Palmitoylation of the human bradykinin B2 receptor influences ligand efficacy.

To investigate the palmitoylation of the human bradykinin B2 receptor, we have mutated individually or simultaneously into glycine two potential acylation sites (cysteines 324 and 329) located in the carboxyl terminus of the receptor and evaluated the effects of these mutations by transfection in COS-7, CHO-K1, and HEK 293T. The wild-type receptor and the single mutants, but not the double mutant, incorporated [3H]palmitate, indicating that the receptor carboxyl tail can be palmitoylated at both sites. The mutants did not differ from the wild-type receptor for the kinetics of [3H]bradykinin binding, the basal and bradykinin-stimulated coupling to phospholipases C and A2, and agonist-induced phosphorylation. The nonpalmitoylated receptor had a 30% reduced capacity to internalize [3H]bradykinin. This indicates that palmitoylation does not influence the basal activity of the receptor and its agonist-driven activation. However, the mutants triggered phospholipid metabolism and MAP kinase activation in response to B2 receptor antagonists. Pseudopeptide and nonpeptide compounds that behaved as antagonists on the wild-type receptor became agonists on the nonpalmitoylated receptor and produced phospholipases C and A2 responses of 25-50% as compared to that of bradykinin. These results suggest that palmitoylation is required for the stabilization of the receptor-ligand complex in an uncoupled conformation.

Activation of mitogen-activated protein kinase by the bradykinin B2 receptor is independent of receptor phosphorylation and phosphorylation-triggered internalization.

Recent evidence suggests that serine/threonine phosphorylation and internalization of beta2-adrenergic receptors play critical roles in signalling to the mitogen-activated protein kinase cascade. To investigate whether this represents a general mechanism employed by G protein-coupled receptors, we studied the requirement of these processes in the activation of mitogen-activated protein kinase by G alpha(q)-coupled bradykinin B2 receptors. Mutant B2 receptors impaired in receptor phosphorylation and internalization are fully capable to activate mitogen-activated protein kinase. Bradykinin-induced long-term effects on mitogenic signalling monitored by measuring the transcriptional activity of Elk1 were identical in cells expressing the wild-type or mutant B2 receptors. Therefore, G protein-coupled bradykinin receptors activate the mitogen-activated protein kinase pathway independently of receptor phosphorylation and internalization.

Bradykinin-induced internalization of the human B2 receptor requires phosphorylation of three serine and two threonine residues at its carboxyl tail.

The binding of bradykinin (BK) to B2 receptor triggers the internalization of the agonist-receptor complex. To investigate the mechanisms and the receptor structures involved in this fundamental process of receptor regulation, the human B2 receptor was mutated within its cytoplasmic tail by complementary strategies of truncation, deletion, and amino acid substitution. Ligand binding, signal transduction, internalization as well as phosphorylation were studied for the mutated receptors expressed in COS, CHO, and HEK 293 cells. Truncation of 44 out of 55 amino acid residues of the receptor's cytoplasmic tail corresponding to positions 321-364 did not alter the kinetics of BK binding and the receptor coupling to phospholipase C and phospholipase A2. By contrast, truncations after positions 320 and 334, deletions within the segment covering positions 335-351, as well as alanine substitution of serine and threonine residues within segment 335-351 diminished the internalization capacity of the mutant receptors. Mutants with a markedly reduced internalization potential failed to produce BK-induced receptor phosphorylation suggesting that phosphorylation may be involved in receptor internalization. The mutagenesis approaches converged at the conclusion that three serines in positions 339, 346, and 348 and two threonines in positions 342 and 345, contained in a sequence segment that is highly conserved between species, have a critical role in the ligand-dependent internalization and phosphorylation of kinin receptors and can intervene in these processes in an alternative manner. However, mutants lacking these residues were still sensitive to dominant-negative forms of beta-arrestin and dynamin, suggesting the existence of additional receptor structure(s) involved in the receptor sequestration through clathrin-coated vesicles.

The Ca2+-sensing receptor in the rabbit cortical thick ascending limb (CTAL) is functionally not coupled to phospholipase C.

The recently cloned rabbit kidney Ca2+-sensing receptor (RabCaR) was functionally characterized in microperfused rabbit cortical thick ascending limb (CTAL) segments. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed that this nephron segment contains mRNAs coding for the RabCaR. Elevation of the extracellular Ca2+ concentration ([Ca2+]e) from 1 to 5 mmol l-1 induced an increase in the fluorescence emission ratio (R), thus reflecting an increase in intracellular Ca2+ activity ([Ca2+]i). This increase was inhibited by verapamil, nifedipine and SKF 96365, and potentiated by a previous application of Bay K 8644. Neither verapamil nor Bay K 8644 modified the resting [Ca2+]i. This suggests that the basolateral Ca2+ influx induced by a high [Ca2+]e occurs via verapamil- and dihydropyridine-sensitive Ca2+ channels, which are not open under resting conditions. In contrast to that evoked by antidiuretic hormone (ADH), the [Ca2+]i increase induced by a high [Ca2+]e did not result from an accumulation of inositol phosphates. Neomycin, Gd3+, Mg2+, commonly used agonists of the Ca2+-sensing receptor, did not increase the [Ca2+]i. In the presence of verapamil, ADH still produced a transient [Ca2+]i increase that was not observed in the presence of an increased [Ca2+]e. These results suggest that the RabCaR in rabbit CTAL cells is not functionally coupled to phospholipase C. In conclusion, the high [Ca2+]e-induced [Ca2+]i increase involves verapamil- and dihydropyridine-sensitive Ca2+ channels and is independent of phosphoinositide metabolism. Whether these channels are activated by the RabCaR remains to be elucidated.

Negative cooperativity in the human bradykinin B2 receptor.

A human kidney bradykinin (BK) B2 receptor cDNA was transfected in CHO-K1 cells to establish cell lines that express stably and at high density a receptor exhibiting B2 receptor properties in terms of coupling to cell signaling effectors, desensitization, and internalization. A cell line with a density of 1.3 x 10(6) receptors/cell allowed us to carry out a detailed study of BK-receptor interaction over a wide range of BK concentrations. A model assuming that BK binds to two receptor affinity states (depending on guanine nucleotide-sensitive coupling) was not sufficient to account for the kinetics of BK binding. Equilibrium kinetic analysis and studies of the effects of receptor occupancy by agonists or antagonists on the kinetics of BK-receptor complex dissociation revealed features typical of negative cooperative binding. The negative cooperativity phenomenon was also observed in isolated membranes in both the presence and absence of guanine nucleotide. Thus, following the interaction with BK, B2 receptor molecules likely interact with each other, resulting in an acceleration of bound ligand dissociation and a decrease in the apparent affinity of the receptor for BK. This phenomenon can participate in the desensitization process.

Cholinergic agonists increase phosphoinositide metabolism and cell calcium in isolated rat renal proximal tubule.

The intracellular signaling involved in cholinergic modulation of renal proximal tubule functions has not been addressed. We report that acetylcholine and carbachol increase the production of inositol phosphates and the intracellular calcium concentration in rat proximal tubule. Muscarinic M3 receptors are involved, given the inhibitory effects of selective antagonists (4-diphenylacetoxy-N-methylpiperidine > > pirenzepine > methoctramine). The muscarinic response is absent in the early part of proximal straight tubule. The response is smaller and more variable in proximal convoluted tubule segments selected at random from cortical tubule suspensions than in the early part of proximal convoluted tubule. This contrasts with the norepinephrine response, which has almost the same magnitude all along the proximal tubule. We conclude that cholinergic agonists activate muscarinic M3 receptors in proximal tubule and that there is a decreasing response gradient from the early convoluted tubule to the early straight tubule.

The parietal sheet of Bowman's capsule of rat renal glomerulus: a target of endothelin and PAF.

On the basis of intracellular calcium concentration ([Ca2+]i) measurements, we have previously reported that the parietal sheet of Bowman's capsule was sensitive to cholinergic agonists. The aim of the present work was to investigate whether this structure could be also a target of endothelin and platelet-activating factor (PAF), since we observed [Ca2+]i increases in response to both agonists in the glomerulus, but which were very different from that induced by carbachol. For this purpose, we measured [Ca2+]i on single microdissected parietal sheets, using a fura 2 microfluorescence technique and compared the effects of maximal concentrations of the three agonists (10(-7), 10(-8), and 10(-4) M for endothelin, PAF, and carbachol, respectively) under various experimental conditions. We observed that, like in the glomerulus, endothelin and PAF induced, in the parietal sheet, [Ca2+]i responses that differed in many respects from those found with carbachol. Thus, in the presence of 2 mM external calcium, 1) endothelin and PAF responses spontaneously declined to basal level, whereas a stationary plateau was observed after a sharp peak of [Ca2+]i with carbachol; 2) the magnitude of [Ca2+]i peak was smaller with endothelin and PAF than with carbachol; and 3) endothelin and PAF, but not carbachol, induced a homologous dose-dependent desensitization. Moreover, in the absence of external calcium, endothelin and PAF responses were smaller than carbachol response, although all three responses apparently resulted from release of calcium ions from the same internal pool. In additional experiments, we observed that, like carbachol, endothelin and PAF contracted the parietal sheet, which is only composed of myoepithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Frog glomerular vasotocin receptors resemble mammalian V1b receptors.

Vasotocin receptors were investigated in glomeruli and nephron segments microdissected from collagenase-treated kidneys of Rana ridibunda, using [d(CH2)5Tyr(Me)2,Thr4,Orn8,125I-Tyr-NH2(9)]vasotocin (125I-OVTA) as a radioligand. Specific 125I-OVTA binding sites were found only in glomeruli and not in all tubule segments tested. Glomerular receptors exhibited the following stereospecificity for recognition of vasotocin analogues: Tyr-NH2(9)-LA-V1a > 125I-OVTA > arginine vasotocin (AVT) > or = [d(CH2)5Tyr-(Me)2]AVP > OVTA > or = [Phe2,Orn8]VT > oxytocin (OT) > or = [d(CH2)5-Sar7]AVP > desGly9[d(CH2)5Tyr(Et)2]VAVP > or = [d(CH2)5Tyr(Et)2]VAVP > AVP > [1-desamino-8-D-arginine]vasopressin (DDAVP) > [Thr4,Gly7]OT. In addition, vasotocin enhanced [3H]inositol phosphate production in sieved glomeruli labeled with myo-[3H]inositol; the rank order of structural vasotocin analogues for stimulation of phosphoinositidase C was [Phe2,Orn8]VT > AVT > OT > AVP > DDAVP, whereas [Thr4,Gly7]OT was almost inactive, and the rank order of antagonists for inhibition of hormone-induced enzyme activation was Tyr-NH2(9)-LA-V1a > [d(CH2)5Tyr(Me)2]AVP = OVTA > [d(CH2)5Sar7]AVP > [d(CH2)5Tyr(Et)2]VAVP > or = desGly9[d(CH2)5Tyr(Et)2]VAVP. Results indicate that the 125I-OVTA-labeled binding sites detected in frog glomeruli reveal the pharmacological properties of mammalian V1b-pituitary vasopressin receptors and might be physiological vasotocin receptors involved in phosphoinositidase C stimulation.

Cholinergic stimulation of phosphoinositide metabolism in isolated rat glomeruli.

Cholinergic effects on kidney function have been observed in some mammals but the intrarenal localization and the cellular mechanisms of these effects are poorly defined to date. The aim of this work was to study the effects of carbachol on phosphoinositide metabolism in freshly isolated rat glomeruli labeled with myo-[3H]inositol. Carbachol rapidly and markedly stimulates phosphoinositide metabolism with a 50% effective concentration of 3 microM. The enormous magnitude of the response is enlightened by the use of 10 mM lithium, which provokes in the presence of the agonist a large accumulation of inositol phosphates and a corresponding depletion of cellular free inositol. The response is inhibited by 85% by pirenzepine, is pertussis toxin insensitive, and shows no desensitization at maximum dose of carbachol up to 40 min of stimulation.

Relationship between cell volume and cation content in thick ascending limb of rat kidney.

We examined the relationship between the cell volume and cation concentration ([Nai] and [Ki]) of isolated segments of rat medullary thick ascending limb (MAL) after incubation at 30 degrees C in various isotonic solutions. When the tubules were incubated in a normal NaCl solution containing 5 mmol/l K+, addition of 1 mmol/l of ouabain increased [Nai] and decreased [Ki] but did not change the total ([Nai] + [Ki]) concentration (about 90 mEq/l) or tubular volume. After incubation in various K+-free solutions, the tubules were almost fully K+-depleted; their volume per unit of length was similar in the three solutions, although the choline Cl-treated tubules had a very low sodium content compared to the NaCl- and Na2SO4-treated tubules (8 vs. 97 and 95 mEq/l respectively). Ouabain altered neither volume nor [Nai] of tubules incubated in choline Cl or Na2SO4 solution. Transfer of tubules from K+-free Na2SO4 or K+-free choline Cl solution into K+-free NaCl solution resulted in an increase in [Nai] (by 29 and 97 mEq/l respectively) without much increase in tubular volume. A marked swelling of the tubules was only observed when the K+-free NaCl solution contained also ouabain. Under this condition, [Nai] was comparable to the Na+ concentration of the incubation medium. After washing and incubation in a normal NaCl solution containing K+, the swollen tubules recovered their initial volume and restored Na+ and K+ concentration gradients across the cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of Na and Cl in ouabain-induced cell swelling in thick ascending limb of rat kidney.

Changes in the volume of isolated segments of rat medullary thick ascending limb (MAL) were studied by a photographic technique, after tubule incubation in isotonic solutions in the absence or presence of ouabain and/or K. When segments were incubated at 30 degrees C in NaCl solution, their volume increased by 75% after removal of external K, and by 170% after removal of external K plus addition of 1 mmol/l ouabain. At steady state, tubular volume was a function of the external K concentration. Resting volume was obtained with external K concentrations higher than 0.1 and 1.0 mmol/l in the absence and presence of ouabain respectively. When MAL samples were incubated in isotonic K-free Na2SO4 or K-free choline Cl solution, their volume per unit of length was similar to that determined in NaCl medium, but there was no swelling after the addition of ouabain. The ouabain-induced swelling was shown to depend on both the Na and Cl concentrations in the incubate (apparent Km of 87 and 80 mmol/l for Na and Cl respectively). Swollen tubules recovered their resting volume when ouabain, Na or Cl was removed from the incubation medium. Recovery of resting volume was also observed after addition of K into the incubation medium. These observations indicate that rat MAL cell volume is the result of coupled passive net fluxes of Na and Cl, which depend on the respective electrochemical gradients for Na or Cl across the cell membranes and the Na-pump activity which continuously extrudes Na.

Effects of temperature, ouabain and diuretics on the cell sodium and potassium contents of isolated rat kidney tubules.

Cell Na+ and K+ contents were measured by flame photometry in single pieces of rat medullary thick ascending limb (MAL) and medullary collecting tubules (MCT) after an overnight incubation at various temperatures. Below 8 degrees C, MCT samples were no more able to sustain high-K+, low-Na+ cell concentrations, and sodium progressively replaced cell potassium as the temperature decreased. The loss of potassium and the accompanying accumulation of sodium by MCT cells occurred at lower or higher temperature when amiloride (20 mumol/l) or ouabain (1 mmol/l) was present respectively in the incubation medium compared to that observed on control non-treated MCT. In contrast, MAL samples maintained normal cation gradients across their membrane at all temperatures, including 0 degree C, even in the presence of ouabain. However, MAL cells lost nearly all their potassium which was replaced by sodium when they were incubated in K+-free solution. These Na+-loaded, K+-depleted MAL cells restored high-K+ and low-Na+ contents similar to those of control samples when they were further incubated for 1 h at 0 degree C in presence of 5 mmol/l external potassium. Even in the presence of 1 mmol/l ouabain and at 0 degree C, a restoration of almost normal cation gradients occurred provided that Na+-loaded MAL were incubated for 24 h after potassium addition to the external medium. The results are discussed in relation to the respective effects of low temperatures on the passive and active components of the cation balance in the cells of the two nephron segments.

Effects of external potassium concentrations on the cell sodium and potassium contents of isolated rat kidney tubules.

The effects of 20 mumol/l amiloride, 10 mumol/l furosemide and 1 mmol/l ouabain on cell Na and K concentrations were investigated by flame microphotometry in isolated rat medullary collecting tubules and medullary thick ascending limbs (MCT and MAL) as a function of the external potassium concentration [Ke]. The results are expressed as Na and K concentrations per liter cell volume ([Nac] and [Kc], mmol/l) and relative sodium content, [Nac]/([Nac] + [Kc]). From the experimental curves, [Ke]1/2 is defined as the [Ke] value corresponding to half maximal exchange of K against Na in cells. When [Ke] was 5 mmol/l, the relative Na content was less than 15% in control and amiloride-treated MCT as well as in control and furosemide-treated MAL, and about 24% in ouabain-treated MCT and MAL. In MCT, relative cell Na content increased up to 90% or more when [Ke] was reduced from 2.5 to 0.25 mmol/l. [Ke]1/2 was 0.55, 0.45 and 1.25 mmol/l for control, amiloride-treated and ouabain-treated MCT respectively. In MAL, similar increases in relative Na content were observed when [Ke] was reduced from 0.5 to 0.05 mmol/l. [Ke]1/2 was 0.25, 0.10 and 1.75 mmol/l for control, furosemide-treated and ouabain-treated MAL respectively. When [Ke] was reduced from 5 to 1 mmol/l, [Nac] dropped from 16.4 to 8.4 mmol/l (P less than 0.01) in control MAL. When [Ke] was 5 mmol/l, [Nac] was lower in furosemide-treated MAL (7.8 mmol/l) than control MAL (P less than 0.01). At 1 mmol/l [Ke], [Nac] was similar in both groups. These results are discussed in terms of the balance between the active and passive components of Na and K fluxes across apical and basolateral cell membranes. They indicate that a K-dependent passive Na entry process exists in the membranes of MAL cells but not of MCT cells. This process was proportionally more inhibited than the active Na pump when [Ke] was reduced from 5 to 1 mmol/l. In addition, it was found sensitive to furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma antidiuretic hormone levels and liver vasopressin receptors in the jerboa, Jaculus orientalis, and rat.

V1 vasopressin, angiotensin, alpha-adrenergic, and glucagon receptors in liver were studied on membrane fractions prepared from two groups of jerboas ( Jaculus orientalis) given dry or water-enriched diets for periods of 4 to 7 weeks, and from rats acutely treated with pharmacological amounts of arginine-vasopressin (AVP) or (1-deamino-8-D-arginine)-vasopressin (dDAVP). Tritiated (8-lysine)-vasopressin ([3H]vasopressin), tritiated (1-asparagine-5-valine)-angiotensin II ([3H]angiotensin II), tritiated dihydroergocryptine ([3H] DHEC ), and iodinated glucagon ([125I]-glucagon) were used as specific labeled ligands of these receptors. The V1 vasopressin, angiotensin, alpha-adrenergic, and glucagon receptors detected in both groups of jerboas were identical to receptors found in rat liver plasma membranes in regard to the apparent dissociation constants for their respective labeled ligands. Furthermore, vasopressin receptors in jerboa liver membranes discriminated as efficiently as rat liver receptors between the natural neurohypophyseal peptides arginine-vasopressin and lysine-vasopressin on the one hand and the structural analogs (1-deamino-8-D-arginine)-vasopressin and (4-valine-8-D-arginine)-vasopressin on the other. The reduction of antidiuretic hormone (ADH) secretion in jerboas fed a water-enriched diet compared to those on a dry diet (75 +/- 25 pM versus 372 +/- 86 pM) was accompanied by an increase in the number of liver vasopressin receptors (2.79 +/- 0.53 versus 1.25 +/- 0.14 pmol [3H]vasopressin bound/mg protein). The modifications observed were specific for vasopressin receptors, as judged by the maximal binding capacities of [3H]angiotensin II, [3H] DHEC , and [125I]-glucagon, which remained unchanged in jerboas whatever the levels of endogenous circulating ADH. Similarly, administration of pharmacological doses of AVP by iv infusion to rats induced, 2 hr later, a loss of about 50% of V1 liver vasopressin receptors, while the numbers and apparent dissociation constants of angiotensin, alpha-adrenergic, and glucagon liver receptors remained unchanged, and V2 kidney vasopressin receptors were almost desensitized. For V1 liver and V2 kidney vasopressin receptors, the desensitization process was strikingly dependent on the antidiuretic/glycogenolytic activity ratio of the peptide used. Thus, im injection to rats of dDAVP (an analog possessing a very high antidiuretic/glycogenolytic activity ratio) induced, 1 hr later, a total loss of V2 kidney receptors without modification of the number and apparent dissociation constant of V1 liver receptors.

Ontogenic development of antidiuretic hormone receptors in rat kidney: comparison of hormonal binding and adenylate cyclase activation.

The development of adenylate cyclase responsiveness to vasopressin and parathyroid hormone was studied using membrane fractions prepared from medullo-papillary and cortical portions of kidneys of 2-46-day-old rats. The development of vasopressin binding capacity was followed on the same preparations, using [3H]vasopressin. The characteristics of medullo-papillary adenylate cyclase response to vasopressin were identical in young and adult control animals as regards apparent Km values for [Lys8]vasopressin (3 X 10(-8) M), specificity towards the natural neurohypophysial peptides and the effects of Mg2+. However, the magnitude of maximal enzyme activation by vasopressin was much lower in very young than adult animals. Accordingly vasopressin responsiveness increased sharply between the 10th and 25th days but the magnitude of the maximal response only reached the adult value between the 30th and 45th days after birth. During both periods basal adenylate cyclase activity was almost independent of age. Specific vasopressin binding sites were detected on kidney medullo-papillary membranes from young animals. Vasopressin binding capacity and adenylate cyclase responsiveness to the hormone followed similar development patterns. However, the appearance of specific binding sites slightly preceded the onset of adenylate cyclase responsiveness. Basal cortical adenylate cyclase activity/mg protein was 12 times higher in 2-day-old rats than in the adult controls. It dropped with age but only fell to the adult value between the 25th and the 35th days after birth. For the youngest animals tested (2 days old), the increase in activity due to parathyroid hormone was about half the increase measured in adults, and gradually rose to about 75% of the adult response between the 2nd and 46th days after birth. Apparent Km values for parathyroid hormone were identical in young and adult animals (3.2 and 3.0 U/ml, respectively).