A cDNA encoding a functional bradykinin receptor was isolated from a rat uterus library by a clonal selection strategy using Xenopus laevis oocytes to assay for expression of bradykinin responses. The predicted protein is homologous to the seven transmembrane G protein-coupled superfamily of receptors. Bradykinin and its analogs stimulate a Cl current in oocytes expressing the receptor with the rank order of potency: bradykinin Lys-bradykinin > [Tyr8]-bradykinin >> [Phe6Jbradykinin. This is the rank order of potency observed for these compounds in competitive binding assays on soluble receptor from rat uterus. Des-Arg9-bradykinin (10 pAM) elicits no response when applied to oocytes expressing the receptor; thus, the cDNA encodes a B2 type bradykinin receptor. [This8,DPhe7Jbradykinin, where Thi is I3-(2-thienyl)-alanine, is a very weak partial agonist and inhibits the bradykinin-mediated ion flux, suggesting the cDNA encodes a smooth muscle, rather than a neuronal, B2 receptor subtype. Receptor message has a distribution consistent with previous reports of bradykinin function and/or binding in several tissues and is found in rat uterus, vas deferens, kidney, lung, heart, ileum, testis, and brain. Receptor subtypes are a possibility because several tissues contain two or three message species (4.0, 5.7, and 6.5 kilobases). Southern blot highstringency analysis demonstrated that the rat, guinea pig, and human genomes contain a single gene. As bradykinin is a key mediator of pain, knowledge of the primary structure of this receptor will allow a molecular understanding of the receptor and aid the design of antagonists for pain relief.The nonapeptide bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-ProPhe-Arg) is a mediator of pain, inflammation, vascular permeability, smooth muscle tone in vascular and other tissues, and gastrointestinal function (1-5). Bradykinin can serve as a growth factor (2, 6, 7). Bradykinin binds to G proteincoupled receptors that activate phospholipase C or phospholipase A2 and increases synthesis of inositol trisphosphate or arachadonic acid (8-10).Bradykinin receptors have been classified as two major subtypes, B1 and B2 (1). The bradykinin metabolite des-Arg9-bradykinin is a B1 receptor agonist with potency greater than bradykinin, whereas it is inactive at B2 receptors. B2 receptors have been subdivided into two subtypes, a "neuronal" form, which is fully activated by [Thi58,DPhe7]bradykinin, where Thi is 83-(2-thienyl)-alanine, and a "smooth muscle" form, which is weakly activated by [This'8,DPhe7]bradykinin (11,12). Other subtypes of the B2 receptor have also been suggested (13,14).

Nicotinic acetylcholine receptor (AChR) genes are expressed in subpopulations of chick dorsal root ganglion (DRG) neurons. In 18-day embryonic ganglia, 19% of the neurons have material homologous to neuronal AChR alpha 3 gene mRNA, and 8% have material homologous to alpha 4 mRNA as seen with in situ hybridization. RNAase protection experiments confirm that DRG RNA contains alpha 3 and alpha 4 transcripts, and Northern blot analysis establishes the size of the transcripts as being 3.5 and 3.3 kb, respectively. The proportion of DRG neurons containing alpha 3 mRNA does not decline up through 1 year post-hatch, indicating that alpha 3 gene expression is not a developmentally transient event in the ganglion. An antiAChR monoclonal antibody detects cross-reacting material in 16% of the DRG neurons from 18-day embryos, indicating that AChR mRNA is translated into protein. Electrophysiological measurements confirm the presence of functional AChRs on DRG neurons freshly isolated from 18-day embryos: 24% of the neurons have substantial ACh sensitivities, whereas another 23% have small but detectable responses. Staining dorsal root ganglion sections with an anticholine acetyltransferase antiserum reveals cross-reactive material localized in axons in the ganglion; no evidence suggests the presence of cholinergic synaptic structures or AChR clusters on neuronal somata in the ganglion. It is possible that AChRs on DRG neurons participate in a diffuse form of transmission between the cholinergic fibers and a subpopulation of neuronal somata in the ganglion. Alternatively, AChRs on the somata may represent an ectopic distribution of receptors whose primary function is at the terminals of central or peripheral DRG processes.

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate chemical transmission through the ganglion, and GABAA receptors of unknown significance. Previous experiments examining the role of cell-cell interactions in regulating neuronal AChRs have shown that postganglionic axotomy of ciliary ganglia in newly hatched chicks causes a 10-fold decline in total AChRs within 5 d compared with unoperated contralateral ganglia and that preganglionic denervation causes a 3-fold decline within 10 d. Many of the AChRs are known to be intracellular; of those present on the cell surface, only a small fraction appears to be functionally available normally. In the present experiments, the effects of the operations on functional AChRs and GABAA receptors in the plasma membrane of the neurons were examined by removing the ganglia 5 d after axotomy or 10 d after denervation, dissociating them into single cells, and immediately measuring their ACh and GABA sensitivities with intracellular recording techniques. The ACh sensitivity of axotomized ciliary ganglion neurons was reduced 10-fold compared with neurons from unoperated contralateral ganglia of the same chicks. The reduction could be largely accounted for by a decrease in the maximum response and did not arise from a change either in the dose-response curve or the acetylcholinesterase activity of the neurons. Autoradiographic studies using a radiolabeled anti-AChR monoclonal antibody also demonstrated a substantial decrease in the total number of surface AChRs associated with axotomized neurons. In contrast, axotomy had no unilateral effect on the GABA response.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract