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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.

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.

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.

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.