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Corticotropin releasing factor (CRF) and Urocortin are important neurotransmitters in the regulation of physiological and behavioral responses to stress. Centrally administered CRF or Urocortin produces anxiety-like responses in numerous animal models of anxiety disorders. Previous studies in our lab have shown that Urocortin infused into the basolateral nucleus of the amygdala produces anxiety-like responses in the social interaction test. Subsequently, in the current study we prepared a specific CRF1 receptor antagonist (N-Cyclopropylmethyl-2,5-dimethyl-N-propyl-N'-(2,4,6-trichloro-phenyl)-pyrimidine-4,6-diamine, NBI3b1996) to examine in this paradigm. This CRF1 receptor antagonist inhibited the ex vivo binding of 125I-sauvagine to rat cerebellum with an ED50 of 6 mg/kg, i.p. NBI3b1996 produced a dose-dependent antagonism of Urocortin-induced anxiety-like behavior in Social Interaction test with an ED50 of 6 mg/kg, i.p. The compound had no effect on baseline social interaction. In addition, the CRF1 receptor antagonist prevented the stress-induced decrease in social interaction. These results provide further support for the CRF1 receptor in anxiety-like behavior and suggest this pathway is quiescent in unstressed animals.

Urocortin, a novel 40 amino acid neuropeptide, is a member of the corticotropin-releasing factor family. With 45% homology to corticotropin-releasing factor, urocortin binds with similar affinity to the corticotropin-releasing factor- and corticotropin-releasing factor-2 receptors and may play a role in modulating many of the same systems as corticotropin-releasing factor. To assess whether urocortin and corticotropin-releasing factor are localized in the same regions of the brain, we compared the distribution of urocortin- and corticotropin-releasing factor-like immunoreactivities in the rat central nervous system. Polyclonal antibodies to rat corticotropin-releasing factor and rat urocortin were generated and utilized to map the distribution of corticotropin-releasing factor- and urocortin-like immunoreactivities throughout the rat forebrain and brainstem. Characterization of the antibodies by radioimmunoassay showed no cross-reactivity with related peptides. Male Sprague-Dawley rats were treated with colchicine for 18-24 h. Following colchicine treatment, the rats were perfused with paraformaldehyde-lysine-periodate fixative and their brains removed. Serial coronal sections were taken throughout the rat brain and processed for either corticotropin-releasing factor- or urocortin-like immunoreactivity. Urocortin-like immunoreactivity shows a discrete localization within several regions including the supraoptic nucleus, the median eminence, Edinger-Westphal nucleus and the sphenoid nucleus. This is in contrast to the more abundant corticotropin-releasing factor-like immunoreactivity. Regions containing high levels of corticotropin-releasing factor immunoreactivity include the lateral septum, paraventricular nucleus of the hypothalamus, median eminence and locus coeruleus. There are a few regions that contain both urocortin-immunoreactive and corticotropin-releasing factor-immunoreactive cells, such as the supraoptic nucleus and the hippocampus. Therefore, urocortin and corticotropin-releasing factor appear to have different distribution patterns which may be indicative of their respective physiological functions.

Through its pro-inflammatory effects on leukocytes, endothelial cells, and keratinocytes, the lipid mediator platelet-activating factor (PAF) has been implicated in cutaneous inflammation. Although the 1-alkyl PAF species has been considered historically the most abundant and important ligand for the PAF receptor (PAF-R), other putative ligands for this receptor have been described including 1-acyl analogs of sn-2 acetyl glycerophosphocholines. Previous bioassays have demonstrated a PAF-like activity in lesions of the autoimmune blistering disease bullous pemphigoid. To assess the actual sn-2 acetyl glycerophosphocholine species that result in this PAF agonistic activity, we measured PAF and related sn-2 acetyl GPCs in fresh blister fluid samples from bullous pemphigoid and noninflammatory (suction-induced) bullae by mass spectrometry. We report the presence of 1-hexadecyl as well as the 1-acyl PAF analog 1-palmitoyl-2-acetyl glycerophosphocholine (PAPC) in inflammatory blister fluid samples. Because PAPC is the most abundant sn-2 acetyl glycerophosphocholine species found in all samples examined, the pharmacological effects of this species with respect to the PAF-R were determined using a model system created by transduction of a PAF-R-negative epidermoid cell line with the PAF-R. Radioligand binding and intracellular calcium mobilization studies indicated that PAPC is approximately 100x less potent than PAF. Though a weak agonist, PAPC could induce PAF biosynthesis and PAF-R desensitization. Finally, intradermal injections of PAF and PAPC into the ventral ears of rats demonstrated that PAPC was 100x less potent in vivo. These studies suggest possible involvement of PAF and related species in inflammatory bullous diseases.