1. A new rat model has been developed allowing body fluid status to be accurately controlled and maintained throughout experimentation by computer-driven, servo-controlled replacement of spontaneous urinary fluid losses. 2. Experiments in vitro were performed to test the accuracy of the servo system, and experiments in vivo were carried out to re-assess basic renal function in servo-controlled vasopressin-replete Long Evans and vasopressin-deficient Brattleboro rats. The model was further evaluated in water-diuretic Wistar rats with or without administration of a vasopressin V2-receptor agonist, 1-desamino-8-D-arginine vasopressin. 3. The data gained from the present study indicate the suitability of the servo-controlled replacement system for conscious renal function studies in three different rat strains. Haemodynamic and renal function variables measured were demonstrated to be stable throughout a 5 h experimental procedure and reproducible between repeated experimental occasions over a 14 day post-operative period. 4. Using the servo-control technique, the expected action of 1-desamino-8-D-arginine vasopressin on renal water handling was demonstrated, but the natriuretic effect reported by some workers was not evident. 5. Since the servo-controlled fluid replacement technique maintains many of the inherent differences between vasopressin-replete Long Evans and vasopressin-deficient Brattleboro rats and eliminates the changes in body fluid volume during transition from a diuretic to an antidiuretic state, the model confers an advantage over previously employed constant infusion protocols.

SUMMARYThe two neurohypophysial hormones arginine vasopressin (AVP) and oxytocin have actions in the inner medullary collecting duct (IMCD) where both peptides induce an increas8e in cAMP accumulation. The present study has employed a novel IMCD cell line to determine whether these two hormones induce cAMP accumulation via common or separate receptors, and to characterize the potential receptors responsible. Equal volumes of vehicle (150 mM NaCI) or hormone/antagonist solutions were added to aliquots of lo4 IMCD cells in the presence of M 3-isobutylmethylxanthine (IBMX) and incubated at 37OC for 4min. cAMP levels were determined by radioimmunoassay and protein concentration by Bradford assay. Both AVP and oxytocin elicited dose-dependent increases in cAMP generation, though oxytocin was less potenlt than AVP (EC,, = 1.6 x lo-' M vs. 7.4 x lo-'' M). AVP at lo-' M and oxytocin at M, concentrations sufficient to elicit near-maximal cAMP accumulation, resulted in cAMP levels of 73.4 k 1.7 and 69.0 k 3.3 pmol (mg protein)-' (4 min)-', respectively (n = lo), compared with the vehicle-treated basal value of 37.7 f 2.2 pmol (mg protein)-' (4 min)-' ( P c 0.001, n = 10). Combined AVP (lo-' M) and oxytocin M) resulted in cAMP accumulation of 63.8 f 3.1 pmol (mg protein)-' (4 min)-' (n = lo), which was not significantly different from the effect of oxytocin alone, but slightly less than that for AVP alone ( P c 0.05). A submaximal concentration of AVP (lo-" M) induced cAMP accumulation of 48.6 f 2.5 pmol (mg protein)-' (4 min)-' ( P c 0.01 compared with basal level of 34.9 f 2.4 pmol (mg protein)-' (4 min)-', n = lo), which was blocked in the presence of a vasopressin V, receptor antagonist M OPC-31260) but not by the oxytocin receptor antagonist ( low6 M [Pen',pMePhe2,Thr4,0rns]oxytocin) ( 3 6 3 k 6.1 and 45.1 f 1.3 pmol (mg protein)-' (4 min)-' respectively, P c 0.05, n = 10). A submaximal concentration of oxytocin M) induced a cAMP accumulation of 45.8 f 1.8 pmol (mg protein)-' (4 min)-' (n = lo), which was reduced by addition of M oxytocin antagonist (36.3 & 2.1 pmol (mg protein)-' (4 min)-', P c 0.05, n = lo), whereas co-incubation with M of the V, receptor antagonist had no effect (43.2 f 1.3 pmol (mg protein)-' (4 min)-', n = 10).These results indicate that AVP and oxytocin induce cAMP accumulation from a common ATP pool in IMCD cells, and that separate vasopressin V, and oxytocin receptor systems are involved, perhaps coupled to a common adenylate cyclase system. INTRODUCTIONThe antidiuretic osmotic water transport (P,) in the inner medullary collecting duct of most mammals, including humans and rats, is regulated by arginine vasopressin (A'VP) via CAMPmediated translocation of aquaporin-2-containing vesicles to the luminal membrane. The other neurohypophysial hormone, oxytocin, has also been demonstrated to have an antit Corresponding author: rnopleetw@stud.rnan.ac.uk , 1984;Lyness et al. 1985) and has also been found to stimulate an increase in osmotic water transport (P,) in microdissected inner medullary colle...

Recent evidence suggests that vasopressin exerts a regulatory influence on transport processes in the rabbit cortical collecting duct via both the baso-lateral and luminal membranes. The present study was undertaken to examine whether luminal vasopressin receptors, coupled to changes in intracellular calcium, were also present in microperfused rat medullary thick ascending limb (mTAL), a key element of the urine concentrating mechanism. Addition of 1 nM vasopressin to the luminal microperfusate elicited a small but significant and sustained rise in intracellular calcium, from basal values of 100.1 ± 20.1 to 169.6 ± 24.1 nM after 250 s. The effect observed following luminal addition of vasopressin was dose-dependent, with a larger increment of 190.2 ± 32.2nM evoked by addition of 1 µM vasopressin. Addition of 1 µM oxytocin to the lumen did not cause a significant increase in intracellular calcium concentration, consistent with the response to vasopressin being mediated by specific luminal vasopressin receptors. In the absence of calcium in the bath and lumen together or in the bath alone, a residual response to 1 µM luminal vasopressin was still evident, suggestive of a small component of release of calcium from intracellular stores. Selective calcium removal from the luminal microperfusate alone left the response intact. These data are congruous with a model of vasopressin-induced entry of calcium which occurs via the basolateral membrane following ligand binding to the apical membrane. These findings, coupled with earlier observations in the collecting duct, suggest that a fundamental re-assessment of where and how vasopressin, and perhaps other hormones, acts in the kidney may be required. Hormonal regulation of distal tubular function may not therefore be determined only by blood-borne delivery of hormone, but may also involve tubular fluid delivery to apical receptors in distal nephron sites.

The renal and endocrine actions of atrial natriuretic peptide (ANP) administered at a rate to induce plasma concentrations within the physiological range have been re-examined in conscious rats in which body fluid volume was maintained by infusion of replacement fluid at a rate to match spontaneous urine losses (servo-controlled replacement) throughout experimentation. The involvement of vasopressin in the actions of ANP was assessed by comparing the responses induced in Brattleboro (DI) and Long-Evans (LE) rats. A rate of ANP administration inducing a less than twofold increment in circulating ANP concentration evoked a small but significant diuresis and natriuresis. In contrast to previous studies during which body fluid balance had not been maintained and the response to ANP was transient, renal responses were rapid in onset and sustained over the period of hormone administration. The change in renal excretion occurred without concomitant changes in mean arterial blood pressure, haematocrit or glomerular filtration rate, and without consistent alterations in the circulating concentrations of angiotensin II, vasopressin, aldosterone or corticosterone. Furthermore, although small differences between the two strains in the character of the response could be demonstrated, the evoked response was of similar magnitude in vasopressin-replete and -deficient animals. In summary, in conscious rats in which body fluid volume was maintained, the profile of the diuretic and natriuretic responses evoked by low-rate ANP administration was different from that previously observed in anaesthetized and/or constantly infused preparations; being rapid in onset and sustained.(ABSTRACT TRUNCATED AT 250 WORDS)