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1.
J Clin Invest ; 76(2): 474-81, 1985 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2993360

RESUMO

Observations in vivo suggest that catecholamines modulate reabsorptive functions of proximal tubules by acting on beta-adrenoceptors. However, beta-catecholamine binding sites or beta-adrenoceptor-sensitive adenylate cyclase (AdC) has not been found in segments of proximal tubules of rat, rabbit, or mouse kidney. In the present study, we investigated the responsiveness of AdC to catecholamines, [8-Arg]vasopressin (AVP), and to parathyroid hormone (PTH) in proximal convoluted tubules (PCT), proximal straight tubules (PST), and in late distal convoluted tubules (LDCT) microdissected from canine kidney. Isoproterenol (ISO) caused a marked and dose-dependent stimulation of AdC in PST (maximum: delta + 850%; half maximum stimulation at 10(-7) M ISO), but ISO had no effect on AdC in PCT. The AdC in both PCT and PST was markedly stimulated by PTH; AVP stimulated the AdC in LDCT but not in PST or in PCT. The stimulatory effect of 10(-5) M ISO in PST (delta + 725%) was significantly greater than in LDCT (delta + 307%); norepinephrine and epinephrine had stimulatory effects in PST similar to ISO. The stimulation of AdC in PST by ISO was blocked by propranolol and by beta 2-blocker ICI-118551. On the other hand, alpha-blocker phentolamine and beta 1-blocker metoprolol did not abolish the stimulation of AdC in PST by ISO. The accumulation of cAMP in intact PCT and PST incubated in vitro was stimulated by PTH both in PST and in PCT, but ISO elevated cAMP (delta + 683%) only in PST. Our results show that proximal tubules of canine nephron, PST but not PCT, contain beta-adrenoceptors of beta 2 subtype coupled to AdC. These observations provide direct evidence that the effects of catecholamines, either released from renal nerve endings or arriving from blood supply, can act directly on beta 2-adrenoceptors located in proximal tubules, and also suggest that at least some of the catecholamine effects in proximal tubules are mediated via cAMP generation.


Assuntos
Adenilil Ciclases/metabolismo , Isoproterenol/farmacologia , Túbulos Renais Proximais/metabolismo , Néfrons/enzimologia , Receptores Adrenérgicos beta/metabolismo , Animais , AMP Cíclico/metabolismo , Cães , Metoprolol/farmacologia , Néfrons/efeitos dos fármacos , Norepinefrina/farmacologia , Propanolaminas/farmacologia , Propranolol/farmacologia
2.
J Clin Invest ; 72(3): 997-1004, 1983 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-6309913

RESUMO

Among other defects in water metabolism, adrenal insufficiency is associated with an inability to concentrate urine maximally in both man and experimental animals. Recent studies in the rabbit cortical collecting tubule have suggested indirectly that this defect may result from impaired cyclic AMP (cAMP) formation in response to antidiuretic hormone stimulation. In the present study, we examined key elements of arginine vasopressin (AVP)-dependent cAMP metabolism in the papillary collecting duct (PCD), microdissected from 8-d adrenalectomized (ADX) and sham-operated control rats. AVP-sensitive adenylate cyclase (ADC) activity in PCD did not differ between control and ADX rats. cAMP-phosphodiesterase activity (cAMP-PDIE), measured at 10(-6) M cAMP substrate concentration, was significantly higher (delta + 31.6%) in PCD of ADX rats compared with controls. Incubation of intact PCD from ADX rats with AVP resulted in an accumulation of cAMP (delta - 48.5%) significantly lower than observed in control PCD. Chronic administration of dexamethasone reduced cAMP-PDIE activity in PCD of ADX rats to levels close to or below those observed in control rat PCD, and also resulted in a restoration of AVP-stimulated cAMP accumulation to levels approaching control values. Results indicate that the impaired maximal urinary concentrating ability associated with adrenal insufficiency may be due, at least in part, to a reduced accumulation of cAMP in response to AVP in the PCD. This decreased cAMP accumulation results from increased cAMP-PDIE activity in the PCD of ADX rats and can be corrected by administration of glucocorticoid.


Assuntos
AMP Cíclico/metabolismo , Capacidade de Concentração Renal , Túbulos Renais Coletores/metabolismo , Túbulos Renais/metabolismo , Vasopressinas/administração & dosagem , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Adrenalectomia , Animais , Arginina Vasopressina , Dexametasona/administração & dosagem , Capacidade de Concentração Renal/efeitos dos fármacos , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/enzimologia , Masculino , Ratos , Ratos Endogâmicos , Vasopressinas/fisiologia
3.
J Clin Invest ; 72(4): 1298-313, 1983 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-6313759

RESUMO

To determine vasopressin (VP)-potentiating effect of chlorpropamide (CPMD), we studied the effect of CPMD in vivo and in vitro in kidneys and in specific tubule segments of rats with hypothalamic diabetes insipidus, homozygotes of the Brattleboro strain (DI rats). Rats on ad lib. water intake were treated with CPMD (20 mg/100 g body wt s.c. daily) for 7 d. While on ad lib. water intake, the urine flow, urine osmolality, urinary excretion of Na +, K +, creatinine, or total solute excretion did not change. However, corticopapillary gradient of solutes was significantly increased in CPMD-treated rats. Higher tissue osmolality was due to significantly increased concentration of Na +, and to a lesser degree urea, in the medulla and papilla of CPMD-treated rats. Consequently, the osmotic gradient between urine and papillary tissue of CPMD-treated rats (delta = 385 +/- 47 mosM) was significantly (P less than 0.001) higher compared with controls (delta = 150 +/- 26 mosM). Minimum urine osmolality after water loading was higher in CPMD-treated DI rats than in controls. Oxidation of [14C]lactate to 14CO2 coupled to NaCl cotransport was measured in thick medullary ascending limb of Henle's loop (MAL) microdissected from control and CPMD-treated rats. The rate of 14CO2 production was higher (delta + 113% +/- 20; P less than 0.01) in CPMD-treated MAL compared with controls, but 14CO2 production in the presence of 10(-3) M furosemide did not differ between MAL from control and from CPMD-treated rats. These observations suggest that CPMD treatment enhances NaCl transport in MAL. Cyclic AMP metabolism was analyzed in microdissected MAL and in medullary collecting tubule (MCT). MCT from control and from CPMD-treated rats did not differ in the basal or VP-stimulated accumulated of cAMP. The increase in cAMP content elicited by 10(-6) M VP in MAL from CPMD-treated rats (delta + 12.0 +/- 1.8 fmol cAMP/mm) was significantly (P less than 0.02) higher compared with MAL from control rats (delta + 5.1 +/- 1.0 fmol cAMP/mm). Preincubation of MAL dissected from Sprague-Dawley rats with 10(-4) M CPMD in vitro increased cAMP accumulation in the presence of VP, but no such enhancement was found in preincubated MCT. Adenylate cyclase activity, basal or stimulated by VP, 5'guanylimidodiphosphate, or by NaF, assayed in isotonic medium did not differ between MAL or MCT from control rats and MAL or MCT from CPMD-treated rats. When assayed in hypertonic medium (800 mosM), the adenylate cyclase activity in the presence of 10(-6) M VP was significantly higher in MAL of CPMD-treated rats. MAL and MCT from control and CPMD-treated rats did not differ in the activities of cAMP phosphodiesterase. The rate of [(14)C]prostaglandin E2 by medullary and papillary microsomes was not different between the control and CPMD-treated rats; likewise, there was no difference in accumulation of immunoreactive prostaglandin E2 in the medium of in vitro incubated medullary or papillary slices prepared from control and CPMD-treated rats. Based on the findings recounted above, we propose a hypothesis that CPMD administration enhances the antidiuretic effect of VP, primarily by increasing medullary and papillary tonicity dye to increased NaCl reabsorption in MAL. There is no evidence that CPMD sensitizes collecting tubules to the action of VP, at least at the camp-generation step. Therefore, increased antidiuretic response to VP in the kidneys of CPMD-treated DI rats is due to enhanced osmotic driving force for water reabsorption (lumen-to-interstitium osmotic gradient) in collecting tubules, rather than due to increased VP-dependent water permeability of tubular epithelium.


Assuntos
Clorpropamida/administração & dosagem , Diabetes Insípido/fisiopatologia , Capacidade de Concentração Renal/efeitos dos fármacos , Animais , Arginina Vasopressina/administração & dosagem , Dióxido de Carbono/metabolismo , Clorpropamida/farmacologia , AMP Cíclico/urina , Diabetes Insípido/urina , Dinoprostona , Diurese/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Alça do Néfron/metabolismo , Masculino , Prostaglandinas E/biossíntese , Ratos , Ratos Brattleboro
4.
Biochim Biophys Acta ; 732(3): 553-61, 1983 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-6871215

RESUMO

The characteristics of nicotinamide adenine dinucleotide (NAD) binding on brush border membranes prepared from rat renal cortex were investigated with the use of radioactively labelled NAD, [adenine-2,8-3H]NAD+, as a ligand. (1) We found that NAD binds on brush border membrane and that the extent of NAD binding is linearly proportional to the brush border membrane protein, and progressively increases with concentration of NAD in the medium. (2) The rate of NAD binding was dependent on temperature. At 20 degrees C, the equilibrium binding was obtained at 15 min, while NAD binding at 0 degree C was slower, but the final level of binding reached at 120 min was similar to that plateau of binding observed at 20 degrees C. Brush border membrane inactivated by heating at 95 degrees C for 3 min did not bind NAD. Binding of NAD on brush border membranes was reversed by simple dilution or by the addition of unlabelled NAD. Both alpha-NAD and beta-NAD stereoisomers displaced bound [3H]NAD. Reduced NAD (NADH) caused less displacement of bound NAD than oxidized NAD+. Adenine, nicotinamide, pyrophosphate, of 5'-AMP did not displace bound NAD. (3) The NAD binding to brush border membranes was nearly saturable, approximating saturation at 10(-4) M NAD. Kinetic analysis by Scatchard plot indicates two sets of NAD binding sites in brush border membranes: a high-affinity binding site (Kd = 1.9 . 10(-5) M) and a low-affinity binding site (Kd = 2.2 . 10(-3) M). (4) Unlike concentrative uptake of D-[14C]glucose by brush border membrane vesicles, binding of NAD was not dependent on the presence of an outside-in sodium gradient [Na+0 greater than Na+i], nor was it abolished by repeated freezing and thawing of brush border membranes. Unlike D-[14C]glucose uptake, NAD binding by brush border membranes did not change upon decrease of intravesicular volume in hypertonic media. These observations indicate that NAD association with brush border membranes is true binding rather than intravesicular uptake of this compound. (5) The presence of specific binding sites in renal brush border membrane capable of binding of NAD with a high degree of affinity suggests that such sites may be involved in previously observed (Kempson, S.A., Colon-Otero, G., Ou, S.L., Turner, S.T. and Dousa, T.P. (1981) J. Clin. Invest. 67, 1347) modulatory effect of NAD on sodium-gradient-dependent uptake of phosphate across luminal brush border membrane of proximal tubules.


Assuntos
Membrana Celular/metabolismo , Córtex Renal/metabolismo , Microvilosidades/metabolismo , NAD/metabolismo , Animais , Sítios de Ligação , Cinética , Masculino , Concentração Osmolar , Ratos , Ratos Endogâmicos , Trítio
5.
J Clin Endocrinol Metab ; 60(5): 914-21, 1985 May.
Artigo em Inglês | MEDLINE | ID: mdl-2984237

RESUMO

The major tubular effects of [8-Arg]vasopressin (AVP) in regulation of renal water excretion are initiated by stimulation of adenylate cyclase (AdC) coupled with V2 receptors. We explored whether the AVP-sensitive AdC is present in both collecting tubules and the thick ascending limb of Henle's loop of human and canine kidney. In cortical collecting tubule (CCT) and medullary collecting tubules (MCT) of human kidney, AdC was markedly stimulated by AVP [maximum change from basal level (delta), +2700%] and the the nonhormonal stimulatory agent forskolin (delta, +2000%). In human CCT, the effects of both compounds were synergistic. In contrast, AVP had no effect on AdC in either the medullary (MAL) or cortical (CAL) segment of the thick ascending limb of Henle's loop of human kidney; AVP also did not stimulate AdC in CAL or MAL in the presence of forskolin. Similar to that in the human kidney, in the canine kidney, AdC in CCT and MCT was markedly stimulated by AVP and forskolin (delta, +1000%), but AVP had no effect on AdC in CAL and MAL of the canine kidney. In intact tubules dissected from dog kidney and incubated in vitro, AVP markedly increased cAMP accumulation in MCT. AVP also elicited a small but detectable increase in cAMP accumulation in MAL. From these observations, we conclude that AVP-sensitive AdC is well developed in collecting tubules, but that AVP-sensitive AdC is absent in MAL and CAL of human kidney. Likewise, in canine nephron, the AVP-sensitive AdC of MAL and CAL is rudimentary or very labile. These findings suggest that the unresponsiveness of the AdC-cAMP system to AVP in segments of the thick ascending limb of Henle's loop may be a factor that accounts for a relatively low maximum osmotic concentration of urine which can be achieved by human or canine kidneys.


Assuntos
Adenilil Ciclases/metabolismo , Arginina Vasopressina/farmacologia , Túbulos Renais Coletores/enzimologia , Túbulos Renais/enzimologia , Alça do Néfron/enzimologia , Adulto , Idoso , Animais , Colforsina , AMP Cíclico/metabolismo , Diterpenos/farmacologia , Cães , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Técnicas In Vitro , Córtex Renal/enzimologia , Medula Renal/enzimologia , Masculino , Pessoa de Meia-Idade , Especificidade da Espécie
6.
Artigo em Inglês | MEDLINE | ID: mdl-6098600

RESUMO

The medullary collecting tubules (MCT) and the medullary thick ascending limb of Henle's loop (MAL) are two major sites of [8-Arg]-vasopressin (AVP) action, via cyclic AMP, on the mammalian nephron. In the present study, the effect of forskolin and forskolin in combination with AVP on accumulation of cyclic AMP in tubular segments of MAL and MCT microdissected from rat kidney was examined in vitro. Under all tested conditions, forskolin stimulated the accumulation of cyclic AMP to a greater degree in MCT than in MAL. In MAL, the effects of forskolin and AVP were synergistic; the increase in cyclic AMP after incubation with forskolin and AVP added together was 150% greater than the arithmetic sum of the increases elicited either by AVP or by forskolin alone. On the other hand, in MCT the stimulatory effects of AVP and forskolin were strictly additive. These results suggest that an intramembranous arrangement of AVP-sensitive adenylate cyclase complex differs in two types of epithelial cells located in the same tissue. The potentiating effect of forskolin on AVP-stimulated cAMP generation in MAL can be helpful in experimental studies of AVP-sensitive cyclic AMP metabolism in this nephron segment, which has a variable sensitivity to AVP in the kidneys of diverse mammalian species.


Assuntos
Arginina Vasopressina/farmacologia , AMP Cíclico/metabolismo , Diterpenos/farmacologia , Túbulos Renais/efeitos dos fármacos , Animais , Colforsina , Sinergismo Farmacológico , Técnicas In Vitro , Medula Renal/efeitos dos fármacos , Medula Renal/metabolismo , Túbulos Renais/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Distribuição Tecidual
7.
Am J Physiol ; 249(6 Pt 2): F956-66, 1985 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-2416223

RESUMO

The modulatory effect of Ca on [Arg8]vasopressin-dependent (AVP) cAMP metabolism was studied in medullary collecting tubules (MCT) and medullary ascending limbs (MAL) microdissected from rat kidney. In MCT segments incubated in vitro with AVP, the accumulation of cAMP was enhanced (delta +59%) when Ca was omitted from the incubation medium compared with a medium with 2 mM of ionized calcium (Ca2+). Ionophore A23187 caused a decrease in AVP-stimulated cAMP accumulation in MCT in the presence of 2 mM Ca2+ but not in a Ca2+-free medium. Diltiazem and verapamil enhanced the AVP-stimulated cAMP accumulation in MCT; PTH had no detectable effect. A23187 caused a dose-dependent inhibition of cAMP accumulation stimulated by AVP with forskolin in both MCT and in MAL. However, in MAL the A23187 concentration needed for half-maximum inhibition (6.3 X 10(-6) M) was higher than for MCT (3.9 X 10(-7) M). The maximum inhibition in MAL (-65%) was less than in MCT (-97%). In the presence of 3-isobutyl-1-methylxanthine, AVP-stimulated cAMP accumulation was inhibited by A23187 in MCT (-45%) but not in MAL. Naproxen or ibuprofen did not relieve the inhibitory action of A23187 in MCT. Added Ca2+ inhibited the AVP-stimulated adenylate cyclase in MCT and MAL (half-maximum approximately equal to 5 X 10(-4) M Ca2+) and stimulated cAMP phosphodiesterase (cAMP-PDIE) in both MCT and in MAL (half-maximum approximately equal to 9 X 10(-5) M Ca2+). Incubation of MCT and MAL with A23187 decreased (-50%) the content of ATP. Results suggest that increased influx of extracellular Ca2+ inhibits the AVP-stimulated cAMP accumulation in MCT and to a much lesser degree in MAL. Deceased cAMP accumulation in MCT is probably due to both stimulation of cAMP-PDIE and the inhibition of adenylate cyclase, whereas in MAL it is due to stimulation of cAMP-PDIE. The results suggest that Ca2+ influx exhibits a negative modulatory effect on AVP-dependent cAMP metabolism mainly in MCT.


Assuntos
Cálcio/farmacologia , AMP Cíclico/metabolismo , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , Vasopressinas/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Trifosfato de Adenosina/metabolismo , Inibidores de Adenilil Ciclases , Animais , Arginina Vasopressina/farmacologia , Calcimicina/farmacologia , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Ibuprofeno/farmacologia , Túbulos Renais Coletores/metabolismo , Masculino , Naproxeno/farmacologia , Ratos , Ratos Endogâmicos , Rotenona/farmacologia
8.
Am J Physiol ; 253(2 Pt 2): F269-76, 1987 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2956891

RESUMO

NAD content and the rate of NAD hydrolysis were determined in proximal convoluted tubules (PCT), proximal straight tubules (PST), and adjacent cortical nephron segments microdissected from kidneys of thyroparathyroidectomized (TPTX) rats. In the basal state, rats fed a normal phosphate diet had an NAD content higher in PCT, PST, and in cortical ascending limb (CAL) than in glomeruli. After intraperitoneal injection of nicotinamide, the NAD content increased significantly in all nephron segments except CAL; the greatest (delta + 277%) increase was found in PCT, with less (delta + 82%) in PST. In experiments conducted on TPTX rats stabilized on a low-phosphorus diet, NAD content increased in response to a nicotinamide injection in PCT (delta + 197%), but did not change significantly in PST. The catabolism of NAD was determined by generation of [3H]adenosine, a major metabolite of [adenine-2,8-3H]NAD. The rate of [3H]adenosine generation from [3H]NAD was significantly (P less than 0.001) higher in PST than in PCT. We conclude that, in response to nicotinamide administration in vivo, the NAD content increases more in PCT than in PST and that this difference may be, at least partly, due to a lower rate of NAD breakdown in PCT. In a state of dietary phosphate deprivation, NAD also increases significantly in response to intraperitoneal nicotinamide in PCT, but it does not increase significantly in PST. The nicotinamide-elicited increase of NAD content in proximal tubules, mainly in PCT, may be related to inhibition of Na+-gradient-dependent inorganic phosphate (Pi) reabsorption across the brush-border membrane of proximal tubules and to the phosphaturic effect of nicotinamide in rats fed normal-Pi diet.


Assuntos
Dieta , NAD/metabolismo , Néfrons/metabolismo , Niacinamida/farmacologia , Fosfatos/farmacologia , Animais , Glândulas Paratireoides/fisiologia , Ratos , Ratos Endogâmicos , Tireoidectomia
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