David A. Jans

Receptor-mediated endocytosis and nuclear transport of a transfecting DNA construct

A soluble construct consisting of a plasmid carrying the gene of the SV40 large T-antigen and an insulin-poly-L-lysine conjugate is able to selectively transfect PLC/PRF/5 human hepatoma cells which possess insulin receptors. Transfection can be efficiently competed by excess free insulin. To examine intracellular transport of the construct, it was fluorescently labeled and its accumulation on and in cells visualized by video-enhanced microscopy and quantitative confocal laser scanning microscopy. After 2 h at 37 degrees C, the labeled construct was found predominantly in intracellular acidic compartments, with a substantial portion of fluorescence localized both near and in the cell nucleus. Binding, endocytosis, and nuclear localization of the labeled conjugate could all be competed by excess free insulin, thus indicating that entry of the conjugate into cells was specifically mediated by the insulin receptor.

THE USE OF INTERNALIZABLE DERIVATIVES OF CHLORIN E6 FOR INCREASING ITS PHOTOSENSITIZING ACTIVITY

Experiments with human hepatoma PLC/PRF/5 cells and human embryo skin fibroblasts involving the use of three different tests (colony formation, Trypan blue exclusion, labeled thymidine incorporation) have demonstrated a significantly higher photosensitizing activity of chlorin e6 conjugates with internalizable ligands as compared to that of chlorin e6 itself. Receptor‐mediated internalization of chlorin e6 conjugates ensures a greater photosensitization of cells than binding of those conjugates to cell surface receptors. The suitability of such conjugates that permit the delivery of a photosensitizer to sensitive intracellular targets is discussed.

A LOW AFFINITY VASOPRESSIN V2-RECEPTOR IN INHERITED NEPHROGENIC DIABETES INSIPIDUS.

Congenital nephrogenic diabetes insipidus (NDI) is an X-linked inherited disorder characterized by renal resistance to the antidiuretic hormonal action of vasopressin. This study describes the molecular basis of nephrogenic diabetes insipidus in a dog family. Kidney membranes prepared from NDI-affected male huskies were examined for vasopressin binding and response. Compared to membranes from unaffected canines, those from the kidney inner medulla of NDI-dogs possessed normal V2-receptor numbers, but with 10-fold lower affinity for [Arg8] vasopressin (AVP). Adenylate cyclase stimulation by AVP in contrast to that by forskolin or GTP-analogues was similarly reduced in a dose responsive manner. The NDI-affected dogs showed antidiuretic responses to very high doses of V2-specific agonists, consistent with their possessing V2-receptors of lower affinity. Prolonged treatment with V2-agonists, 1-deamino [D-Arg8] VP (dDAVP) and 1-deamino [Val4, Sar7] AVP (dVSAVP), rendered the NDI-affected dogs near normal in terms of water intake and urine osmolality.

Ammonium chloride affects receptor number and lateral mobility of the vasopressin V2-type receptor in the plasma membrane of LLC-PK1 renal epithelial cells: Role of the cytoskeleton

The acidotropic agent ammonium chloride (NH4Cl) not only affects receptor metabolism by inhibiting lysosomal acidification, but can also affect the targeting of proteins to specific membranes in polarized cells, possibly through effects mediated by the cytoskeleton. The present study examines the effects of NH4Cl and perturbers of cytoskeleton structure on vasopressin V2 receptor expression in LLC-PK1 renal epithelial cells. Surprisingly, long-term pretreatment of cells with NH4Cl or short-term treatment with the actin perturber cytochalasin B resulted in an up to 70% increase in specific Arg-8-vasopressin binding compared to control cells, which was independent of the presence of NH4Cl in the binding test, and apparently the result of increased V2 receptor expression. Perturbers of microtubules such as colchicine and vinblastine had no such effect. A rhodamine-labeled analog of vasopressin was used to fluorescently label the V2 receptor of LLC-PK1 cells, and microscopic measurements of membrane-localized fluorescence confirmed the increased V2 receptor expression in the basal plasma membrane subsequent to NH4Cl pretreatment. Lateral mobility of the V2 receptor was measured in living cells using the technique of microphotolysis (photobleaching). The fraction of mobile receptors was 0.2 in cells pretreated with NH4Cl, markedly reduced compared to that of 0.9 in untreated cells. The apparent lateral diffusion coefficient D was about 3 x 10(-10) cm2/s in both pretreated and untreated cells. Results for fluorescence labeling of the actin cytoskeleton indicate that NH4Cl pretreatment of LLC-PK1 cells results in perturbation of microfilament structure. All results imply that the cytoskeleton plays a central role in V2 receptor expression and lateral mobility.

N-glycosylation plays a role in biosynthesis and internalization of the adenylate cyclase stimulating vasopressin V2-receptor of LLC-PK1 renal epithelial cells: An effect of concanavalin A on binding and expression☆

The role of N-glycosylation in the function and biosynthesis of the vasopressin V2-receptor in LLC-PK1 renal epithelial cells was examined using various lectins and inhibitors operating at different steps of the glycosidic pathway. Tunicamycin, which blocks all N-glycosylation, and castanospermine, which inhibits glycosidase I and hence blocks formation of high-mannose-type N-glycosylated intermediates, resembled one another in affecting V2-receptor biosynthesis and internalization in a concentration-dependent manner. In contrast, swainsonine, an inhibitor of mannosidase II and hence of complex-type oligosaccharide formation, had no effect. Interestingly, the alpha-D-mannose/alpha-D-glucose-specific lectin concanavalin A, (Con A), in contrast to the beta-D-galactose-specific lectin ricin, had a marked effect on the V2-receptor in LLC-PK1 cells, increasing both receptor numbers up to twofold in vivo and specific [3H]AVP binding up to 50% in vitro in a concentration-dependent manner. The concentrations inducing half-maximal response were about 0.2 and 20 micrograms/ml for the in vivo and in vitro responses, respectively, implying distinct effects on V2-expression and ligand binding. That the in vitro effect on binding was due to a direct effect on the V2-receptor could be shown by the lack of a Con A effect on [3H]AVP binding in membranes prepared from LLC-PK1 cells down-regulated for the V2-receptor or from cells of the LLC-PK1 V2-receptor deficient mutant M18. All results were consistent with a functional role for N-glycosylation of the V2-receptor in LLC-PK1 cells.

cAMP-dependent protein kinase activation affects vasopressin V2-receptor number and internalization in LLC-PK1 renal epithelial cells

The relationship between activation of the cAMP‐dependent protein kinase (cAMP‐PK) and ligand binding and internalization by the vasopressin renal (V2‐type) receptor of LLC‐PK1 renal epithelial cells was examined. Upon cAMP‐PK activation through 1 h treatment with the cAMP analogue 8‐bromo‐cAMP (BrcA), a marked reduction in V2‐receptor steady state number and internalization in LLC‐PK1 cells was effected. In cells treated for 17 h with BrcA and hence down‐regulated for cAMP‐PK1 the V2‐receptor number was normal but internalization was markedly reduced. Cells of the LLC‐PK1 mutant FIB4, which possesses about 10% parental cAMP‐PK catalytic subunit activity, exhibited lower V2‐receptor steady state number and internalization in comparison to untreated LLC‐PK1 cells. A negative correlation was thus evident between cAMP‐PK activation and V2‐receptor number and internalization. Phosphorylation by cAMP‐PK may effect ligand‐independent removal of receptor from the plasma membrane.

Oxytocin induced cAMP-dependent protein kinase activation and urokinase-type plasminogen activator production in LLC-PK1 renal epithelial cells is mediated by the vasopressin V2-receptor

Using a variety of peptide analogues of oxytocin (OT) and Arg8‐vasopressin (AVP), OT‐mediated induction of urokinase‐type plasminogen activator (uPA) was examined in LLC‐PK1 renal epithelial cells, which possess distinct high‐affinity receptors of both the OT‐ and vasopressin renal (V2‐) types. OT or OT‐receptor specific agonists induced concentration‐dependent cAMP synthesis, activation of the cAMP‐dependent protein kinase (cAMP‐PK) and uPA production consistent with their respective binding affinities for the V2‐ and not the OT‐receptor. OT‐mediated uPA induction could be inhibited in a concentration‐dependent fashion by coincubation with a V2/V1‐receptor specific antagonist, but not by an OT‐receptor specific antagonist. Results implied that stimulation of cAMP‐ and uPA responses in LLC‐PK1 cells by OT was V2‐receptor‐mediated.

An inverse relationship between receptor internalization and the fraction of laterally mobile receptors for the vasopressin renal-type V2-receptor: An active role for receptor immobilization in down-regulation?

Lateral mobility of the vasopressin renal‐type V2‐receptor was investigated in LLC‐PK1, porcine epithelial cells using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine‐labelled vasopressin analogue. At various times after ligand addition, cells were analyzed for both receptor lateral mobility and ligand internalization. The V2‐receptor mobile fraction diminished from 0.9 to 0.43 over 60 min at 37°C. whereas the apparent lateral diffusion coefficient remained essentially unchanged (2–3 × 10−10cm2s). Interestingly, the fraction of immobile V2‐receptors corresponded exactly with the fraction of internalized receptors, implying a functional relationship. These observations together with comparable results reported for other polypeptide hormone receptors indicate a possible machanistic role for receptor immobilization in the desensitization of hormonal response.

A System to Select for Mutant LLC-PK1 Cells Affected in camp Mediated Hormonal Response Using a Photoactivatable Analogue of Vasopressin

The photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenyl-amidino)lysine] vasopressin (apa-LVP) could be used to elicit stimulation of cAMP production in LLC-PK renal epithelial cells, detectable up to 24 h after photoactivation by flash photolysis. This is in contrast to cells treated with vasopressin, or apa-LVP without photoactivation, where cAMP synthesis is down regulated within 4 h. The prolonged stimulation of cAMP production induced by photoactivation of apa-LVP was demonstrated to be cytotoxic to LLC-PK1 cells, whereas the vasopressin receptor negative LLC-PK1 mutant M18 was resistant to the cytotoxic effect. A selection strategy was developed for mutants resistant to this long-term stimulation of cAMP production, whereby multiple cycles of treatment with apa-LVP and photoactivation were used. Mutants so selected were then characterized using a novel screening system for detection of the production of urokinase-type plasminogen activator in response to cAMP agonists. One mutant was examined and found to be impaired in hormonal responsiveness, whereby hormone and forskolin stimulated cAMP-mediated responses were markedly reduced. It exhibited resistance to the long-term stimulation of cAMP production elicited by apa-LVP and photoactivation. This implies that apa-LVP can be used to select for novel mutants specifically impaired in cAMP metabolism and in particular down-regulation of cAMP response.

Generation of anti-idiotypic monoclonal antibodies recognizing vasopressin receptors in cultured cells and kidney sections

To produce anti-idiotypic antibodies against receptors for the neurohypophyseal hormone vasopressin, an anti-vasopressin monoclonal antibody with a ligand specificity similar to that of vasopressin receptors was employed for immunization. Three anti-idiotypic monoclonal antibodies were obtained which induced, like vasopressin, plasminogen activator production in the renal epithelial cell line LLC-PK1 (expressing V2-receptors). Induction of plasminogen activator synthesis by the anti-idiotypic antibodies could be inhibited by coincubation with a vasopressin antagonist. In a fashion similar to that of vasopressin itself, the anti-idiotypic antibodies induced receptor down-regulation. The anti-idiotypic antibodies were employed to visualize vasopressin receptors on LLC-PK1 and A7r5 (V1-receptor-expressing) smooth muscle cells by immunofluorescence. Antibody-mediated fluorescence was not observed in receptor-deficient mutant cell lines or vasopressin-receptor-down-regulated cells. Furthermore, these antibodies were used for immunohistochemical localization of vasopressin receptors in rat and bovine kidney preparations. In accordance with earlier physiological and biochemical observations, vasopressin receptors were detected predominantly in collecting ducts in cortex and medulla. On the cellular level, a differential staining pattern was observed.