摘要:

This study reports the synthesis of 1‐desamino‐7‐lysine‐ (fluorescein)‐8‐arginine‐vasotocin (7‐lys(flu) dAVT), and describes its biological activity in the isolated urinary bladder of the toadBufo marinus. 7‐lys(flu)dAVT was fully active in increasing bladder permeability to water. A half‐maximal hydroosmotic response was obtained at a concentration of 3×10−8M. A unique feature of this analog was that its reponse was not readily reversed after reoval of the analog from the serosal bathing solution. The residual response to 7‐lys(flu) dAVT was abolished (reversibly) by reducing serosal bath pH from 7.4 to 6.0, suggesting that acidification inhibits the response to analog at a step after the interaction of the ligand with its receptor. Although 8‐arginine‐vasopressin (AVP) was about 20 times more potent than 7‐lys(flu)dAVT in increasing membrane permeability to water, the response to AVP was readily reversed. Preincubation of bladders with 7‐lys(flu)dAVT in the presence of AVP blocked the residual response to 7‐lys(flu) dAVT. These studies suggest that 7‐lys(flu)dAVT forms a stable and physiologically active complex with hydrosmotic toad bladder receptors, and it may, therefore, serve as a useful fluorescent marker for receptors in tissues from this and other species that use vasotocin

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00808.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Toad urinary bladder epithelial cells grown in culture (primary) show a significant increase in water‐soluble inositol phosphates when treated with 10−8M vasopressin (AVP), but not with (3‐deamino‐8‐d‐arginine)vasopressin (ddAVP), a V2‐agonist. The increase in inositol phosphates was blocked by the V1‐antagonist, d(CH2)5Tyr(Me)AVP, suggesting a V1‐coupled phosphoinositide breakdown. The V1‐antagonist had no effect on basal adenylate cyclase activity nor on that stimulated by AVP. However, the V1‐antagonist was found to attenuate the hydrosmotic response of AVP, suggesting some role of the V1‐receptor cascade in the water flow response. Mezerein (MZ), a non‐phorbol activator of protein kinase C (PKC) increased osmotic water flow response. Mezerein (MZ), a non‐phorbol activator of magnitude and occurred over a longer period (90 min) than that observed with AVP. In an attempt to emulate the V1‐response, activation of PKC, and an increase in intracellular calcium, toad bladders were incubated with MZ and the calcium ionophore A23187 (IP). It was found that IP enhanced the water flow response to MZ at all times measured. Mz and IP were also found to enhance cAMP‐mediated water flow, suggesting that apical membrane permeability may be regulated in part through V1‐receptor stimulation and its respective second messengers. Collectively, these observations suggest that the V1receptor may play a role not only as part of a negative feedback system, but also as an integral component of the enhanced water permeability t

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00809.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Toad bladder epithelial cells were homogenized and fractionated by Percoll density‐gradient centrifugation. Binding of tritium‐labeled vasopressin ([3H]AVP) was measured in surface membranes (SM), microsomes (M), and a 100,000g(60‐min) micrsomal supernatant fraction (S). More than two‐thirds of the total receptors were in S. Receptors in SM — but not in S — were tightly coupled to G‐protein as suggested by inhibition of [3H]AVP binding by GTP. GTP‐sensitivity of [3H]AVP binding was not altered by vasotocin (AVT) stimulation, although the distribution of receptors shifted from SM to S.Intact bladders, exposed on the serosal side to 1‐desamino, 7‐lysine‐(4‐azidobenzoyl), 8‐arginine vasotocin (d7‐N3‐AVT) in the presence of UV light, exhibited a persistent hydroosmotic response compared to controls stimulated with photoaffinity analog in the dark. Cell fractions from the irradiated bladders showed a reduction in [3H]AVP binding in SM and S.Intact bladders, exposed on the mucosal side to d7‐N3‐AVT in the presence of UV light (while stimulated from the serosal side with AVP) exhibited a decrease in [3H]AVP binding in SM compared to controls without d7‐N3‐AVT.These findings suggest the following working hypothesis for the hydroosmotic action of vasopressin: (1) Vasopressin binds to receptors at the basolateral membrane, which is water‐permeable in the unstimulated state; (2) The hormone‐receptor complex is endocytosed and, together with other components of the basolateral membrane containing “water channels”, appears in “light vesicles”; (3) These light vesicles (and/or similar vesicles in a cytoplasmic reservoir) move to the apical membrane where they fuse with the water‐impermeable apical membrane and thereby

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00810.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Monensin, a highly selective sodium ionophore, inhibits vasopressin‐stimulated water flow in toad urinary bladder pretreated with naproxen, an inhibitor of prostaglandin synthesis. Inhibition is partially dependent on the presence of sodium in the serosal medium, but not on serosal calcium. We have found that monensin does not inhibit water flow generated by forskolin, cyclic AMP, or isobutyl methyl xanthine (MIX); indeed, an enhancement of water flow was seen following cAMP and MIX, as well as following 0.2 μM forskolin. Our findings suggest that monensin uncouples the vasopressin‐receptor‐G protein‐adenylate cyclase sequence at some early step, by a mechanism that remains unknown, but that may directly or indirectlyinvolve intracellula

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00811.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Although it is well accepted that vasopressin (ADH) increases the permeability to water of the toad bladder granular cell's luminal membrane, recent studies have suggested that regulation also takes place at an additional “postluminal” site within the epithelial granular cell. These studies are based upon the observation that a number of experimental maneuvers can alter tissue permeability to water, but do not change the number of particle aggregates observed on the protoplasmic face of the granular cell's luminal membrane with freeze‐fracture electron microscopy. These aggregates are believed by many investigators to mediate the transport of water across the luminal membrane.The dissociation between permeability and aggregate frequency described above has been variously interpreted as the consequence of changes in the permeability of the aggregates themselves, or of changes in the permeability of a “postluminal” barrier that is functionally in series with the luminal membrane.We attempted to distinguish between these 2 possibilities by studying paired toad bladders during 3 protocols that alter vasopressin‐stimulated water flow across the intact tissue without altering aggregate frequency. Estimates of the permeability of postluminal barriers were obtained by exposing the luminal surface to amphotericin B, an antibiotic that forms water‐permeant channels in the luminal membrane.Of the 3 protocols, only diminishing bladder filling volume decreased the water flow elicited by luminal amphotericin B, suggesting that only that protocol indeed decreased the permeability of some postluminal barrier. The other 2 protocols, increasing PCO2and repeatedly stimulating the bladder with vasopressin, did not alter amphotericin B‐elicited flow, suggesting that postluminal barriers were not altered by these 2 protocols. We thus conclude that a decrease in luminal membrane aggregate permeability or a decreased frequency of fusion of aggregate‐rich tubules to the luminal membrane mediates the decreased flow‐response in the latter 2 protocols, and not modulation of a postlumin

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00812.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

3,3′‐diallyldiethylstilbestrol (DADES), a blocker of the facilitated diffusion of glucose, was found to interfere markedly with the hydrosmotic response to antidiuretic hormone and its related agonists. Frog urinary bladders were isolated and monitored for transmural net water flow. DADES was added either to the serosal or to the apical medium at concentrations ranging from 10−4M to 10−6M. Pretreatment for 30 min with apical 10−4M DADES drastically reduced the subsequent hydrosmotic response:(a)to oxytocin (4.4×10−8M) by 91.7±17.6% versus 6.2±7.8 in control;(b)to 8‐bromo 3′,5′‐cyclic AMP by 93.5±19.4% versus 19.4±11.4%;(c)to serosal hyperosmolarity (mannitol 220 mOsm) by 99.3±0.5% versus 12.3±18.2%.This effect was dose‐dependent. Inhibitory action of DADES was more effective on the apical side than on the serosal side (97.0±1.5 versus 45.8±10.8). Freeze‐fracture studies revealed a modified distribution of the particles and unusual endocytotic pits and vesicles in the apical membrane of both granular and mitochondria‐rich epithelial cells. These observations point to multiple and complex effects of the drug. Thus, it seems that DADES has numerous effects on urinary epithelium, which makes it a nonspecific inhibitor of water permeation. Conclusions on its use should theref

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00813.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Some features of oxytocin stimulation of a cation‐selective channel of the apical membrane of amphibian tight epithelia were examined in an attempt to understand the channel's role in the regulation of epithelial transport. We first examined the ability of the channel to pass alkaline‐earth cations. We found that oxytocin can stimulate the movement of alkaline‐earth ions through the channel. This stimulation became greatly enhanced by treatment with Ag+. The stimulation of alkaline‐earth movements is discussed together with recently reported experiments which suggest that the channel may be involved in K+secretion. In addition we carried out comparative studies of the effects of oxytocin on the channel in a variety of epithelia obtained from different amphibians to examine whether the stimulation of ionic currents through the channel and the enhancement of hydrosmotic permeability caused by the hormone are linked. The results of our experiments showed that oxytocin activates the channel in the urinary bladders ofBufo marinus, andRana catesbeianaas well as in the skin ofB. marinus. It is well known that in all these tissues the hormone increases water permeability of the apical membrane. On the other hand, in skins ofRana catesbeiana, Rana pipiens, andRana temporaria, where oxytocin does not have a hydrosmotic effect, the hormone did not increase the currents through the cation‐selectiv

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00814.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

In agreement with previous reports, we found that absence of K+from the serosal bath of the toad urinary bladder substantially impairs vasopressin and cAMP‐stimulated water flow. The decreased response to vasopressin appears unrelated to prostaglandin production since inhibition of endogenous prostaglandins by pretreatment with naproxen 10−5M failed to prevent the effect seen with K+‐free Ringer's. The resistance to vasopressin does not appear to be directly related to epithelial K+concentrations, in that maneuvers leading to decreased intracellular K+failed to produce a similar effect. A more likely explanation appears to be that K+‐free Ringer's induces an increased cytosolic Ca++which, in turn, decreases the hydrosmotic effects of vasopressin. Several lines of evidence argue in favor of such an explanation: (a) Increased cytosolic Ca++had been found in other tissues with low extracellular K+; (b) The resistance to vasopressin decreases with decreased serosal Ca++; (c) The effects of K+‐free Ringer's are not additive in situations believed to have increased epithelial Ca++,i.e.replacement of serosal Na+with choline; (d) The effects of K+‐free serosal bathing medium could be both prevented and/or reversed if already established by increasing serosal bath, and presumably intracellular, pH, which is believed to decrease intrace

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00815.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

We measured the effects of oxytocin on capacitance and hydroosmotic water flow in the urinary bladder of the toadBufo marinusand the skins ofRana pipiensandRana temporaria. Oxytocin increased capacitance in all these tissues but stimulated hydroosmotic water flow only in the urinary bladder. We also measured the effects of oxytocin and PMA on the capacitance and hydroosmotic water flow of the toad urinary bladder. Both agents produced increases in membrane capacitance that were additive, however, PMA produced a stimulation of water flow that was only a fraction of that caused by oxytocin. Comparison of the effects of PMA and insulin in toad urinary bladder showed that in contrast with PMA, insulin did not increase membrane capacitance in this tissue. Moreover, insulin stimulated Iscin the urinary bladder while PMA produced an inhibition of variable magnitude. These results suggest that: (1) oxytocin can promote the fusion with the apical membrane of cytoplasmic membranes with or without water channels; (2) oxytocin and PMA stimulate the fusion with the apical membrane of cytoplasmic membranes originating in different pools; membranes in each pool have different water permeabilities and their insertion is controlled by different signals; (3) PMA and insulin act through different mechanisms in the toad urinary bladder.

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00816.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

The water permeability of collecting ducts is greatly increased by the antidiuretic hormone, vasopressin (VP). Freeze‐fracture studies were carried out to test if this permeability increase is associated with the appearance of intramembrane particle (IMP) aggregates and whether increased doses of VP lead to an increase in the number and size of particle aggregates in the luminal membrane of principal cells in the isolated cortical collecting duct. Unstimulated cells expressed 17±6.5 particle aggregates per 100 μm2. Stimulation with VP at concentrations of 20 or 200 μU/ml increased the number of particle aggregates significantly to 129±15.8 and 324±45.8, respectively. The size of the particle aggregates increased from 0.0012 μm2under control conditions to 0.025 μm2at 20 μU/ml VP and to 0.063 μm2at 200 μU/ml VP. In addition, the total area occupied by the IMP increased from 0.02 μm2/100 μm2(controls) to 3.17% and 20.38% (after 20 and 200 μU ADH/ml, respectively). Particle aggregates were also observed in the luminal plasma membrane of isolated collecting ducts fixed immediately after dissection, resembling thein vivostatus. These results demonstrate that a dose‐dependent relationship exists between the concentration of the applied VP and the number of particle aggregates, as well as the size of the aggregates. Cytoplasmic tubular vesicles in fusion with the apical membra

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00817.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY