Daniel Butlen

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).

Kinetic and pharmacological characterization of vasopressin membrane receptors from human kidney medulla: Relation to adenylate cyclase activation

Abstract Membranes from the medullopapillary portions of three human kidneys unsuitable for transplantation were prepared and stored in liquid nitrogen. Specific vasopressin binding sites were detected on these membranes and characterized using tritiated lysine vasopressin. The biological activities of vasopressin and of structurally related peptides were estimated on the same membrane preparations by measuring dose-dependent adenylate cyclase activation. The population of vasopressin binding sites was homogeneous and displayed the following characteristics: equilibrium dissociation constant K D 27 ± 7 nM (for lysine vasopressin); maximal specific binding capacity 0.7±0.1 pmol [ 3 H]LVP bound/mg protein. Maximal adenylate cyclase activation by lysine vasopressin was 5.4 times the basal value of 50 ± 4 pmol cAMP formed/5 min per mg protein. Half-maximal activation (K act ) was obtained at a concentration of 34 ± 5 nM. Both lysine vasopressin binding and hormone-induced adenylate cyclase activation were sensitive to guanyl nucleotides. 5′-Guanylyl imidodiphosphate reduced K act by a factor of 3 and increased K D by a factor of about 8. K D and K act were determined for a series of peptides including natural vasopressins, oxytocin and their structural analogues which were found to have enhanced or reduced antidiuretic activities in the rat. The concentration range of the K D and K act values covered a concentration range of more than three orders of magnitude. The most active peptide was the natural arginine vasopressin (K D 4.4 ± 0.7 nM; K act 3.1 ± 1.6 nM). There was a highly significant correlation between paired K D and K act values. The two vasopressin structural analogues [1-(β- mercapto-β, β-cyclopentamethylene propionic acid), 4-valine, 8-D-arginine]vasopressin and [1-β-mercapto-β, β-cyclopenthamethylene propionic acid), 2-O-ethyltyrosine, 4-valine]arginine vasopressin, behaved like competitive inhibitors of vasopressin-induced adenylate cyclase activation (K i = 65 and 59 nM respectively). Freezing the membranes in liquid nitrogen reduced the affinity of vasopressin receptors but fully preserved their selectivity towards vasopressin structural analogues. It is concluded that the vasopressin binding and/or adenylate cyclase assays might be useful for estimating several aspects of the pharmacological activities of vasopressin structural analogues in man.

Insulin receptors along the rat nephron: [125I] insulin binding in microdissected glomeruli and tubules.

Binding of [125I] Tyr A14 human insulin ([125I] insulin) was measured at 4°C in glomeruli and pieces of tubule microdissected from collagenase-treated rat kidneys. For glomeruli and all segments tested, total and non specific binding increased linearly with glomeruli number or tubular length. When determined with 4.0 nM labelled hormone, the distribution of specific binding sites (expressed as 10−18 mol [125I] insulin bound per glomerulus or mm tubule length) was as follows: glomerulus, 2.5±0.3; proximal convoluted tubule (PCT), 12.6±0.6; pars recta (PR), 4.0±2.3; thin descending limb (TDL), 0.6±0.2; thin ascending limb (TAL), 0.6±0.2; medullary thick ascending limb (MAL), 0.8±0.1; cortical ascending limb (CAL), 2.1±0.1; distal convoluted tubule (DCT), 5.6±1.1; cortical collecting tubule (CCT), 3.2±0.3 and outer medullary collecting tubule (MCT), 2.3±0.1. Specific [125I] insulin binding to glomeruli and tubule segments was time- and dose-dependent, saturable, reversible after elimination of free labelled ligand, and inhibited by unlabelled human insulin. When analysed in Scatchard and Hill coordinates, the binding data revealed a negative cooperation in the interaction processes between [125I] insulin and glomerular and tubular binding sites, with apparent dissociation constants and Hill coefficients of the following values: glomerulus, 0.6 nM and 0.60; PCT, 10.0 nM and 0.55; MAL, 4.3 nM and 0.80; CAL, 2.0 nM and 0.74; CCT, 7.6 nM and 0.80 and MCT, 1.0 nM and 0.57 respectively. The stereospecificity of nephron binding sites was assessed in competitive experiments showing that unlabelled bovine and procine insulins were as efficient as human insulin for displacing [125I] insulin, whereas A and B chains of insulin and unrelated peptide hormones were almost inactive. These results indicate that the detected [125I] insulin binding sites may correspond to physiological insulin receptors.

Atrial natriuretic peptide effects on cGMP and cAMP contents in microdissected glomeruli and segments of the rat and rabbit nephrons

A microradioimmunoassay has been developed in order to measure the changes in cGMP cell content induced in vitro by atrial natriuretic peptides (ANP) in either glomeruli or defined portions of tubules microdissected from collagenase treated rat and rabbit kidneys. When tested at 0.1 μM or 1 μM, all ANP analogues used produced in rat glomeruli a 20–25 fold increase in cGMP accumulation compared to basal values. Threshold responses were obtained with about 1 nM ANP and apparentKa values ranged between 5 and 50 nM. Atriopeptin III led to similar results in glomeruli isolated from rabbit. Under the same experimental conditions, no cGMP could be detected in any ANP-treated nephron segment from the rat kidney (namely, from the proximal convoluted tubule up to the outer medullary collecting tubule) nor in cortical collecting tubules isolated from the rabbit kidney. Moreover, ANP did not after the forskolin-induced increase in cAMP content in glomeruli or collecting tubules, nor the AVP-induced increase in cAMP content in collecting tubules. Our data confirm the marked effect of ANP on cGMP generation by isolated glomeruli from rat and rabbit; however, they are not competible with a direct action of ANP stimulating cGMP generation in tubules or inhibiting vasopressin-induced cAMP generation in collecting tubules.

Glucagon receptors along the nephron: [125I]glucagon binding in rat tubules

Binding of [125I]glucagon was measured in microdissected pieces of tubules from the rat nephron. Specific glucagon binding sites were found only in nephron segments containing a glucagon-sensititive adenylate cyclase activity. At 7.5 nM labelled hormone, higher levels of specific binding (16–27×10−18 mol mm−1) were found in the thick ascending limb of the Henles loop and in the distal convoluted tubule and lower binding levels (2–5×10−18 mol mm−) in the collecting tubule whereas specific binding could not be detected in the proximal tubule and in the thin segments of the Henles loop. In the medullary thick ascending limb, Scatchard analysis of specific [125I]glucagon binding indicated an apparent equilibrium dissociation constant of 2.4 nM. The stereospecificity of binding sites in medullary thick ascending limbs and medullary collecting tubules, was assessed by competition experiments using unlabelled glucagon, enteroglucagon and unrelated hormones (vasopressin, calcitonin, parathyroid hormone and insulin); in both segments, glucagon was more active than enteroglucagon in displacing labelled glucagon from its tubular binding sites, whereas all other hormones tested were inactive. These results indicate that tubule binding sites might be the physiological receptors for glucagon involved in adenylate cyclase activation.

Atrial natriuretic peptide receptors along the rat and rabbit nephrons: [125I] α-rat atrial natriuretic peptide binding in microdissected glomeruli and tubules

Binding of [125I] α-rat atrial natriuretic peptide ([125I] α-RANP) was measured in glomeruli and pieces of tubule microdissected from rat and rabbit nephrons. High densities of specific ANP binding sites were found only in the glomeruli (10–30×10−18 mol·glom−1), whereas no specific binding could be detected in the proximal tubule, the thin segments of the Henles loop, the thick ascending limb, the distal tubule and the cortical and outer medullary collecting tubules. Rising the temperature from 4° C to 35° C resulted in biphasic kinetics of binding, suggesting a temperature-dependent inactivation of labelled hormone by glomeruli. At 4° C, specific binding of [125I] α-RANP was time and dose-dependent and Scatchard analysis of data indicated an apparent equilibrium dissociation constant of 0.63 nM. Competition experiments revealed the following sequence of stereospecificity for binding to rat glomeruli: RANP 3–28>[125I] α-RANP=[125I] α-HANP=α-RANP=atriopeptin III > atriopeptin II, whereas binding was unaffected by pharmacological doses of unrelated peptide hormones, prostaglandins, adrenergic agonists, dopamine, histamine and carbamylcholine. The results indicate that glomerular binding sites might be the physiological ANP receptors.

Comparison of the developmental patterns of vasopressin, glucagon and α-adrenergic receptors from rat-liver membranes ☆

Abstract The ontogenic developmental patterns of vasopressin, glucagon and α-adrenergic receptors in liver were studied by using membrane fractions prepared from rats aged from 19 days postcoitum to 29 days post-partum. Tritiated lysine-vasopressin ([ 3 H] vasopressin), iodinated glucagon ([ 125 I]glucagon) and tritiated dihydroergocryptine ([ 3 H]DHEC) were used as specific labelled ligands of these receptors. Vasopressin, glucagon and α-adrenergic receptors detected on liver membranes from foetuses or young rats had characteristics identical with those found on liver membranes from adult animals as regards apparent dissociation constants for their respective specific ligands. Furthermore, vasopressin liver receptors from young animals discriminated as efficiently as liver receptors from adult rats between the 3 natural neurohypophyseal peptides: lysine-vasopressin, arginine-vasopressin and oxytocin. Liver α-adrenergic receptors from young and adult rats were mainly of the α 1 type as judged by their high affinity for prazosin and much lower affinity for yohimbine. Membrane contents in vasopressin, glucagon and α-adrenergic receptors expressed in terms of pmoles per mg membrane protein exhibited marked variations during ontogenic development. The respective developmental patterns for the 3 receptors studied were different. Thus, vasopressin receptors were undetectable in liver membranes from foetuses and only few at birth. Membrane content in vasopressin receptors rose rapidly up to the adult level between the 6th and 21st days after birth. The number of glucagon receptors measured in the youngest foetuses tested (19 days) represented about 15% of that determined in adult rats; it rose progressively to reach 75% of the adult rat level in the 29-day-old animals. The evolutionary pattern of α-adrenergic receptors was biphasic. The number of receptors found in 19-day-old foetuses was about 1.5 times the number observed in adult rats; during the later foetal period and the first 2 weeks after birth, this number dropped to about one quarter and then rose to reach the adult level at the end of the weaning period. For vasopressin receptors, a close resemblance was noted between the pattern observed in the liver and that previously described for the rat kidney.

Receptors for neurohypophyseal hormones along the rat nephron: 125I-labelled d(CH2)5[Tyr(Me)2, Thr4, Orn8, Tyr-NH(2)9] vasotocin binding in microdissected tubules.

A microassay was developed to measure the binding of the labelled monoiodinated analogue [1-(βmercapto-β,β-cyclopentamethylenepropionic acid), 2-O-mithyltyrosine, 4-threonine, 8-ornithine, 9-125I-tyrosylamide]vasotocin 125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH29]OVT to isolated nephron segments microdissected from collagenase-treated rat kidneys. When determined using 1.7 nM labelled ligand at 4° C, specific binding sites (expressed at 10−18 mol 125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH29]OVT bound/mm tubule length) were found in medullary thick ascending limbs (MTAL), 1.67±0.49; cortical thick ascending limbs, 2.20±0.80; cortical collecting ducts, 2.39±0.86; outer medullary collecting ducts (OMCD), 2.54±0.53 and inner medullary collecting ducts, 5.33±0.40, whereas no specific binding could be detected in glomeruli and proximal tubules. Specific 125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH29]OVT binding to OMCD was saturable with incubation time and reversible after elimination of free labelled ligand (the association and dissociation rate constants at 4° C were 1.06×107 M−1 min−1 and 1.95×10−2 min−1 respectively). The stereospecificity of MTAL and OMCD binding sites was assessed in competitive experiments revealing the following recognition pattern for a series of eight vasopressin analogues:ddAVP>AVP>d(CH2)5-[Tyr (Me)2, Thr4, Tyr-NH29]OVT=AVT=OT>d(CH2)5[Tyr(Me)2]AVP=[Thr4, Gly7]OT>[Phe2, Orn8]VT, whereas pharmacological concentrations of insulin and glucagon did not impair radioligand binding. These results indicate that the detected labelled binding sites might correspond mainly to physiological V2 vasopressin receptors.

Developmental patterns of renal atrial natriuretic peptide receptors: [125I]alpha-rat atrial natriuretic peptide binding in glomeruli and inner medullary collecting tubules microdissected from kidneys of young rats.

The ontogenic developmental patterns of atrial natriuretic peptide (ANP) receptors of glomeruli and inner medullary collecting tubules (IMCT) were studied by measuring the specific binding of [125I]alpha-rat ANP 1-28 ([125I]alpha-RANP) to isolated glomeruli and IMCT microdissected from collagenase-treated kidneys of young rats aged from 2 to 35 days post-partum. For glomeruli and IMCT from young and adult animals, total and non-specific binding increased linearly with glomerulus number or tubular length. ANP receptors detected in glomeruli and IMCT from young rats showed the same stereospecifities as those from adult rats for recognition of ANP analogues (alpha-RANP 1-28, ANP 3-28, atriopeptin III and atriopeptin II). The numbers of ANP receptors in glomeruli and IMCT (expressed in terms of 10(-18) mol labelled ANP bound per glomerulus or per mm IMCT length, respectively) exhibited marked variations during postnatal ontogenesis; they were low after birth and rose progressively with age up to the corresponding adult levels (20 +/- 2 X 10(-18) mol.glom-1 and 4.4 +/- 0.8 X 10(-18) mol.mm-1) at the end of the 5th week of postnatal life.

Postnatal ontogenesis of vasopressin receptors in the rat collecting duct.

The ontogenesis of vasopressin receptors in the rat collecting duct was studied by measuring the binding of [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-O-methyltyrosine,4-threonine,8-ornithine,9-125I-tyrosylamide+ ++]-vasotocin (125I-d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH(9)2]-OVT) to isolated cortical collecting ducts (CCD), outer medullary collecting ducts (OMCD) and inner medullary collecting ducts (IMCD) microdissected from collagenase-treated kidneys of 2- to 34-day-old rats and adult animals. The stereospecificity for recognition of a series of seven vasopressin structural analogues by CCD and OMCD receptors reveals that the labeled binding sites identified in 11- to 16-day-old and adult rats are homologous respectively and contain a major population of V2 type and a minor population of V1a type of vasopressin receptors. At all postnatal stages examined, the receptor density (expressed as 10(-18) mol radioligand bound per square millimeter tubular outer surface area) decreases gradually from the CCD to the IMCD. For the three segments, the numbers of receptors detected remained constant during the first 2 weeks after birth and increased sharply after 20 days to reach the corresponding adult levels during the fifth week.