Stimulated Water Flow Research Articles

Bradykinin B2 type receptor activation regulates fluid and electrolyte transport in the rabbit kidney

Bradykinin is an important autacoid produced in the kidney, regulating both renal function and blood pressure. In vivo studies in anesthetized rabbits, revealed that BK induced diuresis (UV), natriuresis (U NaV) and was not associated with renal hemodynamic changes. These diuretic and natriuretic effects were blocked by the BK-B 2 antagonist HOE-140. BK also inhibits vasopressin (AVP)-stimulated water flow ( L p) in microperfused rabbit cortical collecting ducts (rCCD), in a concentration-dependent fashion, consistent with its in vivo diuretic effects. BK-B 1 antagonist Leu 8-des-Arg 9-BK did not alter the effect of BK on L p, but HOE-140 completely blocked the inhibitory effects of BK on L p. While BK did not increase [Ca 2+] i in fura-2 loaded freshly microdissected rCCD, BK increased [Ca 2+] i in immortalized cultured rCCD cells demonstrating different signaling mechanisms are activated by BK in microdissected versus cultured rCCD. In microperfused rCCD, neither the protein kinase C inhibitor staurosporine nor the phospholipase C (PLC) inhibitor U-73,122 attenuated the BK response arguing against activation of PLC/PKC by BK in rCCD. We conclude: (1) BK induces UV and U NaV by a BK-B 2 receptor; (2) BK inhibits AVP-stimulated L p by a BK-B 2 receptor suggesting that its effects on L p are not via a PLC/PKC; (3) finally, BK raises [Ca 2+] i in rCCD cells by a BK-B 2 receptor mechanism.

Role of PKC isozyme III (γ) in water transport in amphibian urinary bladder

Vasopressin stimulated water flow across renal epithelia is thought to occur through a V2 receptor coupled to adenylcyclase. The increase in water flow occurs as a result of a fusion of water channels with the apical membrane and is indicative of an increase in membrane capacitance following hormone addition.What controls the cycling of water channels and their insertion into the membrane is uncertain. Our laboratory has demonstrated that renal epithelia as well as amphibian urinary bladder membranes, contain a vasopressin V1 receptor which upon activation results in the breakdown of phosphoinositide and the formation of inositol triphosphate and diacylglycerol, the latter an activator of protein kinase C (PKC). The initiation of transepithelial water flow also appears to involve V1 receptors and possibly activation of PKC. To test this hypothesis, we have been using activators of PKC, such as phorbol esters and mezerein, as pharmacological tools to determine if PKC activation results in similar physiological responses as the hormone. Several PKC isozymes, upon activation, are known to be translocated to the apical membrane as visualized by FITC immunofluorescence. Previously, we reported co-localization of PKC subtypes I (γ) and II (β) in toad urinary bladders using monoclonal antibodies and protein A-gold probes. This report includes the localization of PKC subtype III (α) and its distribution pattern using immunogold labeling.

Changes in intracellular sodium during the hydroosmotic response to vasopressin

During vasopressin (VP)-induced water movement, toad urinary bladder epithelial cells undergo unique morphological changes. The osmolality within these responding cells remains relatively stable despite the large transcellular transport of water. We hypothesized that the hydroosmotic response to VP may be associated with a net increase in sodium either as an aid in maintaining the intracellular osmolality or as part of a Na-Ca exchange process. Changes in intracellular sodium (Nai) were monitored over time in individual hemibladders using 23Na NMR. Hemibladders were mounted as bags on glass pipets and filled with deionized water. During NMR studies, the serosal bath consisted of aerated 2.4 mM HCO3 amphibian Ringer's (pH 8.1) made up with 15% D2O containing the shift reagent, dysprosium tripolyphosphate (1 mM). This reagent allowed for visualization of Nai by shifting the extracellular Na signal; it did not affect basal or VP stimulated water flow, short-circuit current, or high energy phosphate metabolism as seen by 31P NMR. Changes in Nai were determined by integrating the area under the unshifted Na peak at each measurement and expressing differences as a ratio relative to baseline. The initial Nai signal from unstimulated hemibladders remained stable in these tissues over at least 180 minutes. Within 30 minutes of VP (20 mU/ml) exposure, however, the Nai peak increased 2.47 times above pretreatment baseline (N = 16, P less than 0.001). The Nai signal returned toward baseline values with removal of VP from the serosal bath but only after approximately 90 minutes. When change in cell shape and water movement were prevented by having isotonic sorbitol in the mucosal bath, VP produced no change in the Nai signal (N = 10).(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancing effects of angiotensin I on the vasopressin-stimulated water flow of toad bladder through increased cyclic AMP in mucosal cells

The effects of angiotensins I and II on 10 mU/ml vasopressin- stimulated water flow across toad bladder were examined. Angiotensin I at concentrations of 10 −6 and 10 −7 M enhanced the water flow, but angiotensin II failed to do so at these concentrations. Angiotensin I had no effect on 5 mM cyclic AMP-stimulated water flow. After being preincubated for 30 min with angiotensin II, angiotensin I failed to have any stimulatory effect on vasopressin-stimulated water flow. At 10 −6 M angiotensin I significantly enhanced vasopressin-stimulated cyclic AMP content in bladder mucosal cells. These results indicate that angiotensin I enhances vasopressin-stimulated water flow by increasing cyclic AMP production in bladder cells and that angiotensin II may possibly interfere with angiotensin I in a competitive manner.

Comparative effects of prostaglandin E 2 and prostaglandin E 3 on water flow and cyclic AMP in the urinary bladder of the frog, [formula omitted

Prostaglandins (PGs) modulate osmotic water flow in amphibian urinary bladders. Gas chromatographic analysis of prostaglandin precursors in bladders showed that arachidonic acid represented 13.0 ± 0.6% and eicosapentaenoic acid 4.3 ± 0.1% of the total fatty acid content. The effects of PGE 2 and PGE 3 on basal and arginine vasotocin (AVT) stimulated water flow were compared. Contol water flow (1.1 ± 0.2 mg/min) was increased to 4.6 ± 0.3 mg/min with AVT (10 −6M) present. PGE 2 (10 −6M) inhibited both basal and AVT stimulated water flow. In contrast, PGE 3 (10 −6M) stimulated basal water flow and further increased AVT stimulated water flow. Basal adenylate cyclase activity (ACA, 59 ± 0.3 pmol cyclic AMP/mg protein/10 min) was stimulated by the addition of AVT in the absence or presence of exogenous guanosine 5′ triphosphate (GTP, 10 −5M). Both PGE 2 and PGE 3 stimulated basal ACA in the absence, but not in the presence of GTP. In the absence of exogenous GTP, PGE 2 increased AVT stimulated ACA, whereas PGE 3 decreased it. Both prostaglandins inhibited AVT stimulation when GTP was added. The effects of PGE 2, PGE 3 and AVT on tissue cycilc AMP levels in whole urinary bladders were similar to the effects seen on ACA in the absence of exogenous GTP. The contrasting effects of PGE 2 and PGE 3 on control water flow appear distinct from their similar effects on ACA. However, PGE 2 and PGE 3 may regulate AVT stimulation through mechanisms involving cyclic AMP.

Effect of vanadate on water transport by the toad bladder

Vanadate increases renal Na and water excretion. The mechanism whereby vanadate impairs water transport was examined in the toad bladder. Vanadate did not alter baseline water transport but caused a significant inhibition of water transport elicited by high doses of AVP. The inhibition of AVP stimulated water flow by vanadate was dose dependent with inhibition present with concentration as low as 10 −7 and maximal inhibition occurring at 10 −5M. Vanadate also inhibited water transport stimulated by cyclic AMP or by phosphodiesterase inhibition indicating that vanadate has an effect beyond cyclic AMP step, in addition to whatever effect it might have on adenylate cyclase. The inhibitory effect of vanadate on AVP stimulated water flow was not altered by prior Na-K-ATPase or prostaglandin inhibition. Since vanadate has been shown to stimulate adenylate cyclase in other tissues we examined whether addition of vanadate 10 minutes after addition of AVP would enhance water transport. Vanadate caused a transient enhancement of AVP stimulated water flow. These data demonstrate that vanadate can inhibit or stimulate water flow in the toad bladder.

Dopaminergic inhibition of vasopressin-stimulated water flow in the toad bladder: Evidence for local formation of dopamine

Dopamine administration increases renal excretion of water and Na. It remains uncertain whether these effects of dopamine are the result of a hemodynamic effect or the consequence of a direct cellular action. We investigated the effect of dopamine on water transport by the isolated toad bladderin vitro. Dopamine failed to alter baseline water flow but caused a significant inhibition of arginine vasopressin (AVP) or cyclic adenosine monophosphate (AMP) stimulated water flow. The effect of dopamine on stimulated water flow was not due to activation of α adrenergic, β adrenergic, or cholinergic receptors. The selective antagonists of dopamine, metoclopramide and apomorphine, prevented the effect of dopamine on AVP-stimulated water flow. These observations suggest the existence of a dopaminergic receptor in the toad bladder.l-Dopa also inhibited AVP-stimulated water flow. The effect ofl-Dopa could be prevented by metoclopramide, thus suggesting thatl-Dopa is converted to dopamine by an aromatic amino acid decarboxylase present in the toad bladder. To investigate this possibility we measured the effect of the decarboxylase inhibitor, carbidopa, on the14CO2 production generated by decarboxylation of14Cl-Dopa in isolated toad bladder epithelial cells. Isolated toad bladder epithelial cells generated significant amounts of14CO2 from14Cl-Dopa. This effect could be blocked by carbidopa, thus suggesting the existence of an aromatic amino acid decarboxylase system in the toad bladder. Carbidopa also prevented the inhibitory effect ofl-Dopa on AVP-stimulated water flow, suggesting thatl-Dopa needs to be converted to dopamine to inhibit water flow. These data suggest the existence of a dopaminergic receptor in the toad bladder. These data also suggest that dopamine can be formed locally in the toad bladder and can thus serve as a local modulator of water transport.

Cellular and membrane events involved in the K-induced increase in water permeability of toad skin.

Exposure of the inner surface of toad skin (Bufo marinus) to high [K+] resulted in a marked (up to 7-fold) increase in water permeability (Pf) that was more marked in KC1-Ringer than in K2SO4-Ringer. Although high [K+] did not elicit a maximal increase in Pf, it blunted the hydrosmotic responses to vasopressin, isoproterenol and cAMP. Both "post-cAMP" inhibitors of stimulated water flow, such as diamide and vanadate, and "pre-cAMP" inhibitors, such as methohexital and propranolol, markedly reduced the K response, while exposure to Ca2+-free, KC1-Ringer did not inhibit water flow. Intramembrane particle aggregates, similar to those induced by cAMP-mediated hydrosmotic agents, were seen in the apical membrane of granular cells, just beneath the stratum corneum, in skins exposed to KC1. Available evidence indicates that cAMP might mediate, at least partially, the hydrosmotic effect of high [K+]. In contrast, a role of voltage-dependent Ca2+ channels, described in other cell systems depolarized with K, was not apparent in toad skin.

Frogs produce a physiologically active compound from eicosapentaenoic acid

Prostaglandins have been shown to modulate water flow in anuran amphibian urinary bladders. These experiments examined which fatty acid precursor could be metabolized by bladders, and the effect of metabolites on osmotic water flow. Hemibladders were incubated with precursors or prostaglandins (1 μM) and water flow measured. In addition, hemibladders were incubated with 14C-labelled eicosatrienoic, arachidonic, or eicosapentaenoic acid, and products identified by thin layer chromatography. Addition of prostaglandins E 1, E 2 and I 2 inhibited water flow. Eicosatrienoic acid did not affect water flow. Arachidonic acid inhibited basal water flow, an effect which was not completely reversed with the addition of indomethacin. Eicosapentaenoic acid stimulated water flow, and the stimulation was blocked with indomethacin. Frog urinary bladder did not synthesize any prostaglandins from 14C-eicosatrienoic acid. 14C-arachidonic acid was converted into PGE 2 and PGD 2. 14C-eicosapentaenoic acid was synthesized into compounds, presumably PGE 3 and PGD 3, with the opposite physiological effects of two-series prostaglandins. The data suggest that effects of prostaglandins on amphibian bladder depend on the substrate which is metabolized.

Thromboxane and stable prostaglandin endoperoxide analogs stimulate water permeability in the toad urinary bladder.

The effects of thromboxane B(2) and the stable prostaglandin endoperoxide analogs (15Z)-hydroxy - 9alpha - 11alpha - (epoxymethano)prosta - 5Z,13E - dienoic acid (U44069) and (15Z)-hydroxy -11alpha,9alpha-(epoxymethano) prosta-5Z,13E-dienoic acid (U46619) were tested on water flow across the toad urinary bladder. In the presence of indomethacin or meclofenamic acid, inhibitors of prostaglandin and thromboxane A(2) synthesis, thromboxane B(2) stimulated water flow in a dose-dependent manner. U44069 (1 muM) stimulated water flow from 3.6+/-0.8 to 12.4+/-1.2 mg/min per 10 cm(2) hemibladder surface area, while U46619 (1 muM) stimulated water flow from 2.8+/-1.0 to 21.8+/-2.0 mg/min per 10 cm(2). The prostaglandin endoperoxide/thromboxane A(2) antagonist trans- 13-azaprostanoic acid, an inhibitor of vasopressin-stimulated water flow, inhibited thromboxane B(2)- and U46619-stimulated water flow in a dose-dependent manner. The inactive cis-13-azaprostanoic acid did not inhibit vasopressin-stimulated water flow in untreated hemibladders and had no effect on U46619-stimulated water flow in indomethacin or meclofenamic acid pretreated hemibladders. U46619 (1 muM) enhanced vasopressin-stimulated water flow in indomethacin pretreated hemibladders, producing a significant parallel shift (P < 0.001) in the dose-response relationship to submaximal concentrations of vasopressin (0.1-0.6 mU/ml), while not affecting water flow stimulated by supramaximal concentrations of vasopressin (10 mU/ml). trans-13-Azaprostanoic acid abolished the potentiating effects of U46619 on vasopressin-stimulated water flow. These results show that thromboxane A(2)-like compounds stimulate water flow in the toad urinary bladder.

Effect of verapamil on the hydroosmotic response to antidiuretic hormone in toad urinary bladder.

To investigate the role of the calcium ion in the hydroosmotic response to antidiuretic hormone (ADH), the effects of verapamil, an inhibitor of calcium ion entry into cells, on stimulated water flow was examined in vitro in the toad urinary bladder. Verapamil, 50 micro M, decreased ADH-stimulated osmotic water flow from 23.4 +/- 4.1 to 9.9 +/- 3.3 mg . min-1 . hemibladder-1 (mean +/- SE, n = 12, P < 0.001). That this inhibitory effect was due to a verapamil-induced alteration in cellular calcium metabolism is suggested by the findings that 45Ca2+ uptake by isolated toad bladder epithelial cells was reduced nearly 50% in the presence of verapamil and that reversibility of verapamil's inhibitory action was calcium dependent. Additionally, verapamil reduced theophylline- (20 mM) stimulated water flow from 22.8 +/- 2.7 to 9.5 +/- 2.9 mg . min-1 . hemibladder-1 (n = 7, P < 0.001) but enhanced cAMP- (10 mM) induced water flow from 12.8 +/- 2.5 to 21.6 +/- 1.1 ng . min-1 . hemibladder-1 (n = 7, P < 0.001). The latter effect was due, at least in part, to a direct inhibitory effect by verapamil on phosphodiesterase activity of toad bladder homogenates. These results, therefore, suggest that the calcium ion is an important coupling factor at the level of the adenylate cyclase enzyme complex for the stimulus-reabsorption coupling between ADH and the transporting epithelia of the toad urinary bladder.

Effect of quinidine on Na, H+, and water transport by the turtle and toad bladders.

The effect of quinidine on Na and H+ transport by the turtle bladder and water transport by the toad bladder was examined. Quinidine inhibited the short-circuit current and the potential difference in a dose-dependent fashion. The effect of quinidine on the short-circuit was not dependent on extracellular calcium concentration and was not reversible with removal of the drug. Quinidine inhibited H+ secretion in a dose-dependent fashion. The effect of quinidine on H+ secretion also was not dependent on extracellular calcium concentration and was not reversible, either with removal of the drug or with stimulation of H+ secretion with 5% CO2. The effect of quinidine on Na or H+ transport could not be elicited by an equivalent dose of tetracaine, suggesting that the inhibitory effect of quinidine is not dependent on its anesthetic properties. Quinidine also inhibited vasopressin and cyclic AMP stimulated water flow in the toad bladder. Quinidine did not alter calcium uptake by the turtle bladder but increased calcium efflux by the turtle and toad bladders. These observations suggest that a rise in cytosolic calcium is responsible for the inhibitory effect of quinidine on Na, H+, and water transport.

The effect of acid-base changes on vasopressin-stimulated water flow in toad urinary bladder

Water flow was measured gravimetrically in the presence and absence of vasopressin across the toad urinary bladder. Four groups of toads in different states of acid-base balance were used; a normal group, a group in NH 4Cl induced metabolic acidosis, respiratory acidosis, and a group in NaHCO 3 induced metabolic alkalosis. Vasopressin induced water flow was significantly reduced during metabolic acidosis and respiratory acidosis. Metabolic alkalosis had no effect on the hydro-osmotic response to vasopressin. Dibutyryl cyclic-AMP-stimulated water flow on the other hand was not affected by either a metabolic or respiratory acidosis. Treatment with indomethacin was able to reverse the observed reduction in the vasopressin-stimulated water flow response in the toad bladder during metabolic and respiratory acidosis. We conclude that the vasopressin stimulated water flow is altered during acidosis and evidence suggests that prostaglandins may be involved in the observed reduction in vasopressin-stimulated water flow.

Temperature dependence of ADH-induced water flow and intramembranous particle aggregates in toad bladder.

Antidiuretic hormone (ADH)-induced luminal intramembranous particle aggregates and hormonally stimulated water flow in toad urinary bladder are reduced simultaneously with a reduction in temperature. When water movement is factored by the aggregation response, the apparent activation energy for this process decreases from 12.1 +/- 1.6 to 3.0 +/- 2.3 kilocalories per mole. The data are consistent with the view that the particle aggregates contain sites for transmembrane water movement and that these sites behave as pores.