Immunolocalization of V1 vasopressin receptors in the rat kidney using anti-receptor antibodies.

By using immunocytochemical techniques we have been able to localize the V1 vasopressin receptor in the rat kidney. Immunoblotting using an antiserum raised against an affinity-purified vasopressin receptor showed a 55,000 daltons protein band that has a molecular mass similar to that of the liver V1 vasopressin receptor, as demonstrated by cross-linking studies. Immunoblotting of the antibody showed a band of 55,000 daltons in A-10 cells, which contains the V1 subtype, whereas it did not stain LLC-PK1 cells, which possess the V2 subtype, showing that the antibody recognizes the V1 vasopressin receptor. The immunostaining of kidney sections with this antiserum showed a strong reaction of the connecting tubules and cortical and medullary collecting ducts. The immunostaining pattern of connecting tubule and collecting duct cells was different, that is, the former showed a staining of both the apical and basal plasma membrane but also in the cytoplasm, whereas the latter showed a strong reaction mainly in the basolateral membrane. Immunostaining of consecutive serial sections with an antiserum raised against tissue kallikrein, an enzyme present exclusively in connecting tubules, and with the anti-receptor serum allowed us to show, for the first time, the presence of the vasopressin receptor in the connecting tubule cells and their absence in intercalated cells, the other cell type present in connecting tubules. These findings support experiments carried in the eighties on the release of renal tissue kallikrein by AVP.

Modulation of the affinity of the vasopressin receptor by magnesium ions.

The binding of [3H]vasopressin (AVP) and the 125I-labelled vasopressin antagonist (VP-AT) d(CH2)5[Tyr2(Me),Tyr9(NH2)]AVP to rat liver membranes was examined with or without the addition of milimolar concentrations of divalent cations. The binding of vasopressin was enhanced by Mg2+ and Co2+ and markedly decreased by EGTA. The addition of EGTA and Mg2+ together restored the binding to a value similar to that of Mg2+ alone. On the contrary, the addition of Mg2+, Co2+, EGTA, and the combination of EGTA and Mg2+ decreased the binding of VP-AT to rat liver membranes. Kinetic analyses showed that Mg2+ increased the Kd twofold for VP-AT; that is from 0.13 nM to 0.28 nM. Moreover, it showed that the receptor with or without the addition of Mg2+ consists of a single population of binding sites, indicating that the receptor is switched from a high affinity to a low affinity state for VP-AT in the presence of 10 mM Mg2+. GTP gamma S was unable to block the effect of Mg2+ on the binding of VP-AT. These results suggest that this divalent cation interacts with receptor itself producing a conformational changes which thus modulates the affinity of the receptor.

Identification of the V1 vasopressin receptor by chemical cross-linking and ligand affinity blotting.

Chemical and photoaffinity cross-linking experiments as well as ligand affinity blotting techniques were used to label the V1 vasopressin receptor. In order to determine the optimal reaction conditions, pig liver membranes were incubated with 5 nM [8-lysine]vasopressin (LVP) labeled with 125I and then cross-linked with the use of DMS (dimethyl suberimidate), EGS [ethylene glycol bis(succinimidyl succinate)] or HSAB (hydroxysuccinimidyl p-azidobenzoate) at different final concentrations. Consistently, EGS was found to label with high yield one band of Mr 60,000 in rat and pig liver membranes when used at a final concentration between 0.05 and 0.25 mM. The protein of Mr 60,000 is labeled in a concentration-dependent manner when pig liver membranes are incubated with increasing concentrations of 125I-LVP and then cross-linked with EGS. The label was displaced by increasing concentrations of unlabeled LVP or d(CH2)5 [Tyr2(Me),-Tyr9(NH2)]AVP (V1/V2 antagonist). A protein band of similar molecular mass was cross-linked with 125I-LVP in rat liver membranes. The reaction was specific since the incorporation of label into the protein of Mr 60,000 was inhibited by LVP, [8-arginine]vasopressin (AVP), the V1/V2-antagonist, and the specific V1-antagonist d(CH2)5 [Tyr2(Me)]AVP, only partially by [des-Gly9]AVP (V2-agonist) and by oxytocin, and not at all by angiotensin II. Incubation of nitrocellulose containing membrane proteins from pig liver with 125I-LVP showed the labeling of a band of Mr 58,000 that is inhibited by an excess of unlabeled LVP. This band of Mr 58,000 seems to correspond with the protein of Mr 60,000 revealed by the cross-linking experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies against specific membrane proteins of neurosecretory granules isolated from bovine neural lobes.

Membrane proteins from bovine neurosecretory granules isolated by density gradient centrifugation were separated by polyacrylamide gel electrophoresis. A doublet of 120 kDa and and 67 kDa bands were identified as specific proteins of the neurosecretory granule membrane. Antibodies against the 120 kDa doublet were raised in rabbits and characterized by western blotting and immunocytochemistry. Analysis of the antiserum by western blotting showed that this recognizes mainly the 120 kDa doublet and some other minor components which seem to be degradation products. The antiserum against the 120 kDa proteins stained, by immunocytochemistry, specifically the supraoptic and paraventricular neurons of the rat hypothalamo-neurohypophysial system. In the neural lobe the immunoreaction was found around blood vessels on structures which appear to be nerve endings and on Herring bodies. Immunoelectron microscopy using protein A-gold showed that the 120 kDa antigens are located on the membrane of neurosecretory granules in sections of rat neural lobes. The presence of the 120 kDa antigens exclusively in the hypothalamo-neurohypophysial system suggests that these proteins are probably not involved in a general secretory mechanism and that they might be a result of the tissue-specific expression of proteins.

Identification and distribution of the cell types in the pituitary gland of Austromenidia laticlavia (Teleostei, Atherinidae).

By using antisera against human pituitary hormones in immunocytochemistry in combination with classical cytochemical techniques, we have been able to identify the different cell types in the adenohypophysis of the Austromenidia laticlavia and to determine their location. Antisera against prolactin and growth hormones did not stain cells in the pituitary of Austromenidia, whereas antisera against beta-endorphin, LH, and beta-TSH selectively cross-reacted with cells which have a specific location within the adenohypophysis. The beta-endorphin antiserum stained the periodic acid-Schiff (PAS)-negative cells in the pars intermedia and also, though faintly, the PAS-negative cells in the internal border of the rostral pars distalis (RPD). Human beta-TSH antiserum showed a discrete population of small PAS-positive cells in the proximal pars distalis (PPD). Antiserum against human LH stained PAS-positive cells located in the most ventral zone of the PPD and around the pars intermedia (PI). The distribution of the different cell types is similar to that of other teleosts. The phylogenetic implications of the degree of cross-reactivity of the antisera against human pituitary hormones with specific cells of the teleost fish pituitary is discussed.

Purification of bovine neurosecretory granule membranes by density gradient centrifugation.

A procedure for the subfractionation of neurosecretory granules into membrane and content components is described. The procedure involves the hypotonic lysis of the secretory granule fraction and further purification of the membranes by centrifugation through a discontinuous sucrose gradient. The neurosecretory granule membranes represented 5.2% of the total proteins of the neurosecretory granule fraction and were highly enriched in cytochrome b561. Electron microscopic analysis of the purified membranes showed vesicles devoid of electrodense content.