Inhibitors of bacterial enoyl acyl carrier protein reductase (FabI): 2,9-disubstituted 1,2,3,4-tetrahydropyrido[3,4-b]indoles as potential antibacterial agents.

An SAR study of a screening lead has led to the identification of 2,9-disubstituted 1,2,3,4-tetrahydropyrido[3,4-b]indoles as inhibitors of Staphylococcus aureus enoyl acyl carrier protein reductase (FabI).

Separate receptors mediate oxytocin and vasopressin stimulation of cAMP in rat inner medullary collecting duct cells.

The two neurohypophysial hormones arginine vasopressin (AVP) and oxytocin have actions in the inner medullary collecting duct (IMCD) where both peptides induce an increase 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 NaCl) or hormone/antagonist solutions were added to aliquots of 10(4) IMCD cells in the presence of 10(-3) M 3-isobutylmethylxanthine (IBMX) and incubated at 37 degrees C for 4 min. 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 potent than AVP (EC50 = 1.6 x 10(-8) M vs. 7.4 x 10(-10) M). AVP at 10(-8) M and oxytocin at 10(-8) M, concentrations sufficient to elicit near-maximal cAMP accumulation, resulted in cAMP levels of 73.4 +/- 1.7 and 69.0 +/- 3.3 pmol (mg protein)-1 (4 min)-1, respectively (n = 10), compared with the vehicle-treated basal value of 37.7 +/- 2.2 pmol (mg protein)-1 (4 min)-1 (P < 0.001, n = 10). Combined AVP (10(-8) M) and oxytocin 10(-6) M) resulted in cAMP accumulation of 63.8 +/- 3.1 pmol (mg protein)-1 (4 min)-1 (n = 10), which was not significantly different from the effect of oxytocin alone, but slightly less than that for AVP alone (P < 0.05). A submaximal concentration of AVP (10(-10) M) induced cAMP accumulation of 48.6 +/- 2.5 pmol (mg protein)-1 (4 min)-1 (P < 0.01 compared with basal level of 34.9 +/- 2.4 pmol (mg protein)-1 (4 min)-1, n = 10), which was blocked in the presence of a vasopressin V2 receptor antagonist (10(-7) M OPC-31260) but not by the oxytocin receptor antagonist (10(-6) M [Pen1,pMePhe2, Thr4,Orn8]oxytocin) (36.3 +/- 6.1 and 45.1 +/- 1.3 pmol (mg protein)-1 (4 min)-1 respectively, P < 0.05, n = 10). A submaximal concentration of oxytocin (10(-7) M) induced a cAMP accumulation of 45.8 +/- 1.8 pmol (mg protein)-1 (4 min)-1 (n = 10), which was reduced by addition of 10(-6) M oxytocin antagonist (36.3 +/- 2.1 pmol (mg protein)-1 (4 min)-1, P < 0.05, n = 10), whereas co-incubation with 10(-6) M of the V2 receptor antagonist had no effect (43.2 +/- 1.3 pmol (mg protein)-1 (4 min)-1, n = 10). These results indicate that AVP and oxytocin induce cAMP accumulation from a common ATP pool in IMCD cells, and that separate vasopressin V2 and oxytocin receptor systems are involved, perhaps coupled to a common adenylate cyclase system.

Interaction between adrenergic agonists and forskolin on adenylate cyclase activity in the rabbit proximal tubule.

Using a micro-radioimmunoassay, cAMP was measured in single, isolated S2 proximal straight tubules dissected from rabbit kidneys to investigate the effects of adrenergic agonists on adenylate cyclase activity. The baseline activity of adenylate cyclase was low and unaffected by either the alpha 2 agonist clonidine, the beta agonist isoprenaline (in the absence or presence of 1 microM forskolin) or 1 microM forskolin. Adenylate cyclase activity was markedly stimulated by 20 microM forskolin, an effect which was inhibited by 1 microM clonidine. The inhibition by clonidine was not apparent in the presence of the alpha 2 antagonist yohimbine. These results confirm the inability of isoprenaline to stimulate adenylate cyclase in the rabbit proximal tubule and demonstrate the coupling of alpha 2 receptors, in an inhibitory fashion, to adenylate cyclase. The inhibitory action of the alpha 2-receptor agonist was independent of other hormone activity in the renal proximal tubule.

Lack of effect of atrial natriuretic peptide and urodilatin on vasopressin-stimulated cyclic AMP production in microdissected rat inner medullary collecting ducts.

It is unclear whether the diuretic effects of atrial natriuretic peptide (ANP) result, in part, from an inhibition of the renal actions of vasopressin. Moreover, accruing evidence suggests that the kidneys themselves may produce an ANP-like peptide, urodilatin, which shares many of the renal actions of ANP. The mechanism underlying the diuretic action of urodilatin has not yet been examined. Accordingly, we have investigated the potential modulatory actions of both ANP and urodilatin on vasopressin-stimulated cyclic AMP (cAMP) production in microdissected inner medullary collecting duct (IMCD) segments of rat kidney. ANP and urodilatin alone (at 10(-8) or 10(-6) M) had no demonstrable effect on cAMP accumulation in IMCD segments. Moreover, neither ANP nor urodilatin (each at 10(-6) M) significantly altered either the profile or the absolute magnitude of the cAMP response stimulated by vasopressin. These findings indicate that neither ANP nor urodilatin interacts with the vasopressin-sensitive adenylate cyclase site in the rat IMCD to contribute to its diuretic actions.

Effects of luminal vasopressin on intracellular calcium in microperfused rat medullary thick ascending limb.

Recent evidence suggests that vasopressin exerts a regulatory influence on transport processes in the rabbit cortical collecting duct via both the basolateral 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 +/- 32.2 nM evoked by addition of 1 microM vasopressin. Addition of 1 nM 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 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The involvement of vasopressin in the renal actions of atrial natriuretic peptide in conscious fluid-balanced rats.

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)

A novel computer-driven, servo-controlled fluid replacement technique and its application to renal function studies in conscious rats.

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.

Plasma atrial natriuretic peptide in vasopressin deficiency: the effects of acute water deprivation in rats.

Plasma concentrations of atrial natriuretic peptide (ANP) and other renally active hormones were measured in Long-Evans (LE) rats and vasopressin-deficient Brattleboro rats with diabetes insipidus (DI) in conditions of water repletion and deprivation, and in DI rats following chronic vasopressin replacement. In water-replete rats, vasopressin deficiency was associated with elevated circulating ANP and angiotensin II (AII) concentrations, while plasma adrenal steroid concentrations were depressed by comparison with LE rats. These differences were fully reversed after 7 days of vasopressin replacement in DI rats to restore normal water turnover. Water deprivation for 4 h had little effect on plasma tonicity or hormone profile in LE rats. In contrast, however, the unreplaced fluid loss during 4-h water deprivation in the DI rat was associated with a marked increase in plasma tonicity evident within 30 min. Plasma ANP concentrations fell substantially to levels below those in LE rats, coincident with a rise in adrenal steroid levels and independent of any clear change in AII. These changes in circulating ANP concentration were directly correlated with changes in plasma Na+ concentration, osmolality and tissue water content in the DI rats, underlining the importance of body fluid status in modulating the secretion of ANP. These data clearly show that plasma ANP concentration is increased in vasopressin deficiency, but emphasize the sensitivity of circulating hormone levels in vasopressin-deficient animals to acute changes in the state of hydration, underscoring the complex and labile interaction between body fluid and hormonal factors involved in the control of ANP secretion.