Phenotypic alterations in keratinocyte behavior are essential for maintaining epidermal integrity during growth and wound repair and rely on co-ordinated cell signaling events. Numerous growth factors and cytokines have been shown to be instrumental in guiding such changes in keratinocyte activity; here we provide evidence which proposes a novel epidermal signaling pathway mediated by the excitatory amino acid glutamate. Glutamate is the major excitatory neurotransmitter at synaptic junctions within the central nervous system; however, we have identified expression in vivo of several regulatory molecules associated with glutamate signaling in keratinocytes. In resting rat skin epidermis, different classes of glutamate receptors, transporters, and a recently described clustering protein were shown to display distinct distribution patterns, supportive of a multifunctional cellular communication pathway. Immunoreactive N-methyl-D-aspartate-type, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type, and metabotropic-type glutamate receptors were colocalized with the specific glutamate transporter EAAC1 in basal layer keratinocytes, and GLT-1, a related transporter, was expressed suprabasally. In full-thickness rat skin wounds, marked modifications in the distribution of N-methyl-D-aspartate receptors and EAAC1 were observed during re-epithelialization, and alterations in N-methyl-D-aspartate receptor expression accompanied embryonic epidermal development, implicating glutamate signaling in these important biologic events. Furthermore, we provide evidence that these receptors are functional in vitro. These data provide strong evidence to support a role for glutamate in the control of epidermal renewal, and therefore suggest potentially novel therapeutic targets for the treatment of skin disease and enhancement of wound healing.

1 The vasodilator properties and photochemical decomposition of two synthetic iron-sulphur-nitrosyl clusters (cluster A: [Fe4S4(NO)4], tetranitrosyl-tetra-j3-sulphido-tetrahedro-tetrairon; and B:[Fe4S3 (NO)7V-1, heptanitrosyl-tri-u3-thioxotetraferrate(-1)) have been investigated. Experiments were carried out on isolated, internally-perfused segments of rat tail artery.2 Bolus injections (10 Ml) of A or B (>0.25 mM) delivered into the internal perfusate generated sustained (or S-type) vasodilator responses, characterized by a persistent plateau of reduced tone due to NO released from clusters which enter and become trapped within endothelial cells. Clusters were therefore irradiated with visible laser light (Z=457.9 or 514.5 nm) either (a) in solution, while passing through a glass tube en route to the artery; or (b) when retained within the endothelium, by illuminating the artery directly during the plateau of an S-type response. Irradiation produced an additional vasodilator response, the magnitude of which depended upon wavelength and laser beam energy. 3 The nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (100 jM), had no effect on lightinduced vasodilator responses. However, they were (a) blocked entirely by adding oxyhaemoglobin (5 ,uM) to the internal perfusate; and (b) greatly enhanced by the enzyme superoxide dismutase (150 u ml-').4 Photolysis of cluster B was measured by absorption spectroscopy and by detecting NO released with an electrochemical sensor. The photochemical reaction was found to be oxygen-dependent. The half-time for inactivation of cluster-derived NO was measured by interposing different lengths of tubing (i.e time delays) between the photolysis tube and NO sensor. The steady-state probe current decayed exponentially with increasing delay time, with a t112 of 21 s. The amplitudes of vasodilator responses of the tail artery also decreased exponentially by increasing the time delay (t112=58 s). Superoxide dismutase (150 u ml-') prevented this from happening, showing that 'inactivation' of cluster-derived NO was caused by reaction with superoxide anions formed during photolysis. 5 We conclude that potentiation of vasodilator responses to iron-sulphur-nitrosyl clusters by visible light is due to an oxygen-dependent photochemical reaction which accelerates the release of ligated nitrosyl groups as free NO. Based on our measurements, we estimate that ca 100 pM NO is sufficient to produce a just-detectable additional vasodilatation and that the ED50 dose is ca 3.7 nM.

Nicotinamide (NA) is currently entering clinical trials as a radiosensitizer. A major component of its activity is the improvement of tumour oxygenation resulting from a reduction in microregional ischaemia. NA is known to reduce arterial blood pressure in rodents, suggesting a vascular component in its mechanism of action. We have used an ex vivo system to study the direct action of NA on the contractile properties of vascular smooth muscle. Isolated pieces of rat tail artery were internally perfused with Krebs' solution at a constant flow rate so that constriction of the arterial smooth muscle could be measured as an increase in perfusion pressure. Transient vasoconstrictor responses lasting up to 10 min were induced with bolus injections (10 microliters) of phenylephrine, at concentrations ranging from 10(-5) to 10(-2) M, into the internal perfusate whereas a constant increase in vasoconstrictor tone, giving perfusion pressures of 43-84 mmHg, was induced by constantly perfusing with PE (5 x 10(-6) M) or raising the K+ concentration of the Krebs' solution to 122 mM. The addition of NA to the perfusate significantly reduced the size of the transient vasoconstrictor responses in a dose-dependent manner and induced the precontracted vessels to relax. This action of NA could not be blocked either by N omega-nitro-L-arginine methyl ester (L-NAME), indomethacin or propranolol. We conclude that direct effects on supplying blood vessels probably contribute to the oxygenating action of NA in tumours, though the precise mechanism remains obscure.

The role of endogenous nitric oxide (NO) in the growth and vascularization of a rat carcinosarcoma (P22) has been investigated. Tumor-bearing animals were treated with (i) nitric oxide synthase (NOS) inhibitors, administered via the drinking water, including N G -nitro-L-arginine methyl ester (L-NAME), a nonisoform-selective inhibitor, and 2 others that target the inducible (NOS II) enzyme preferentially, namely 1-amino-2-hydroxyguanidine or N-[3-(aminomethyl)benzyl]acetamidine hydrochloride; or (ii) daily injections (intraperitoneally) of 2 Ru(III) polyaminocarboxylates, AMD6221 and AMD6245, both of which are effective NO scavengers. L-NAME, AMD6221, and AMD6245 reduced tumor growth by approximately 60% to 75% of control rates. Tumor sections stained with abs to CD-31/platelet endothelial cell adhesion molecule-1 or NOS III showed that this was associated with a marked reduction (60%-77%) of tumor microvascular densitiy (MVD). Tumors resumed growing promptly when treatment was discontinued, accompanied by partial or complete restoration of MVDs. In contrast, NOS-II selective inhibitors had no effect on tumor growth or vascularization, indicating that both responses require complete blockade of NO production. The results corroborate the view that endogenous NO facilitates tumor development. We suggest that NO deprivation causes tumor feeder vessels to constrict, reducing tumor blood flow. The delivery of oxygen and essential nutrients to the developing tumor is impaired as a consequence, hampering further growth. Normalizing NO levels by withholding treatment causes tumor feeder vessels to dilate, increasing tumor perfusion and reestablishing conditions that allow tumors to begin growing again.

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