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.

Bradykinin attenuates the [Ca(2+)](i) response to angiotensin II of renal juxtamedullary efferent arterioles via an EDHF.

1. Bradykinin (BK) effect on the [Ca(2+)](i) response to 1 nM angiotensin II was examined in muscular juxtamedullary efferent arterioles (EA) of rat kidney. 2. BK (10 nM) applied during the angiotensin II-stimulated [Ca(2+)](i) increase, induced a [Ca(2+)](i) drop (73+/-2%). This drop was prevented by de-endothelialization and suppressed by HOE 140, a B2 receptor antagonist. It was neither affected by L-NAME or indomethacin, nor mimicked by sodium nitroprusside, 8-bromo-cyclic GMP or PGI(2). The BK effect did not occur when the [Ca(2+)](i) increase was caused by 100 mM KCl-induced membrane depolarization and was abolished by 0.1 microM charybdotoxin, a K(+) channel blocker. 3. Although proadifen prevented the BK-caused [Ca(2+)](i) fall, more selective cytochrome P450 inhibitors, 17-octadecynoic acid (50 microM) and 7-ethoxyresorufin (10 microM) were without effect. 4. Increasing extracellular potassium from 5 to 15 mM during angiotensin II stimulation caused a [Ca(2+)](i) decrease (26+/-4%) smaller than BK which was charybdotoxin-insensitive. Inhibition of inward rectifying K(+) channels by 30 microM BaCl(2) and/or of Na(+)/K(+) ATPase by 1 mM ouabain abolished the [Ca(2+)](i) decrease elicited by potassium but not by BK. 5. A voltage-operated calcium channel blocker, nifedipine (1 microM) did not prevent the BK effect but reduced the [Ca(2+)](i) drop. 6. These results indicate that the BK-induced [Ca(2+)](i) decrease in angiotensin II-stimulated muscular EA is mediated by an EDHF which activates charybdotoxin-sensitive K(+) channels. In these vessels, EDHF seems to be neither a cytochrome P450-derived arachidonic acid metabolite nor K(+) itself. The closure of voltage-operated calcium channels is not the only cellular mechanism involved in this EDHF-mediated [Ca(2+)](i) decrease.

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.

Calcium-sensing receptor: regulation of electrolyte transport in the thick ascending limb of Henle's loop.

A calcium-sensing receptor (CaR) has functionally been described in the cortical thick ascending limb of Henle's loop (CTAL) of rat and mouse. This G protein-coupled receptor activates phospholipase C and increases the intracellular Ca2+ concentration. We observed that in the mouse CTAL cAMP formation, induced by 10(-8) mol/l AVP, was inhibited by more than 90% when the extracellular Ca2+ concentration ([Ca2+]e) was increased from 0.5 to 3 mmol/l. Measurements of transepithelial potential difference (PDte) in rat and mouse CTAL and medullary thick ascending limb (mTAL) segments and of transepithelial ion net fluxes in the mouse CTAL (isotonic perfusion conditions: 150 mmol/l NaCl in the lumen and bath) showed that an increase in the [Ca2+]e had no effect on basal and arginine vasopressin (AVP, 10(-10) mol/l)-stimulated transepithelial PDte, NaCl and Mg2+ transport. However, Ca2+ reabsorption was strongly inhibited by increased [Ca2+]e. Addition of AVP reversed this inhibitory effect of increased [Ca2+]e. Under hypotonic perfusion conditions (lumen 50 mmol/l NaCl; bath 150 mmol/l NaCl), a high [Ca2+]e induced a 50% decrease in Mg2+ reabsorption which was restored by AVP. Under these conditions, the effects on Ca2+ transport described above were still observed. In conclusion, activation of the CaR in the mouse TAL has no effect on basal and AVP-stimulated transepithelial NaCl reabsorption despite its large inhibitory effect on cAMP synthesis. The CaR, however, could play a role in the regulation of transepithelial Ca2+ and Mg2+ reabsorption.

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.

Regulation of renal Na+,K(+)-ATPase in rat thick ascending limb during K+ depletion: evidence for modulation of Na+ affinity.

1. NaCl reabsorption along the loop of Henle is reduced in K(+)-depleted rats. Because Na+,K(+)-ATPase energizes this transport and because K+ depletion is known to induce an upregulation of Na+,K(+)-ATPase in most tissues, the regulation of this enzyme was investigated at the level of single thick ascending limbs of the loop of Henle freshly microdissected from rats fed either a normal (control rats) or a low-K+ diet (LK rats). 2. Within 2 weeks of K+ depletion, Na+,K(+)-ATPase activity and [3H]ouabain binding were increased by 30-50% in the medullary portion of the thick ascending limb (MTAL). 3. Despite this increase in the number of Na+,K(+)-ATPase units, the transport capacity of the Na+,K+ pump, determined by ouabain-sensitive Rb+ uptake in the presence of an extracellular concentration of Rb+ mimicking the kalaemia determined in control (4.0 mM Rb+) and LK rats (2.3 mM Rb+), was reduced in MTAL from LK rats. 4. Inhibition of the Na+,K+ pump was not accounted for by changes in either extracellular K+ or intracellular Na+ concentrations, but by a decrease in the pump affinity for Na+. 5. Because this change in the apparent affinity of the Na+,K+ pump for Na+ was detectable in intact but not in permeabilized MTAL cells, it is probably induced by a rapidly reversible cytosolic factor.

Effect of insulin on Na+,K(+)-ATPase in rat collecting duct.

1. The collecting duct is involved in the whole antinatriuretic effect of insulin, as indicated in vitro by the stimulatory effect of the hormone on ouabain-sensitive 86Rb+ uptake. Since Na+,K(+)-ATPase drives Na+ reabsorption, the contribution of the Na+ pump to the effect of insulin was investigated in rat isolated cortical and outer medullary collecting duct. 2. Insulin enhanced ouabain-sensitive 86Rb+ uptake in the absence, as well as in the presence, of either 5 x 10(-4) M amiloride or 10(-3) M hydrochlorothiazide (HCT). Maximal ouabain-sensitive 86Rb+ uptake, measured in Na(+)-loaded tubules, was also enhanced by insulin. The insulin effect persisted both in the absence of external Na+, when the Na+,K(+)-ATPase operates in a Rb(+)-Rb+ exchange mode, and in tubules depolarized by a high external concentration (20 mM) of Rb+ or by addition of 3 mM Ba2+. 3. Insulin treatment did not alter the intracellular Na and K concentrations, the specific binding of [3H]ouabain measured in intact tubules, or the hydrolytic activity of Na+,K(+)-ATPase measured after permeabilization of the tubule cells. 4. In conclusion, in the rat collecting duct, insulin increased Na+,K(+)-ATPase-mediated cation transport independently of Na+ availability, membrane potential and recruitment of pump units. The effect of insulin was lost after cell permeabilization, suggesting the presence of a cytosolic factor which controls the turnover of Na+,K(+)-ATPase.

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)

Ouabain-sensitive and -insensitive K-ATPases in rat nephron: effect of K depletion.

Because a ouabain-sensitive H-K-adenosinetriphosphatase (H-K-ATPase) has been identified recently in the amphibian bladder, we evaluated whether such an ATPase might exist also in the mammalian kidney, along with the ouabain-insensitive H-K-ATPase previously described in the collecting duct. For this purpose, we searched for an Na-independent, K-stimulated, ouabain- and Sch-28080-inhibitable ATPase activity in single segments of rat nephron. Ouabain-sensitive K-stimulated ATPase activity was detected in the absence of Na+ in rat proximal convoluted and straight tubules and in medullary and cortical thick ascending limbs of Henle's loop but not in collecting ducts. This K-ATPase differs from Na-K-ATPase by 1) its absence of requirement for Na, 2) its sensitivity to Sch-28080, 3) its higher sensitivity to ouabain, and 4) its absence in the collecting duct. It differs from the collecting duct H-K-ATPase by 1) its distribution along the nephron, 2) its sensitivity to ouabain, and 3) its lower sensitivity to Sch-28080. Furthermore, in rats fed a K-depleted diet for 2 wk, ouabain-sensitive K-ATPase activity was markedly reduced in both proximal tubules and thick ascending limbs, whereas collecting duct H-K-ATPase was upregulated.

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)

Evidence for two molecular forms of solubilized vasopressin receptors in rat kidney membranes. Regulation by guanyl nucleotides.

As previously reported, it is possible to solubilize vasopressin-receptor complexes formed in rat kidney membranes by the use of Triton X-100. Ultracentrifugation on sucrose gradients and elution through molecular sieving columns of these soluble extracts revealed the existence of two molecular forms of vasopressin-receptor complexes (molecular weight = 200,000 and 100,000, respectively). These two molecular forms of vasopressin-receptor complexes can be partially purified and exhibit different properties: (a) The light form is more sensitive to thermal dissociation than is the heavy form. (b) The presence of guanyl nucleotide affects the dissociation rate of only the heavy form of the vasopressin-receptor complex. (c) The light form seems to be convertible to the heavy form by increasing the duration of incubation between the membranes and the tritiated hormone. (d) Guanyl nucleotides affect the distribution of the two molecular forms of the receptor (decrease of the relative amount of the heavy form). These data provide evidence for interaction between vasopressin-receptor complexes (light form) and another protein component, which may be a GTP-binding protein.

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.