containing a moderately electron-dense core filling the entire vesicle. They rather resembled nerves designated as "p-type" fibres 2 because of the similarity of the large granular vesicles to those of the polypeptidestoring neurosecretory nerves 1. This led us to search for vasoactive peptides in perivascular nerves of the brain. Vasoactive intestinal polypeptide (VIP), a potent hypotensive and vasodilatory agent 11, has been isolated from the porcine small intestines le,17. In a recent study we were able to demonstrate the occurrence of VIP, not only in endocrine-like cells, but also in a rich system of gastrointestinal nerves, some of which had a perivascular localization 12. It was therefore decided to examine, by immunohistochemistry, whether VIP occurred in the perivascular pial nerves. The tissues were taken from various regions at the base of the brain of adult cats, and included large pial arteries with underlying brain tissue. The material consisted of both unoperated and sympathectomized animals (bilateral removal of the superior cervical ganglia 1 week prior to killing). The preparations were frozen to the temperature of liquid nitrogen in a propane-propylene mixture, freeze-dried, vapour-fixed with diethylpyrocarbonate 15, embedded in paraffin in vacuo, sectioned at 5/~m thickness, and subjected to an indirect immunofluorescence method 3,1z for the demonstration of VIP immunoreactivity.

title: VIP – a ’Very Important Peptide’ in the sympathetic nervous system?
abstract: Vasoactive intestinal peptide (VIP) is involved in the control of smooth muscle activity, blood flow and exo- as well as endocrine secretion. More recent work has elucidated the effects of this peptide on central and peripheral neurons. These studies suggest that VIP is an important modulator of cell growth, differentiation and neuronal survival during development of the sympathetic nervous system. VIP is also expressed in a subset of adult postganglionic sympathetic neurons. Furthermore, VIP is induced in an additional neuronal subpopulation of the rat superior cervical ganglion after axotomy. The mechanisms leading to increased VIP expression and its possible role during sympathetic nerve regeneration are currently being elucidated. This review summarizes the distribution, regulation and functions of VIP in cervical sympathetic ganglia of higher vertebrates.

title: Vasoactive intestinal peptide: cardiovascular effects.
abstract: Vasoactive intestinal peptide (VIP) is present in the peripheral and the central nervous systems where it functions as a nonadrenergic, noncholinergic neurotransmitter or neuromodulator. Significant concentrations of VIP are present in the gastrointestinal tract, heart, lungs, thyroid, kidney, urinary bladder, genital organs and the brain. On a molar basis, VIP is 50-100 times more potent than acetylcholine as a vasodilator. VIP release in the body is stimulated by high frequency (10-20 Hz) nerve stimulation and by cholinergic agonists, serotonin, dopaminergic agonists, prostaglandins (PGE, PGD), and nerve growth factor. The VIP peptide combines with its receptor and dose-dependently activates adenylyl cyclase. The vasodilatory effect of VIP in different vascular tissues or species also may be due to increases in nitric oxide, cyclic GMP, and other signaling agents. In the heart, VIP immunoreactive nerve fibers are present not only in the epicardial coronary arteries and veins, but also the sinoatrial node, atrium, interatrial septum, atrioventricular node, intracardiac ganglia, and ventricles (right ventricle >> left ventricle). In the coronary arterial walls, VIP may contribute to the regulation of normal coronary vasomotor tone. In research animals and in humans, VIP, administered into the coronary artery or intravenously, increases the epicardial coronary artery cross-sectional area, decreases coronary vascular resistance, and significantly increases coronary artery blood flow. High frequency parasympathetic (vagal) nerve stimulation also releases endogenous VIP in the coronary vessels and heart and significantly increases coronary artery blood flow. In addition, the release of VIP in the heart is increased during coronary artery occlusion and during reperfusion where VIP may promote local blood flow and may have a free-radical scavenging effect. VIP also has a primary positive inotropic effect on cardiac muscle that is enhanced by its ability to facilitate ventricular-vascular coupling by reducing mean arterial pressure by 10-15%. In concentrations of 10(-8)-10(-5) mol, VIP augments developed isometric force and increases atrial and ventricular contractility. The presence of VIP-immunoreactive nerve fibers in and around the sinus and the atrioventricular nodes of mammals strongly suggests that this peptide can affect the heart rate. In this regard, endogenously released or exogenous VIP can significantly increase the heart rate and has a more potent effect on heart rate than does norepinephrine. The presence and significant cardiovascular effects of VIP in the heart suggests that this peptide is important in the regulation of coronary blood flow, cardiac contraction, and heart rate. Current investigations are defining the physiological role of VIP in the regulation of cardiovascular function.

title: Simultaneous detection of receptor mRNA and ligand protein in human skin tissues
abstract: In situ hybridization techniques allow a cell‐type‐specific messenger RNA (mRNA) analysis in complex tissues such as human skin.

title: Immunohistochemical detection of human skin nerve fibers.
abstract: The autonomic nervous system is involved in different functions such as transduction of afferent sensory inputs, trophic actions, modulation of immunologic events and thermoregulation. In the present investigation, we studied the pattern of human autonomic skin innervation with special reference to its relation to blood vessels, hair follicles, sweat glands and sensory receptors. For the first time, two clinically important areas have been compared: the skin of the forearm and of the face. Using indirect immunohistochemistry, we analyzed the distribution of calretinin (CR), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP), neurokinin A (NKA), vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), tyrosine hydroxylase (TH), histamine, serotonin, enkephalin, and, enzyme histochemically, NADPH-diaphorase (NADPH-d). In the epidermis, we found nerve fibers containing SP, NKA and CGRP. In the dermis, SP-, CR-, VIP-, CGRP- and NKA-positive nerve fibers were detected. Particularly the large nerve fibers contained CR. VIP-positive fibers occurred especially around hair follicles and sweat glands. CGRP-positive nerve fibers were located close to the epidermal basal membrane, in the wall of blood vessels, and to a lesser extent around hair follicles. Immunoreactivity for SP and NKA in the dermis was observed predominantly in the papillary layer near the epidermal basal membrane. All neuropeptides tested in this study were also detected in the nerve fibers of the subcutis. Most of them were CGRP- and VIP-positive. They occurred in association with sweat glands and large arteries. NPY-positive nerve fibers are predominant in the wall of arteries, arterioles and veins. Nerve fibers containing NKA and SP were less common and identified only in the walls of large arteries in deeper dermal layers. In double-staining experiments, the NADPH-d reaction and reactivity to tubulin revealed a partial co-localization in nerve fibers, blood vessel walls, around glands and ganglionic cells. VIP-positive fibers were more common in the face skin than in the forearm. However, in forearm we detected more NPY-, CGRP-, NKA- and SP-positive nerve fibers than in face skin. These findings are important for future studies on skin disorders, such as sensory neuropathies, inflammatory reactions or allergic responses of human skin.

title: Vasoactive intestinal polypeptide receptor-like immunoreactivity in human sweat glands
abstract: Human sweat and sebaceous glands were studied immunohistochemically with a monoclonal antibody recognizing a vasoactive intestinal polypeptide (VIP) receptor. The staining pattern correlated well with the known distribution of sympathetic VIP-containing nerves in the human skin. The luminal cell layer of the sweat gland ducts and some acinar cells of the secretory coil of eccrine sweat glands were the major sites of VIP receptor-like immunoreactivity. From these findings and the known pharmacological actions of VIP it is concluded that a major role of VIP released from sympathetic nerves in the skin is to regulate chloride reabsorption from the primary sweat at the ductal segment.

title: Down-regulation of vasoactive intestinal polypeptide receptor expression in atopic dermatitis.
abstract: Receptors for vasoactive intestinal polypeptide (VIP) have recently been suggested to play a key role in immunomodulation with genetically modified mice. However, it is not known whether changes in receptor gene regulation are involved in the pathogenesis of human immune disorders.



We studied the expression of VPAC(2) in acute lesions of the human immune disease atopic dermatitis.



By using nonradioactive in situ hybridization, quantitative immunohistochemistry, RT-PCR, and gene array studies, the expression status of VPAC(2) was assessed in atopic dermatitis and control tissues and in the human mast cell line HMC-1.



In situ hybridization and immunohistochemistry demonstrated VPAC(2) mRNA and protein expression in human mast cells surrounded by VIP positive nerve fibers. Gene array experiments and RT-PCR studies showed high levels of VPAC(2) mRNA expression in mast cells that were increased compared to other receptors such as VPAC(1) or VIP in the human mast cell line HMC-1. Stimulation of HMC-1 cells led to a downregulation of VPAC(2). Similarly, quantitative immunohistochemistry for VPAC(2) in acute atopic dermatitis lesions showed a significantly decreased VPAC(2) immunoreactivity in mast cells.



The downregulation of VPAC(2) in human mast cells in acute lesions of atopic dermatitis suggests a role of this G-protein;coupled receptor in the pathophysiology of the disease.

title: Topical capsaicin pretreatment inhibits axon reflex vasodilatation caused by somatostatin and vasoactive intestinal polypeptide in human skin
abstract: 1 Wheal and flare reactions are described following intradermal injections of somatostatin, vasoactive intestinal polypeptide, substance P and histamine in normal human forearm skin. Bombesin failed to produce a significant wheal and flare. 2 Pretreatment of skin with capsaicin in all cases dramatically inhibited the flare but not the wheal. This result is in accord with the hypothesis that capsaicin blocks the effector side of the axon reflex, perhaps by depleting nerve terminals of vasodilatory peptide(s).

title: Neuropeptide- and capsaicin-induced histamine release in skin monitored with the microdialysis technique.
abstract: Mast cells are thought to be involved in neurogenic inflammation in skin, and numerous neuropeptides are known to degranulate mast cells. We monitored histamine release in skin in situ with the microdialysis method after skin challenge with neuropeptide injections (10 microM substance P, vasoactive intestinal peptide and calcitonin gene-related peptide), capsaicin injection (30 microM) and 0.1% capsaicin cream with a moist compress. Fractions were collected for 15 min each at 3.0 microliter/min. One hour after insertion of the probe, the baseline histamine level was 4.5 +/- 4.5 nM (mean +/- SD, n = 20). Substance P (250 pmol) induced histamine release peak (66.1 +/- 52.5 nM, n = 8) in the 0-15 or 15-30 min fraction. Thereafter, the histamine concentration declined steadily and rapidly and no second rise was observed. A single substance P injection was sufficient to induce major histamine release in three out of four experiments; and the release kinetics of the second injection (1 h later) mimicked that of the first injection. Vasoactive intestinal peptide (100 and 250 pmol) induced a rapid release of histamine in 4 subjects comparable to substance P, whereas calcitonin gene-related peptide (250 pmol) did not release detectable amounts of histamine in 2 subjects tested. Capsaicin induced a low and rather non-significant release of histamine in 4 out of 5 patients who received capsaicin injection and in 2 out of 5 who were treated with capsaicin cream. The present study shows that neuropeptides substance P and vasoactive intestinal peptide, but not calcitonin gene-related peptide, can induce activation of mast cells and release of histamine into the extracellular space. The low release of histamine by capsaicin suggests low levels of neuropeptides or infrequent morphological contacts between mast cells and sensory nerves in normal human skin. The microdialysis method can be used for studying skin inflammatory reactions involving mast cells.

title: Characterization of neuropeptide‐induced histamine release from human dispersed skin mast cells
abstract: 1 Human skin mast cells, unlike other human mast cells so far studied, released histamine in a concentration‐related manner in response to substance P, vasoactive intestinal peptide (VIP) and somatostatin (1 μM to 30 μM). In contrast, eledoisin, physalaemin, neurokinin A, neurokinin B, calcitonin gene‐related peptide (CGRP), neurotensin, bradykinin and Lys‐bradykinin induced negligible histamine release. 2 The low histamine releasing activity of physalaemin, eledoisin, neurokinin A and neurokinin B relative to substance P suggests that the human skin mast cell activation site is distinct from the tachykinin NK‐1, NK‐2 or NK‐3 receptors described in smooth muscle. 3 The relative potencies of substance P and its fragments SP2–11, SP3–11, SP4–11 and SP1–4 in releasing histamine from human skin mast cells suggests that both the basic N‐terminal amino acids and the lipophilic C‐terminal portion of substance P are essential for activity. 4 Peptide‐induced histamine release, like that induced by compound 48/80, morphine and poly‐L‐lysine, is rapid, reaching completion in 10–20 s, is largely independent of extracellular calcium but requires intact glycolysis and oxidative phosphorylation. 5 The substance P analogue, [D‐Pro4, D‐Trp7,9,10] SP4–11 (SPA), not only reduced substance P‐induced histamine release in a concentration‐related manner but also inhibited that induced by VIP, somatostatin, compound 48/80, poly‐L‐lysine and morphine but not anti‐IgE. 6 The similar characteristics of histamine release induced by substance P, VIP, somatostatin, compound 48/80, poly‐L‐lysine and morphine suggest that they share a common pathway of activation‐secretion coupling distinct from that of IgE‐dependent activation. Furthermore, the ability of human skin mast cells to respond to basic non‐immunological stimuli including neuropeptides may reflect a specialised function for these cells.

title: On the actions of substance P, somatostatin, and vasoactive intestinal polypeptide on rat peritoneal mast cells and in human skin
abstract: Summary1)Substance P (SP), somatostatin (Som), and vasoactive intestinal polypeptide (VIP) induced a concentration-dependent release of histamine from isolated rat peritoneal mast cells.2)The release of histamine induced by these neuropeptides was inhibited by preincubation of the cells with the SP analogue [d-Pro4,d-Trp7,9,10]-SP4–11 (SP-A) (10 μM), and also by benzalkonium chloride (10 μM). In addition, SP-A inhibited histamine release induced by compound 48/80, whilst that induced by goat anti-(rat-IgE) was unaffected.3)In human skin, intradermal injection of SP, Som, or VIP produced flare and wheal responses. The flares to all three peptides were inhibited by preinjection of the skin with SP-A (25 pmol), whilst the wheal responses were unaffected.4)It is concluded that the receptors mediating histamine release and the flare response are similar, and that SP, Som, and VIP are acting at a similar receptor to produce these effects. It is probable that this receptor is also the site of action of compound 48/80.

title: Pituitary adenylate cyclase activating polypeptide (PACAP) is localized in human dermal neurons and causes histamine release from skin mast cells
abstract: Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide homologous with vasoactive intestinal polypeptide (VIP) which is known to induce histamine release in human skin mast cells. PACAP has not been detected in human skin. The purposes of the study were to investigate the occurrence of PACAP in human skin and to evaluate the histamine releasing activity of the two common pro-PACAP products, PACAP-27 and PACAP-38.¶Material: Fourteen human surgical skin samples were obtained. PACAP and VIP were visualized by immunohistochemistry. A microdialysis technique was used to measure histamine release in intact skin samples following intradermal injections of the peptides.¶Results: PACAP and VIP were localized in dermal nerves in connection with sweat glands. Intradermal injection of 3 or 10 μm PACAP significantly released histamine. Kinetics of histamine release showed peak release 2–4 min after skin challenge. Ten μm of PACAP-27, VIP and somatostatin caused histamine release with similar efficacy, whereas PACAP-38 was less effective. Substance P was twice as efficient as PACAP-27, whereas calcitonin gene-related peptide did not release histamine.¶Conclusions: PACAP is found in human skin and is capable of releasing histamine from skin mast cells.

title: Potent vasodilator activity of calcitonin gene-related peptide in human skin.
abstract: We have recently shown that the novel neuropeptide calcitonin gene-related peptide, CGRP, is a potent vasodilator. In this paper we report a detailed study of the effects of CGRP in human skin. CGRP induces a clearly defined, long-lasting erythema. We have measured the effect of CGRP on blood flow in human skin using a laser Doppler technique and have demonstrated increased local blood flow that persists for a number of hours. We compared the response of CGRP with other known vasodilators [histamine, prostaglandin (PG) E2, PGI2, substance P, and vasoactive intestinal peptide (VIP)] in the skin, and in all subjects the erythema induced by CGRP was more persistent than that induced by the other mediators tested. Except at high doses the local vasodilatation induced by CGRP was not associated with a wheal and flare as seen with histamine, substance P, and VIP. CGRP is an extremely potent vasodilator and if released into the circulation, or locally from peripheral nerve endings, it could have a role in the regulation of blood flow in both physiologic and pathologic conditions; CGRP may be the endogenous mediator of the flare in the triple response. A deficiency in CGRP secretion or action could be an important component of peripheral vascular disease. Some flushing reactions (e.g., those associated with medullary thyroid carcinoma) may result from circulating CGRP.

title: Studies on pruritogenic and histamine-releasing effects of some putative peptide neurotransmitters.
abstract: Pruritus, whealing and axon-reflex erythema appeared in human skin after intradermal injection of (i) several peptides with a putative transmitter function, i.e. vasoactive intestinal polypeptide (VIP) (10(-7)--10(-4) M), [Gln4]-neurotensin (10(-7)--10(-4) M), neurotensin (10(-5)--10(-4) M) and secretin (10(-5)--10(-4) M), which were compared with substance P (10(-7)--10(-5) M) previously shown to be one of the most potent histamine liberators when administered intradermally in humans; (ii) the basic polypeptide protamine (10(-7)--10(-4) M); and (iii) histamine (0.3-10 micrograms/ml) and the histamine liberator compound 48/80 (0.3-10 micrograms/ml). The reactions were inhibited in a dose-related manner by the antihistamine mepyramine, indicating that the peptide-induced responses were mediated by released histamine. This was further confirmed by the histamine release observed when the peptides were incubated with rat peritoneal mast cells. In human skin, VIP was more potent than the other neuropeptides and had roughly the same potency as substance P. The two adjacent basic amino-acid residues and the amide substitution of the terminal C-group of VIP, in addition to its strong net basic charge, may explain its potency as a histamine releaser.

title: Role of nitric oxide in the regulation of microvascular perfusion in human skin in vivo
abstract: 1 Nitric oxide (NO) concentrations were measured in dialysate from healthy human skin, in vivo, both at rest and during the inflammatory response to intradermal histamine or bradykinin. Changes in dialysate NO concentration, measured by electrochemical detection, were related to changes in dermal vascular perfusion, measured using scanning laser Doppler imaging. 2 Basal NO concentration in dermal microdialysate was 0·60 ± 0·14 μM (mean ± s.e.m.). Following the intradermal injection of histamine, a transient, time‐dependent increase in NO concentration was measured in areas of skin incorporating the weal and in others incorporating the flare. The increase in NO concentration was associated with an increase in dialysate cGMP concentration in both the weal and flare areas. 3 Addition of NG‐nitro‐l‐arginine‐methyl ester (L‐NAME, 5 mM) to the probe perfusate resulted in an inhibition of the histamine‐induced increase in NO and cGMP. Moreover, the reduction in dialysate NO concentration was associated with a reduction in dermal vascular flux, both under basal conditions and within the weal and flare response. 4 These results demonstrate, by the use of microdialysis, that vasoactive mediators can be measured in healthy human skin in vivo. They provide direct evidence that endogenous concentration of NO increases during the inflammatory weal and flare response to histamine and that the increase in dermal NO concentration is associated with increases in cGMP concentration and dermal vascular perfusion, thus confirming a role for NO in vasoregulation in human skin.

title: Vascular responses in the skin: an accessible model of inflammation.
abstract: The skin is the most accessible organ of the human body in which to study the inflammatory process. Until recently, changes in vascular perfusion and permeability could only be superficially visualized. A novel combination of techniques has now provided the appropriate tools with which to study this more extensively in humans.

title: RECEPTORS FOR HISTAMINE IN HUMAN SKIN BLOOD VESSELS: A REVIEW
abstract: Histamine was one of the first of the drugs occurring naturally in man to be discovered, and it is surprising that even now the pharmacological characterization of such a simple, abundant and ubiquitous amine still remains incomplete. Histamine was first identified by Dale & Laidlaw (1910; 1911) in extracts of ergot. They emphasized its oxytocic actions but also drew attention to its inhibitory properties. The identification of histamine in animal tissues had to await studies of lung and liver extracts by Best, Dale, Dudley & Thorpe (1927) who also highlighted the inhibitory and vasodilator actions of histamine. At about the same time Lewis (1927) adduced considerable indirect evidence that histamine was liberated from human skin following injury and was an important mediator of skin inflammation. However direct evidence of the release of histamine from skin following injury had to await skin perfusion studies (Greaves & Sondergaard 1970; Sondergaard & Greaves, 1971) in which increased amounts of histamine were recovered in perfusates from urticated skin. More recently increased histamine concentrations have been found in venous blood draining cold-challenged skin in cold urticaria (Bentley-Phillips, Black & Greaves, 1976). The frequent failure of conventional antihistamine given systemically to produce reliable suppression of urticarial reactions has led to the suggestion that histamine may be only one of several mediators responsible for the observed vascular changes. However an alternative explanation is now afforded by the discovery of the existence of subclasses of histamine receptors. Ash & Schild (1966) by studying the responses ofa wide range of preparations to analogues of histamine were able to delineate two subclasses of receptors. Subsequent work by Black et al. (1972) defined H|-receptors which were involved in actions of histamine antagonized by conventional (H,) antihistamines and H, receptors which were unaffected by H,-antihistamines, but were antagonized by burimamide which was thus defined as an H2-receptor antagonist. Actions of histamine involving Hj-receptors include stimulation of gastric acid secretions, the chronotropic action of histamine on the heart and inhibition of contraction of the rat uterus The possibility that further subclasses of histamine receptors exist has not been excluded. Exploration of the possibility that human skin blood vessels possess both classes of receptor was prompted hy the observation that the vascular flushing in skin of human volunteers who had received histamine intravenously could be suppressed by administration of H2-receptor antagonists (Wyllie, Hesselbo & Black, 1972; Burland et ah, 1975) and by the identification of two synthetic analogues of histamine, 2-methyl histamine and 4-methyl histamine with predominantly H,-agonist and Hiagonist activity respectively (Black et al., 1972). 2-Methyl histamine evokes a weal and a bright red surrounding fiare due at least in part to an axon refiex. 4-Methyl histamine also causes a weal but the surrounding erythema is less bright and does not involve an axon reflex. The magnitude of the erythema response, but not the weal, is dose-related for each drug. Itching and pain are variable and occur in response to both compounds (Robertson & Greaves, 1977). These observations suggest that the axon refiex and vasodilator actions of histamine are predominantly due to H, and H2 receptors respectively. The actions of chlorpheniramine, an H,-antihistamine, and cimetidine, a highly selective Hiantihistamine (Brimblecombe et al, 1975), on erythema and wealing due to three doses of both agonists are of great interest (Figs i and 2). Chlorpheniramine inhibited erythema due to 2-methyl histamine but cimetidine had no effect on this reaction. By contrast the erythema reaction due to 4-

title: Involvement of H1- and H2-receptors in triple response to histamine in human volunteers.
abstract: Histamine injected intradermally produces the characteristic 'triple response', initial redness being due to direct vasodilation, the following irregular, brighter, 'flush' (flare) being due to histamine-induced axon reflexes. and the 'wheal' due to increased capillary permeability. While both He and H2-receptors have been shown to be involved in capillary dilatation and Hereceptors alone in increased capillary permeability (6), the role of Hs-receptors is uncertain, A recent report suggesting anti-inflammatory role for Hz-antagonists (2) led us to revaluate the role of H2-receptors in 'triple response',

title: The effect of iontophoresis on the cutaneous vasculature: evidence for current-induced hyperemia.
abstract: Combining laser-Doppler blood flux measurements of the skin microcirculation with iontophoresis of vasoactive agents is a promising noninvasive tool for pharmacological studies. However, preliminary observations in our laboratories suggested significant current-associated vasodilation when an expected vasoconstrictor (NG-monomethyl-L-arginine acetate) was iontophoresed. The present study was designed to define nonspecific current-related versus specific pharmacological effects of iontophoretically administered ions on the cutaneous vasculature. Dose-response studies to a series of anions (nitrite, chloride, acetate, and bicarbonate) and cations (sodium, lithium, and acetylcholine) were carried out in six healthy volunteers (three male) by iontophoresis to the forearm skin on separate days. Laser-Doppler flux was measured at the same sites. All ions caused dose-dependent vasodilation. There was no difference in the response between chloride, bicarbonate, or acetate and nitrite, the nitric oxide donor. The acetylcholine dose response was shifted rightward after atropine pretreatment. Cutaneous vascular responses to iontophoresis comprise nonspecific, current-induced hyperemia and specific effects of the administered agent. Acetylcholine appears to cause muscarinic and current-induced dilatation. Nitrite may cause current-induced hyperemia alone. Current-induced hyperemia should be considered in interpreting the acute cutaneous vascular responses to iontophoretically administered agents in humans.

title: Histamine response and local cooling in the human skin: involvement of H1‐ and H2‐receptors
abstract: Histamine may contribute locally to cutaneous blood flow control under normal and pathologic conditions. The objective of this study was to observe the influence of skin temperature on histamine vasodilation, and the roles of H1-and H2-receptors using novel noninvasive methods. 
 
 
 
Methods Eleven healthy subjects received, double-blind, single doses of the H1-receptor antagonist cetirizine (10 mg), cetirizine (10 mg) plus the H2-receptor antagonist cimetidine (400 mg), or placebo on separate occasions. Histamine was dosed cumulatively by iontophoresis to the forearm skin at 34° C and 14° C. Laser-Doppler flux (LDF) was measured at the same sites using customised probeholder/iontophoretic chambers with Peltier cooling elements. Finger mean arterial pressure (MAP) was measured and cutaneous vascular conductance calculated as LDF/MAP. 
 
 
 
Results Histamine vasodilation was reduced in cold skin. Cetirizine shifted the histamine dose-response at both temperatures: statistically significantly at 14° C only. Combined H1- and H2-receptor antagonism shifted the response significantly at both temperatures. 
 
 
 
 H1- and H2-receptors mediate histamine-induced skin vasodilation. The sensitivity of these receptors, particularly the H1- receptor, is attenuated at low skin temperature. Whether the reduced effect in cold skin represents specific receptor or postreceptor desensitization, or nonspecific attenuation of cutaneous vasodilation remains to be elucidated.

title: Histamine receptors in human skin: indirect evidence
abstract: Use of differential effects of agonists and antagonists has provided an experimental basis for subdivision of histamine receptors into H1 and H2. This has enabled classification of the responses to histamine in a wide range of organs and tissues.

title: Effect of H1- and H2-receptor blockade on the inhibition of immediate cutaneous reactions.
abstract: The effects of cyproheptadine, cimetidine, and their combination on the wheal-and-flare skin-test response was evaluated using an intradermal skin-test technique in a double-blind, placebo-controlled study. Sixteen volunteers with known positive skin-test reactions to at least two antigens were divided into four treatment groups in a four-way crossover design. The patients received baseline intradermal skin tests on the back consisting of dilutions of mixed grasses and weeds, histamine diphosphate and codeine phosphate, and buffered-saline control. Following the baseline testing, the patients received cyproheptadine (4 mg q.i.d.), cimetidine (300 mg q.i.d.), the combination, and placebo in a double-blind trial. Of the 16 patients, 12 completed all treatment courses. Following pretreatment with cyproheptadine, cimetidine, and the combination, areas of wheal-and-flare response for each test substance were significantly suppressed. Although it appeared that the combination augmented wheal-and-flare suppression, the observed difference, compared with the cyproheptadine pretreatment sequence, was not statistically significant. The administration of H1 and H2 antagonists should be discontinued before the diagnostic use of immediate skin tests.

title: Predominant role for nitric oxide in the relaxation induced by vasoactive intestinal polypeptide in human uterine artery.
abstract: It has been previously shown that vasoactive intestinal polypeptide (VIP) induces endothelium-dependent relaxation of the human uterine artery. However, the nature of the mediator of the VIP-induced endothelium-dependent relaxation of the human uterine artery has not yet been determined. Therefore these experiments were undertaken to examine the effects of VIP on human uterine arteries and to establish the role of various endothelial factors on the relaxation induced by VIP. The experiments were performed on isolated human uterine arterial rings. VIP (0.3-100 nM) induced a concentration-dependent relaxation of human uterine arteries with intact endothelium (pEC50 = 8.06+/-0.14, n = 28). After the removal of the endothelium this relaxation was abolished (n = 6). Indomethacin (10 microM), a cyclooxygenase inhibitor, and diethylcarbamazine (100 microM), a lipoxygenase blocker, had no effects on VIP-induced relaxation. In contrast, methylene blue (10 microM), a blocker of guanylate cyclase, NG-monomethyl-L-arginine (10 microM), an inhibitor of nitric oxide (NO) synthase, and 4-aminopyridine (1 mM), a non-selective blocker of K+ channels, antagonized the effect of VIP with suppression of maximal VIP-induced relaxation. Non-competitive antagonism with methylene blue revealed that the pKa value for VIP-receptor complex was 8.10+/-0.10 (n = 6) and the receptor reserve expressed as KA/EC50 was 0.89+/-0.11, where pKa = log10KA, and KA is the dissociation constant of VIP-receptor complex. Therefore, on the basis of the results presented, we can conclude that VIP induces endothelium-dependent relaxation in human uterine arteries, acting as a partial agonist on this blood vessel. It appears that endothelium-dependent relaxation induced by VIP in human uterine artery can be entirely explained by the release of NO from endothelial cells.

title: Interplay of VIP and nitric oxide in regulation of the descending relaxation phase of peristalsis.
abstract: Involvement of vasoactive intestinal peptide (VIP) and nitric oxide (NO) in neurally induced relaxation was examined in smooth muscle from rat colon. Relaxation induced by field stimulation or radial stretch (i.e., descending relaxation phase of the peristaltic reflex) was accompanied by VIP release and NO production. NG-nitro-L-arginine (L-NNA) abolished NO production in both preparations but only partly inhibited VIP release (45 +/- 8% at 8 Hz and 59 +/- 10% at 10 g stretch) and relaxation (62 +/- 5% and 35 +/- 6%); the effect of L-NNA was reversed by L-arginine but not D-arginine. The pattern implied that NO production normally acts to enhance VIP release. In addition, VIP induced relaxation and stimulated NO production in muscle strips and isolated colonic muscle cells: L-NNA abolished NO production but only partly inhibited relaxation (58 +/- 6%); oxyhemoglobin had no effect. The effect of L-NNA on relaxation was reversed by L-arginine but not by D-arginine. The protein kinase A inhibitor (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS] and the protein kinase G inhibitor KT5823 inhibited VIP-induced relaxation by 76 +/- 5 and 35 +/- 4%, respectively; a combination of the two inhibitors abolished relaxation.

title: Stoichiometry of neurally induced VIP release, NO formation, and relaxation in rabbit and rat gastric muscle.
abstract: Vasoactive intestinal peptide (VIP) release, nitric oxide (NO) formation, and relaxation induced by nerve stimulation were examined in rabbit and rat gastric muscle. VIP stimulated NO formation in muscle strips, whereas NO stimulated VIP release. Nerve stimulation (0.025-16 Hz or 2-940 pulses) elicited frequency-dependent stimulation of VIP release, NO formation, and relaxation, all significant at two to three pulses. NG-nitro-L-arginine (L-NMA) abolished NO formation, abolished VIP release and relaxation at low frequencies, and partly inhibited them at higher frequencies. Oxyhemoglobin (oxy-Hb) inhibited VIP release and relaxation by 80% at low frequencies and 20-30% at higher frequencies. VIP-(10-28) abolished NO formation and relaxation at lower frequencies and partly inhibited them at higher frequencies; in contrast, VIP-(10-28) augmented VIP release in both species. The pattern of inhibition was similar in both species. Inhibition of maximal NO formation by VIP-(10-28) (82% in rabbit; 48% in rat) implied that a major component of NO is formed in muscle cells by the action of VIP. Thus 1) inhibition of relaxation by L-NNA reflects suppression of NO and VIP release from nerve terminals and NO formation in muscle cells, 2) inhibition by VIP-(10-28) partly reflects suppression of NO formation in muscle cells, and 3) inhibition by oxy-Hb reflects neutralization of extracellular NO and suppression of VIP release. The study demonstrates the dual origin of NO from nerves and muscle and its interplay with VIP in regulating relaxation.

title: Interplay between nitric oxide and vasoactive intestinal polypeptide in the pig gastric fundus smooth muscle.
abstract: The aim of this study was to investigate the exact mechanism of interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) as inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitters in isolated smooth muscle cells and smooth muscle strips of the pig gastric fundus. In isolated smooth muscle cells, the maximal relaxant effect of VIP (10(-9) M) was inhibited by 94% by the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine (L-NA, 10(-4) M) and by 85% by the inducible NOS (iNOS)-selective inhibitor N-(3-(aminomethyl)-benzyl)acetamide (1400W; 10(-6) M). The relaxant effect of VIP was reduced by more than 70% by the guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; 10(-6) M), the glucocorticoid dexamethasone (10(-5) M) and three protein kinase A inhibitors: (R)-p-cyclic adenosine-3', 5'-monophosphothioate ((R)-p-cAMPS; 10(-6) M), ¿(8R,9S, 11S)-(-)-9-hydroxy-9-n-hexylester-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a, g]cycloocta[cde]-trin-den-1-one¿ (KT5720; 10(-6) M) and N-(2-(p-bromo-cinnamylamino)ethyl))-5-isoquinoline sulfonamide dihydrochloride (H-89; 10(-5) M). In contrast, no influence of the NOS inhibitors, ODQ, dexamethasone, nor the protein kinase A inhibitors could be observed on the relaxant effect of VIP in smooth muscle strips. These data demonstrate that the experimental method completely changes the influence of NOS inhibitors on the relaxant effect of VIP in the pig gastric fundus. The isolation procedure of the smooth muscle cells might induce iNOS that can be activated by VIP.

title: Stimulation of nitric oxide from muscle cells by VIP: prejunctional enhancement of VIP release.
abstract: The source of nitric oxide (NO) and its role in neurally induced relaxation was examined in smooth muscle of the stomach and tenia coli. Field stimulation of gastric muscle strips was accompanied by frequency-dependent relaxation, vasoactive intestinal peptide (VIP) release, and NO production: the NO synthase inhibitor, NG-nitro-L-arginine (L-NNA) completely inhibited NO production and partly inhibited VIP release (52-54%) and relaxation (58-88%); inhibition of all three functions was reversed by L-arginine but not by D-arginine. In isolated gastric muscle cells, VIP caused relaxation and stimulated NO production: L-NNA completely inhibited NO production and partly inhibited relaxation; the inhibition was reversed by L-arginine but not by D-arginine. Abolition of NO production with only partial inhibition of relaxation implied that NO production from muscle cells induced by the action of VIP was partly responsible for relaxation. By contrast, field stimulation of tenia coli was accompanied by relaxation and VIP release but not by NO production. Neither VIP release nor relaxation was affected by L-NNA. In isolated muscle cells of tenia coli, VIP caused relaxation but did not stimulate NO production; relaxation in these cells was not affected by L-NNA. We conclude that 1) VIP is the primary relaxant transmitter in both gastric muscle and tenia coli, 2) the release of VIP in gastric muscle but not in tenia coli stimulates NO production from target muscle cells, and 3) NO amplifies the relaxant effect of VIP in muscle cells and acts presynaptically to enhance the release of VIP.

title: Synergistic effect of nitric oxide and vasoactive intestinal peptide on bronchoprotection against histamine in anesthetized guinea pigs.
abstract: Vasoactive intestinal peptide (VIP) and nitric oxide (NO) are considered to be nonadrenergic, noncholinergic (NANC) inhibitory neurostransmitters in the airways. It seems likely that these neurotransmitters may be coreleased and act as functional antagonists against bronchoconstrictor stimuli. In the present study, we examined the synergistic effect of NO and VIP on bronchoprotection against histamine in anesthetized guinea pigs. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited histamine-induced bronchoconstriction in a dose-dependent manner. VIP also inhibited histamine-induced bronchoconstriction in a dose-dependent manner, but this bronchoprotective effect was short-lived. Additionally, VIP (10(-9) M) had no significant bronchoprotective effect, but a subthreshold dose of SNAP (10(-7) M) significantly potentiated VIP (10(-9) M)-induced bronchoprotection against histamine. Moreover, SNAP (10(-7) M) significantly enhanced VIP (10(-7) M)-induced bronchoprotection for a longer period of time. On the other hand, VIP (10(-9) M) also significantly potentiated SNAP-induced bronchoprotection against histamine. In conclusion, combination therapy with NO donor and VIP receptor agonist may have important advantages in the treatment of bronchial asthma, and both NO and VIP may contribute in complementary fashion to the NANC-induced relaxant response in guinea pig airways.

title: Nitric oxide is not permissive for cutaneous active vasodilatation in humans
abstract: The precise role of nitric oxide (NO) in cutaneous active vasodilatation in humans is unknown. We tested the hypothesis that NO is necessary to permit the action of an unknown vasodilator. Specifically, we investigated whether a low‐dose infusion of exogenous NO, in the form of sodium nitroprusside (SNP), would fully restore vasodilatation in an area of skin in which endogenous NO was inhibited during hyperthermia. This finding would suggest a ‘permissive’ role for NO in active vasodilatation. Eight subjects were instrumented with three microdialysis fibres in forearm skin. Sites were randomly assigned to (1) Site A: control site; (2) Site B: NO synthase (NOS) inhibition during established hyperthermia; or (3) Site C: NOS inhibition throughout the protocol. Red blood cell flux was measured using laser‐Doppler flowmetry (LDF) and cutaneous vascular conductance (CVC; LDF/mean arterial pressure) was normalized to maximal vasodilatation at each site. In Site B, NG‐nitro‐l‐arginine methyl ester (l‐NAME) infusion during hyperthermia reduced CVC by ∼32 % (65 ± 4 % CVCmaxvs. 45 ± 4 % CVCmax; P < 0.05). Vasodilatation was not restored to pre‐NOS inhibition values in this site following low‐dose SNP infusion (55 ± 4 % CVCmaxvs. 65 ± 4 % CVCmax; P < 0.05). CVC remained significantly lower than the control site with low‐dose SNP infusion in Site C (P < 0.05). The rise in CVC with low‐dose SNP (ΔCVC) was significantly greater in Site B and Site C during hyperthermia compared to normothermia (P < 0.05). No difference in ΔCVC was observed between hyperthermia and normothermia in the control site (Site A). Thus, NO does not act permissively in cutaneous active vasodilatation in humans but may directly mediate vasodilatation and enhance the effect of an unknown active vasodilator.

title: Role of nitric oxide in the vascular effects of local warming of the skin in humans.
abstract: Local warming of skin induces vasodilation by unknown mechanisms. To test whether nitric oxide (NO) is involved, we examined effects of NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (L-NAME) on vasodilation induced by local warming of skin in six subjects. Two adjacent sites on the forearm were instrumented with intradermal microdialysis probes for delivery of L-NAME and sodium nitroprusside. Skin blood flow was monitored by laser-Doppler flowmetry (LDF) at microdialysis sites. Local temperature (Tloc) of the skin at both sites was controlled with special LDF probe holders. Mean arterial pressure (MAP; Finapres) was measured and cutaneous vascular conductance calculated (CVC = LDF/MAP = mV/mmHg). Data collection began with a control period (Tloc at both sites = 34 degrees C). One site was then warmed to 41 degrees C while the second was maintained at 34 degrees C. Local warming increased CVC from 1.44 +/- 0.41 to 4.28 +/- 0.60 mV/mmHg (P < 0.05). Subsequent L-NAME administration reduced CVC to 2.28 +/- 0.47 mV/mmHg (P < 0.05 vs. heating), despite the continued elevation of Tloc. At a Tloc of 34 degrees C, L-NAME reduced CVC from 1.17 +/- 0.23 to 0.75 +/- 0.11 mV/mmHg (P < 0.05). Administration of sodium nitroprusside increased CVC to levels no different from those induced by local warming. Thus NOS inhibition attenuated, and sodium nitroprusside restored, the cutaneous vasodilation induced by elevation of Tloc; therefore, the mechanism of cutaneous vasodilation by local warming requires NOS generation of NO.

title: Nitric oxide and neurally mediated regulation of skin blood flow during local heating.
abstract: The mechanisms underlying the skin blood flow (SkBF) response to local heating are complex and poorly understood. Our goal was to examine the role of axon reflexes and nitric oxide (NO) in the SkBF response to a local heating protocol. We performed 40 experiments following a standardized heating protocol with different interventions, including blockade of the axon reflex (EMLA cream), antebrachial nerve blockade (0.5% bupivacaine injection), and NO synthase (NOS) inhibition (> or =10 mM N(G)-nitro-L-arginine methyl ester; microdialysis). Appropriate controls were performed to verify the efficacy of the various blocks. Values are expressed as a percentage of maximal SkBF (SkBF(max); 50 mM sodium nitroprusside). At the initiation of local heating, SkBF rose to an initial peak, followed by a brief nadir, and a secondary, progressive rise to a plateau. Axon reflex block decreased the initial peak from 75+3 to 32 +/- 2% SkBF(max) (P < 0.01 vs. control) but did not affect the plateau. NOS inhibition before and throughout local heating reduced the initial peak from 75 +/- 3 to 56 +/- 3% SkBF(max) (P < 0.01) and the plateau from 87 +/- 4 to 40 +/- 5%. NOS inhibition during axon reflex block did not further reduce the initial SkBF peak compared with axon reflex block alone. Antebrachial nerve block did not affect the local heating SkBF response. The primary finding of these studies is that there are at least two independent mechanisms contributing to the rise in SkBF during nonpainful local heating: a fast-responding vasodilator system mediated by the axon reflexes and a more slowly responding vasodilator system that relies on local production of NO.

title: Cystic fibrosis, vasoactive intestinal polypeptide, and active cutaneous vasodilation.
abstract: The transmitter substance for the active cutaneous vasodilation that accompanies sweating during hyperthermia in humans is unknown. Hökfelt et al. ( 515-521, 180) hypothesized that it is vasoactive intestinal polypeptide (VIP) that is cotransmitted with acetylcholine. Heinz-Erian et al. (Science Wash. DC 229: 1407-1408, 1985) reported that VIP innervation is sparse in the skin of persons with cystic fibrosis (CF). A corresponding attenuation of active vasodilation in these subjects would be evidence that VIP is involved in this effector mechanism of human thermor-regulation. Immunocytochemical analysis of skin biopsies from four men with CF confirmed that VIP innervation was sparse. We also analyzed immunoreactivity for calcitonin gene-related peptide (CGRP; normal), substance P (normal), and neuropeptide Y (low). VIP-immunoreactive Merkel cells were abnormal. Despite sparse VIP-immunoreactive innervation, our CF subjects' cutaneous vascular responses to hyperthermia were normal. Because VIP was not completely absent, this evidence is insufficient to rule out VIP as the vasodilator transmitter. However, the CGRP and substance P innervation we observed could mean that release of one or both of these peptides was the mechanism of the fully developed active cutaneous vasodilation.

title: Cutaneous reactions and sensations after intracutaneous injection of vasoactive intestinal polypeptide and acetylcholine in atopic eczema patients and healthy controls
abstract: We analysed vasoreactions and sensations of atopic eczema (AE) patients and healthy controls after intracutaneous (i.c.) injection of vasoactive intestinal polypeptide (VIP) and acetylcholine (ACh). Blood flow was measured by laser Doppler flowmetry (LDF). Plasma extravasation and flare size were evaluated planimetrically, and sensations were recorded using visual analog scales. Three groups of subjects (controls, AE patients suffering from acute eczema and AE patients during a symptom-free period) were investigated. We administered VIP separately at concentrations of 1.5 × 10 –7 , 1.5 × 10 –6 and 1.5 × 10 –5 M and in combination with ACh (5.5 × 10 –6 M ) into the volar forearm of the subjects. Both substances led to an increase in LDF measurements and induced a wheal and flare reaction. Blood flow was elevated as a function of dose after a single VIP application in all groups. Compared with healthy controls, a significant increase in blood flow was measured after combined VIP and ACh administration in AE patients suffering from acute AE, whereas flare area and plasma extravasation were significantly reduced after single VIP and combined VIP and ACh injections, respectively. In all groups, VIP induced dose-dependent pruritus. Compared with a control stimulus (0.9% sodium chloride and ACh), combined injections of VIP and ACh had no additional effect on the magnitude of the sensation. In AE patients, the intensity was similar to that experienced by the control subjects, but the quality of sensation was different: ACh induced pain in the control subjects, pruritus in AE patients, and a mixture of pain and itching in AE patients showing no symptoms. Our results suggest that VIP- and ACh-induced skin reactions and the quality of the sensations depend on the activity of the atopic eczema. Confirming our former studies, AE patients develop a different quality of sensation after ACh administration and also after administration of VIP combined with ACh. Therefore, we suggest that ACh might be involved in the pathomechanisms of pruritus in AE.

title: Evidence for a Role for Vasoactive Intestinal Peptide in Active Vasodilatation in the Cutaneous Vasculature of Humans
abstract: Active vasodilatation (AVD) in human, non‐glabrous skin depends on functional cholinergic fibres but not on acetylcholine (ACh). We tested whether AVD is a redundant system in which ACh and vasoactive intestinal polypeptide (VIP) are co‐released from cholinergic nerves. (1) We administered VIP by intradermal microdialysis to four discrete areas of skin in the presence of different levels of the VIP receptor antagonist, VIP(10−28), also delivered by microdialysis. Skin blood flow (SkBF) was continuously monitored by laser Doppler flowmetry (LDF). Mean arterial pressure (MAP) was measured non‐invasively and cutaneous vascular conductance (CVC) calculated as LDF/MAP. Subjects were supine and wore water‐perfused suits to control whole‐body skin temperature (Tsk) at 34 °C. Concentrations of 54 μm, 107 μM, or 214 μM VIP(10−28) were perfused via intradermal microdialysis at 2 μl min−1 for approximately 1 h. Then 7.5 μM VIP was added to the perfusate containing VIP(10−28) at the three concentrations or Ringer solution and perfusion was continued for 45‐60 min. At the control site, this level of VIP caused approximately the vasodilatation typical of heat stress. All VIP(10−28)‐treated sites displayed an attenuated dilatation in response to the VIP. The greatest attenuation was observed at the site that received 214 μM VIP(10−28) (P < 0.01). (2) We used 214 μM VIP(10−28) alone and with the iontophoretically administered muscarinic receptor antagonist atropine (400 μA cm−2, 45 s, 10 mM) in heated subjects to test the roles of VIP and ACh in AVD. Ringer solution and 214 μM VIP(10−28) were each perfused at two sites, one of which in each case was pretreated with atropine. After 1 h of VIP(10−28) treatment, individuals underwent 45−60 min of whole‐body heating (Tsk to 38.5 °C). VIP(10−28), alone or in combination with atropine, attenuated the increase in CVC during heat stress, suggesting an important role for VIP in AVD.

title: Nitric oxide and cutaneous active vasodilation during heat stress in humans.
abstract: Whether nitric oxide (NO) is involved in cutaneous active vasodilation during hyperthermia in humans is unclear. We tested for a role of NO in this process during heat stress (water-perfused suits) in seven healthy subjects. Two forearm sites were instrumented with intradermal microdialysis probes. One site was perfused with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) dissolved in Ringer solution to abolish NO production. The other site was perfused with Ringer solution only. At those sites, skin blood flow (laser-Doppler flowmetry) and sweat rate were simultaneously and continuously monitored. Cutaneous vascular conductance, calculated from laser-Doppler flowmetry and mean arterial pressure, was normalized to maximal levels as achieved by perfusion with the NO donor nitroprusside through the microdialysis probes. Under normothermic conditions, L-NAME did not significantly reduce cutaneous vascular conductance. During hyperthermia, with skin temperature held at 38-38.5 degreesC, internal temperature rose from 36.66 +/- 0.10 to 37.34 +/- 0.06 degreesC (P < 0.01). Cutaneous vascular conductance at untreated sites increased from 12 +/- 2 to 44 +/- 5% of maximum, but only rose from 13 +/- 2 to 30 +/- 5% of maximum at L-NAME-treated sites (P < 0.05 between sites) during heat stress. L-NAME had no effect on sweat rate (P > 0.05). Thus cutaneous active vasodilation requires functional NO synthase to achieve full expression.

title: Nitric oxide concentration increases in the cutaneous interstitial space during heat stress in humans.
abstract: To examine the role of nitric oxide (NO) in cutaneous active vasodilation, we measured the NO concentration from skin before and during whole body heat stress in nine healthy subjects. A forearm site was instrumented with a NO-selective, amperometric electrode and an adjacent intradermal microdialysis probe. Skin blood flow (SkBF) was monitored by laser-Doppler flowmetry (LDF). NO concentrations and LDF were measured in normothermia and heat stress. After heat stress, a solution of ACh was perfused through the microdialysis probe to pharmacologically generate NO and verify the electrode's function. During whole body warming, both SkBF and NO concentrations began to increase at the same internal temperature. Both SkBF and NO concentrations increased during heat stress (402 +/- 76% change from LDF baseline, P < 0.05; 22 +/- 5% change from NO baseline, P < 0.05). During a second baseline condition after heat stress, ACh perfusion led to increases in both SkBF and NO concentrations (496 +/- 119% change from LDF baseline, P < 0.05; 16 +/- 10% change from NO baseline, P < 0.05). We conclude that NO does increase in skin during heat stress in humans, attendant to active vasodilation. This result suggests that NO has a role beyond that of a permissive factor in the process; rather, NO may well be an effector of cutaneous vasodilation during heat stress.

title: Effects of atropine and L-NAME on cutaneous blood flow during body heating in humans.
abstract: We sought to investigate further the roles of sweating, ACh spillover, and nitric oxide (NO) in the neurally mediated cutaneous vasodilation during body heating in humans. Six subjects were heated with a water-perfused suit while cutaneous blood flow was measured with a laser-Doppler flowmeter. After a rise in core temperature (1. 0 +/- 0.1 degrees C) and the establishment of cutaneous vasodilation, atropine and subsequently the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) were given to the forearm via a brachial artery catheter. After atropine infusion, cutaneous vascular conductance (CVC) remained constant in five of six subjects, whereas L-NAME administration blunted the rise in CVC in three of six subjects. A subsequent set of studies using intradermal microdialysis probes to selectively deliver drugs into forearm skin confirmed that atropine did not affect CVC. However, perfusion of L-NAME resulted in a significant decrease in CVC (37 +/- 4%, P < 0.05). The results indicate that neither sweating nor NO release via muscarinic receptor activation is essential to sustain cutaneous dilation during heating in humans.

title: Neuronal Nitric Oxide Synthase Activation by Vasoactive Intestinal Peptide in Bovine Cerebral Arteries
abstract: The participation of nitric oxide and vasoactive intestinal peptide (VIP) in the neurogenic regulation of bovine cerebral arteries was investigated. Nitrergic nerve fibers and ganglion-like groups of neurons were revealed by NADPH-diaphorase staining in the adventitial layer of bovine cerebral arteries. NADPH diaphorase also was present in endothelial cells but not in the smooth muscle layer. Double immunolabeling for neuronal nitric oxide synthase and VIP indicated that both molecules co-localized in the same nerve fibers in these vessels. Transmural nerve stimulation (200 mA, 0.2 milliseconds, 1 to 8 Hz) of endothelium-denuded bovine cerebral artery rings precontracted with prostaglandin F2α, produced tetrodotoxin-sensitive relaxations that were completely suppressed by NG-nitro-l-arginine methyl ester (l-NAME) and by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline (ODQ), but were not affected by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), nor by VIP tachyphylaxis induced by pretreatment with 1 μmol/L VIP. Transmural nerve stimulation also elicited increases in intracellular cyclic GMP concentration, which were prevented by l-NAME, and small decreases in intracellular cyclic AMP concentration. Addition of VIP to bovine cerebral artery rings without endothelium produced a concentration-dependent relaxation that was partially inhibited by l-NAME, ODQ, and SQ 22,536. The effects of l-NAME and SQ 22,536 were additive. VIP induced a transient increase in intracellular cyclic GMP concentration, which was maximal 1 minute after VIP addition, when the highest relaxation rate was observed, and which was blocked by l-NAME. It is concluded that nitric oxide produced by perivascular neurons and nerve fibers fully accounts for the experimental neurogenic relaxation of bovine cerebral arteries and that VIP, which also is present in the same perivascular fibers, acts as a neuromodulator by activating neuronal nitric oxide synthase.

title: Mechanisms of endothelium-dependent vascular smooth muscle relaxation elicited by bradykinin and VIP.
abstract: The objective of this study was to elucidate the mechanisms by which bradykinin and vasoactive intestinal polypeptide (VIP) relax bovine intrapulmonary artery and bradykinin, but not VIP, relaxes intrapulmonary vein. Bradykinin and VIP elicited entirely endothelium-dependent relaxation of phenylephrine-precontracted arterial rings, and this was associated with arterial accumulation of both guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). Bradykinin, but not VIP, relaxed precontracted venous rings and increased cGMP, but not cAMP levels, by endothelium-dependent mechanisms. Neither arteries nor veins relaxed in response to substance P, thrombin, bombesin, arginine vasopressin, or angiotensin II. Methylene blue or indomethacin each partially antagonized, whereas both, when together, abolished arterial relaxant responses to bradykinin and VIP. Methylene blue or indomethacin, respectively, abolished arterial cGMP or cAMP accumulation elicited by bradykinin and VIP. Venous relaxation and cGMP accumulation elicited by bradykinin was abolished by methylene blue but was unaltered by indomethacin. Thus bradykinin and VIP relaxed bovine intrapulmonary artery by endothelium-dependent mechanisms involving the actions of cGMP and cAMP whose formation may be stimulated by endothelium-derived relaxing factor and prostacyclin, respectively. In contrast, bradykinin relaxed intrapulmonary vein by endothelium-dependent mechanisms involving only cGMP.

title: Neuropeptides induce release of nitric oxide from human dermal microvascular endothelial cells.
abstract: Nitric oxide is a potent mediator of endothelium-dependent vasodilation, the synthesis of which is catalyzed by the constitutively expressed enzyme endothelial nitric oxide synthase. In this study we have investigated whether human dermal microvascular endothelial cells express endothelial oxide synthase and whether the vasodilator neuropeptides, calcitonin gene-related peptide and substance P, stimulate the release of nitric oxide from these cells. Endothelial nitric oxide synthase was identified by immunohistochemistry in the blood vessels in both the papillary and deep dermis of normal skin, and also in monolayers of human dermal microvascular extracts prepared from both the dermis of normal human skin and human dermal microvascular endothelial cells, a 135-kDa band corresponding to endothelial nitric oxide synthase was identified. Nitric oxide was released from unstimulated human dermal microvascular endothelial cells as assessed by inhibition of platelet aggregation and nitrate formation. Endothelial cell-mediated inhibition of platelet aggregation was blocked by hemoglobin. Calcitonin gene-related peptide, (100 pM to 100 nM) directly inhibited platelet aggregation, and this direct effect was not modulated by microvascular endothelial cells. Substance P (10 nM to 1 muM) and calcitonin gene-related peptide (100 pM to 10 nM) significantly (p<0.05) increased nitrite formation, and this increase was blocked by the competitive nitric oxide synthase antagonist, NG-monomethyl-L-arginine. These results demonstrate that endothelial nitric oxide synthase is expressed in the microvascular endothelium of normal human skin and that human dermal microvascular endothelial cells release nitric oxide constitutively and in response to vasodilator neuropeptides.

title: Role of endothelium and nitric oxide in histamine‐induced responses in human cranial arteries and detection of mRNA encoding H1‐ and H2‐receptors by RT‐PCR
abstract: Histamine induces relaxation of human cranial arteries. Studies have revealed that the relaxant histamine H1‐receptor predominates in human cerebral and the H2‐receptor in temporal arteries, while H1‐ and H2‐receptors are of equal importance in the middle meningeal artery. The purpose of the present study was to examine the role of the endothelium and nitric oxide in histamine‐induced responses and to show the presence of mRNA encoding H1‐ and H2‐receptors in human cranial arteries. Electrophoresis of polymerase chain reaction (PCR) products from human cerebral, middle meningeal and temporal arteries, demonstrated products corresponding to mRNA encoding both H1‐ and H2‐receptors in arteries with and without endothelium. The amplified PCR products were sequenced and showed 100% homology with the published sequences of these histamine receptors. A sensitive in vitro system was used to study vasomotor responses to histamine. In precontracted cerebral, middle meningeal and temporal arteries with and without endothelium, histamine caused a concentration‐dependent relaxation with Imax values between 87% and 81% and pIC50 values between 8.14 and 7.15. In arteries without endothelium the histamine‐induced relaxation was significantly less potent (Imax values between 87% and 66% and pIC50 values between 7.01 and 6.67) than in cranial arteries with an intact endothelium. The addition of histamine to arteries without endothelium and pretreated with the histamine H2‐antagonist, cimetidine (10−5 M), caused a concentration‐dependent contraction of the cranial arteries with Emax values between 86% and 29% and pEC50 values between 7.53 and 6.77. This contraction was blocked by the histamine H1‐receptor antagonist, mepyramine (10−7 M), and even turned into a relaxation with Imax values between 84% and 14% and pIC50 values between 7.42 and 5.86. The nitric oxide synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME, 3×10−5 M) significantly inhibited the relaxant response to histamine in cerebral and temporal arteries (pIC50 values between 7.43 and 7.13). The combined treatment with L‐NAME (3×10−5 M) and cimetidine (10−5 M) caused a further displacement of the concentration‐response curve (pIC50 values between 7.14 and 6.57) and decreased the maximum relaxant responses in all three cranial arteries (Imax values between 62% and 39%). In conclusion, this is the first study which show mRNA encoding histamine H1‐ and H2‐receptors in human cranial arteries. The results indicate that histamine‐induced relaxation of human cranial arteries is partially mediated via an endothelial H1‐receptor coupled to the production of nitric oxide and partially via a H2‐receptor associated with the smooth muscle cells. In addition, there is evidence for a contractile H1‐receptor in the smooth muscle cells in these arteries.

title: Differential Activation of Dual Signaling Responses by Human H1 and H2 Histamine Receptors
abstract: Stimulation of human H1 and H2‐histamine receptors (HRs) primarily activates signaling pathways to increase intracellular calcium [Ca2+]i and cyclic AMP (cAMP), respectively. Activation of H2‐HR in human embryonic kidney (HEK) cells by histamine and dimaprit increases both cAMP formation and [Ca2+]i, as determined by cAMP‐scintillation proximity assays and fluorescence imaging plate reader (FLIPR) assays. In HEK cells expressing relatively high levels of H2‐HR (Bmax = 26 pmol/mg protein), histamine and dimaprit are full agonists in eliciting cAMP responses with pEC50 values of 9.30 and 7.72 that are 1000‐fold more potent than their respective pEC50 values of 6.13 and 4.91 for increasing [Ca2+]i. The agonist potencies decrease for both responses at lower H2‐HR density (5 pmol/mg protein) and dimaprit exhibits partial agonist behavior for the [Ca2+]i response. The inverse agonists ranitidine and cimetidine more potently inhibit cAMP production in the higher expressing H2‐HR line. Histamine also activated both signaling pathways via human H1‐HRs highly expressed (Bmax = 17 pmol/mg protein) in HEK cells, with a 1000‐fold greater potency for [Ca2+]i vs. cAMP responses (pEC50 = 7.86 and 4.82, respectively). These studies demonstrate a markedly different potency for activation of multiple signaling pathways by H1‐ and H2‐HRs that may contribute to the selectivity of histamine responses in vivo.

title: Histamine receptors in the smooth muscle of human internal mammary artery and saphenous vein.
abstract: The effects of histamine were characterized and compared in the vascular smooth muscle of two human isolated blood vessels, the human internal mammary artery (HIMA) and the human saphenous vein (HSV). Segments of these vessels were obtained during aortocoronary bypass surgery and their intimal surface was rubbed in order to eliminate any possible influence of the endothelium. Histamine contracted both types of vessels in a concentration-dependent manner and this effect was antagonized by the H1 receptor antagonists mepyramine and cicletanine. In the case of HIMA only this antagonism was found to be competitive (pA2 values of 9.3 and 7.7 for mepyramine and cicletanine, respectively). Histamine-induced contractions were not significantly affected by phentolamine (0.3 microM). In HSV, but not HIMA, indomethacin (5 microM) significantly depressed histamine-induced contractions (by about 30%). In the presence of the H2 receptor antagonist cimetidine (10 microM), concentration-response curves of histamine-induced contractions were significantly shifted to the left in both HIMA and HSV, suggesting the presence of H2 receptors mediating relaxation. HIMA and HSV precontracted by noradrenaline could be partially and concentration dependently relaxed by histamine, only in the presence of a H1 receptor antagonist. This relaxation was inhibited by cimetidine. The results show that in de-endothelialized HIMA and HSV histamine induced mainly contraction which is sensitive to the H1 receptor antagonists. Only in HIMA, nevertheless, was competitive antagonism established. In addition, histamine-induced relaxation, antagonized by cimetidine, could be demonstrated in both precontracted vessels, indicating the presence of H2 receptors.