ABSTRACT
BACKGROUND: The ion channel TRPV1 is mainly expressed in small diameter dorsal root ganglion (DRG) neurons, which are involved in the sensation of acute noxious thermal and chemical stimuli. Direct modifications of the channel by diverse signalling events have been intensively investigated, but little is known about the composition of modulating macromolecular TRPV1 signalling complexes. Here, we hypothesize that the novel adaptor protein ankyrin-rich membrane spanning protein/kinase D interacting substrate (ARMS) interacts with TRPV1 and modulates its function in rodent DRG neurons. METHODS: We used immunohistochemistry, electrophysiology, microfluorimetry and immunoprecipitation experiments to investigate TRPV1 and ARMS interactions in DRG neurons and transfected cells. RESULTS: We found that TRPV1 and ARMS are co-expressed in a subpopulation of DRG neurons. ARMS sensitizes TRPV1 towards capsaicin in transfected HEK 293 cells and in mouse DRG neurons in a PKA-dependent manner. Using a combination of functional imaging and immunocytochemistry, we show that the magnitude of the capsaicin response in DRG neurons depends not only on TRPV1 expression, but on the co-expression of ARMS alongside TRPV1. CONCLUSION: These data indicate that ARMS is an important component of the signalling complex regulating the sensitivity of TRPV1. SIGNIFICANCE: The study identifies ARMS as an important component of the signalling complex regulating the sensitivity of excitatory ion channels (TRPV1) in peripheral sensory neurons (DRG neurons) and transfected cells.
Subject(s)
Membrane Proteins/metabolism , Nociceptors/metabolism , TRPV Cation Channels/metabolism , Animals , Capsaicin/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , HEK293 Cells , Humans , Mice , Nociceptors/drug effectsABSTRACT
We have determined the effect of ovariectomy and hypophysectomy on prolactin receptors in 7,12-dimethylbenz(a)anthracene-induced mammary tumors. Growing tumors from intact rats show a wide range in the number of prolactin receptors. Ovariectomy causes a slight (approximately 30%) decrease in receptors regardless of whether tumors regress or continue to grow, while the affinity of the receptor for prolactin remains unchanged. Hypophysectomy, which causes a prompt 10-fold decrease in prolactin receptors in rat liver, causes only a slight reduction in prolactin receptors in tumors from these same animals. We conclude that autonomous and ovariectomy responsive 7,12-dimethylbenz(a)anthracene-induced mammary tumors cannot be distinguished on the basis of prolactin receptor sites and that endocrine regulation of prolactin receptors is distinctly different in normal liver and neoplastic mammary tissue.
Subject(s)
Castration , Hypophysectomy , Mammary Neoplasms, Experimental/metabolism , Prolactin/metabolism , Receptors, Cell Surface , 9,10-Dimethyl-1,2-benzanthracene , Animals , Depression, Chemical , Female , Liver/metabolism , Mammary Neoplasms, Experimental/chemically induced , RatsABSTRACT
Prolactin reverses the inhibitory effects of pharmacological doses of androgen on 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth (Quadri, S.K., Kledzik, G.S., and Meites, J.J. Natl. Cancer Inst., 52: 875-878,1974). To determine whether this effect is due to an alteration in the ability of the tumor cell to bind prolactin, we have quantitated prolactin receptors in androgen-responsive and nonresponsive tumors. Prolactin receptors were measured with 125I-labeled ovine prolactin in a subcellular fraction which reproducibly contained 60 to 80% of the total receptor present in tumor homogenates. Prolactin binding was reversible, reached a steady state in 9 hr, and was completed by excess unlabeled prolactin. Prolactin bound to its receptor with a Kd of approximately 1 X 10(-10) M. Growing tumors were biopsied, and rats bearing regrown tumors were given injections of 4 mg testosterone propionate twice a week. Prolactin receptors were reduced in most of the tumors, which regressed after testosterone treatment by an average of 63% compared to the pretreatment biopsy specimens. Nonresponsive tumors and vehicle-injected controls showed no signifcant alterations in receptor content. This reduction of prolactin receptors is probably insufficient to account for androgen-induced mammary tumor regression.
Subject(s)
Mammary Neoplasms, Experimental/metabolism , Prolactin/metabolism , Testosterone/pharmacology , 9,10-Dimethyl-1,2-benzanthracene , Animals , Female , Mammary Neoplasms, Experimental/chemically induced , Mammary Neoplasms, Experimental/drug therapy , Prolactin/pharmacology , Rats , Receptors, Cell Surface/drug effects , Testosterone/therapeutic useABSTRACT
Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we described the optimal conditions for the measurement of 125I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (Kd approx. 2.36 - 10(-9) M), hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lactation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7--8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in a prompt 3--6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eighth and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.
Subject(s)
Lactation , Mammary Glands, Animal/metabolism , Prolactin/metabolism , Animals , Binding Sites , Estradiol/pharmacology , Female , In Vitro Techniques , Pregnancy , Progesterone/pharmacology , Prolactin/pharmacology , Rats , Sheep , Temperature , Time FactorsSubject(s)
Liver/metabolism , Prolactin/pharmacology , Receptors, Cell Surface/drug effects , Animals , Estradiol/pharmacology , Female , Growth Hormone/pharmacology , Hydrocortisone/pharmacology , Liver/drug effects , Pituitary Gland/physiology , Progesterone/pharmacology , Prolactin/metabolism , Rats , Stimulation, Chemical , Triiodothyronine/pharmacologyABSTRACT
A hormone-dependent subline of the transplantable rat mammary tumor MTW9 contains binding sites for both prolactin and estrogen. Prolactin binding is saturable (K-d similar to 2 times 10-9 M), hormone specific, and destroyed by proteases. By contrast, an autonomous subline derived from the same parent tumor has lost more than 75% of both prolactin- and estrogen-binding sites, although binding affinities for both hormones are unchanged. This reduction in binding sites for both prolactin and estrogen in the autonomous line may result in an incomplete recognition of the tumor cells as a target for the circulating hormones with a subsequent loss of hormone-dependent growth characteristics.
Subject(s)
Estrogens/metabolism , Mammary Neoplasms, Experimental/metabolism , Prolactin/metabolism , Receptors, Cell Surface , Animals , Cell Line , Cell Nucleus/metabolism , Cytosol/metabolism , Estradiol/metabolism , Female , Peptide Hydrolases , Rats , Rats, Inbred WFABSTRACT
We demonstrate for the first time the presence of specific high-affinity receptors for prolactin in rat mammayy carcinoma. There appear to be no significant differences between normal rat mammary tissue and the transplantable, estrogen receptor-deficient R3230AC tumor with regard to the number of binding sites, the affinity of the receptor for prolactin, or the specificity of binding.
