ABSTRACT
In this study, we have mapped the immunoreactivity and the binding sites for bufokinin, a tachykinin peptide from the toad intestine. Dense bufokinin-immunoreactive fibers were present at the myenteric plexus, but no cell bodies were stained, suggesting an extrinsic origin. Bufokinin nerve fibers were also associated with submucosal blood vessels and mesenteric arteries. Autoradiographic binding sites for [(125)I]Bolton-Hunter-bufokinin were densely localized over the intestinal circular and longitudinal muscle, submucosal blood vessels and the endothelium of mesenteric arteries. Mesenteric veins had minimal immunoreactivity and binding sites. In the anesthetized toad, topical application of bufokinin onto the mesentery caused a 2.7-fold increase in arterial blood flow, observed using intravital microscopy. This study supports a role for bufokinin as an endogenous spasmogen and hemodynamic regulator in the toad intestine.
Subject(s)
Carrier Proteins/isolation & purification , Intercellular Signaling Peptides and Proteins , Intestines/chemistry , Receptors, Tachykinin/isolation & purification , Splanchnic Circulation , Tachykinins/isolation & purification , Animals , Binding Sites , Bufonidae , Carrier Proteins/pharmacology , Dose-Response Relationship, Drug , Immunohistochemistry , Mesenteric Arteries/chemistry , Mesenteric Arteries/drug effects , Mesenteric Veins/chemistry , Microcirculation , Succinimides , Tachykinins/pharmacology , Tissue DistributionABSTRACT
There have been proposals that the tachykinin receptor classification should be extended to include a novel receptor, the "neurokinin-4" receptor (NK-4R), which has a close homology with the human NK-3 receptor (hNK-3R). We compared the pharmacological and molecular biological characteristics of the hNK-3R and NK-4R. Binding experiments, with (125)I-[MePhe(7)]-NKB binding to HEK 293 cell membranes transiently expressing the hNK-3R (HEK 293-hNK-3R) or NK-4R (HEK 293-NK-4R), and functional studies (Ca(2+) mobilization in the same cells) revealed a similar profile of sensitivity to tachykinin agonists and antagonists for both receptors; i.e., in binding studies with the hNK-3R, MePhe(7)-NKB > NKB > senktide >> NKA = Substance P; with the NK-4R, MePhe(7)-NKB > NKB = senktide >> Substance P = NKA; and with antagonists, SB 223412 = SR 142801 > SB 222200 >> SR 48968 >> CP 99994 for both hNK-3R and NK-4R. Thus, the pharmacology of the two receptors was nearly identical. However, attempts to isolate or identify the NK-4R gene by using various molecular biological techniques were unsuccessful. Procedures, including nested polymerase chain reaction studies, that used products with restriction endonuclease sites specific for either hNK-3R or NK-4R, failed to demonstrate the presence of NK-4R in genomic DNA from human, monkey, mouse, rat, hamster, or guinea pig, and in cDNA libraries from human lung, brain, or heart, whereas the hNK-3R was detectable in the latter libraries. In view of the failure to demonstrate the presence of the putative NK-4R it is thought to be premature to extend the current tachykinin receptor classification.
Subject(s)
Receptors, Neurokinin-3/metabolism , Receptors, Tachykinin/metabolism , Binding, Competitive , Biological Transport , Calcium/metabolism , Cells, Cultured , DNA, Complementary/analysis , Humans , Polymerase Chain Reaction , Radioligand Assay , Receptors, Neurokinin-3/drug effects , Receptors, Neurokinin-3/genetics , Receptors, Tachykinin/drug effects , Receptors, Tachykinin/genetics , Receptors, Tachykinin/isolation & purification , Restriction Mapping , Tachykinins/metabolismABSTRACT
The autoradiographic localization of binding sites for [125I]BH-[Sar9,Met(O2)11]SP, [125I]NKA, and [125I]CGRP was investigated in adjacent sections of urinary bladder body, from adult rats pretreated 14 days before with capsaicin or vehicle. Location of silver grains was assessed both qualitatively and quantitatively using computerized densitometry. Dense labeling of smooth muscle was seen with both [125I]BH-[Sar9,Met(O2)11]SP ([125I]BHSar-SP) and [125I]NKA; in addition, [125I]BHSar-SP labeled submucosal blood vessels. For these radioligands, no differences were apparent between sections from capsaicin- and vehicle-pretreated rats. Specific binding of [125I]CGRP was observed over the epithelium and weakly over submucosal arterioles, but not over smooth muscle. The density of [125I]CGRP binding sites on the epithelium, but not blood vessels, was increased (p < 0.05) by 22% after chronic capsaicin pretreatment, suggesting receptor upregulation. This study demonstrates that although all three peptides are colocalized in primary afferent sensory fibers in rat urinary bladder, the receptors for these neuropeptides are located on different cell types and may be subject to different neural influences.
