Role of osmotic forces in exocytosis: studies of ADH-induced fusion in toad urinary bladder.

Antidiuretic hormone (ADH) treatment of toad urinary bladder activates an exocytotic-like process by which intramembrane particle aggregates are transferred from membranes of elongated cytoplasmic tubules to the luminal-facing plasma membrane. We find that the number of these ADH-induced fusion events, and the number of aggregates appearing in the luminal membrane, are reduced when the luminal bathing medium is made hyperosmotic. As an apparent consequence of the inhibition of their fusion with the luminal membrane, the elongated cytoplasmic tubules become enormously swollen into large, rounded vesicles. These results are consistent with the view that osmotic forces are essential to the basic mechanism of exocytosis.

Evidence that ADH-stimulated intramembrane particle aggregates are transferred from cytoplasmic to luminal membranes in toad bladder epithelial cells.

In freeze-fracture (FF) preparations of ADH-stimulated toad urinary bladder, characteristic intramembrane particle (IMP) aggregates are seen on the protoplasmic (P) face of the luminal membrane of granular cells while complementary parallel grooves are found on the exoplasmic (E) face. These IMP aggregates specifically correlate with ADH-induced changes in water permeability. Tubular cytoplasmic structures whose membranes contain IMP aggregates which look identical to the IMP aggregates in the luminal membrane have also been described in granular cells from unstimulated and ADH-stimulated bladders. The diameter of these cytoplasmic structures (0.11 +/- 0.004 micrometers) corresponds to that of tubular invaginations of the luminal membrane seen in thin sections of ADH-treated bladders (0.13 +/- 0.005 micrometers). Continuity between the membranes of these cytoplasmic structures (which are not granules) and the luminal membrane has been directly observed in favorable cross-fractures. In FF preparations of the luminal membrane, these apparent fusion events are seen as round, ice-filled invaginations (0.13 +/- 0.01 micrometer Diam), of which about half have the characteristic ADH-associated aggregates near the point of membrane fusion. They are less numerous than, but linearly related to, the number of aggregates counted in the same preparations (n = 78, r = 0.71, P less than 0.01). These observations suggest that the IMP aggregates seen in luminal membrane after ADH stimulation are transferred preformed by fusion of cytoplasmic with luminal membrane.

Stabilization of vasopressin-induced membrane events by bifunctional imidoesters.

Vasopressin increases the water permeability of the luminal membrane of the toad bladder epithelial cell. This change in permeability correlates with the occurrence in luminal membranes of intramembrane particle aggregates, which may be the sites for transmembrane water flow. Withdrawal of vasopressin is ordinarily associated with a rapid reduction of water flow to baseline values and a simultaneous disappearance of the particle aggregates. The bifunctional imidoesters dithiobispropionimidate (DTBP) and dimethylsuberimidate (DMS), which cross-link amino groups in membrane proteins and lipids, slow the return of water flow to baseline after vasopressin withdrawal. Cross-linking is maximal at pH 10, and is reduced as pH is lowered. Freeze-fracture studies show persistence of luminal membrane particle aggregates in cross-linked bladders and a reduction in their frequency as water flow diminishes. Fusion of aggregate-containing cytoplasmic tubular membrane structures with the luminal membrane is also maintained by the imidoesters. Reductive cleavage of the central S-S bond of DTBP by beta-mercaptoethanol reverses cross-linking, permitting resumption of the rapid disappearance of the vasopressin effect. Bladders that have undergone DTBP cross-linking and beta-mercaptoethanol reduction respond to a second stimulation by vasopressin. Thus, the imidoesters provide a physiologic and reversible means of stabilizing normally rapid membrane events.

Vasopressin: induced structural change in toad bladder luminal membrane.

Freeze-fracture electron microscopy demonstrates that vasopressin stimulation of isolated toad bladder alters the structure of the luminal membrane of granular cells. This alteration consists of an ordered aggression of intramembranous particles, and appears to be of functional significance, since the frequency of aggregation sites per area of membrane is closely correlated with vasopressin-induced osmotic 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.

Effects of trifluoperazine on function and structure of toad urinary bladder. Role of calmodulin vasopressin-stimulation of water permeability.

Calcium ion plays a major regulatory role in many hormone-stimulated systems. To determine the site of calcium's action in the toad urinary bladder, we examined the effect of trifluoperazine, a compound that binds specifically to the calcium binding protein, calmodulin, and thereby prevents activation of enzymes by the calcium- calmodulin complex. 10 microM trifluoperazine inhibited vasopressin stimulation of water flow, but did not alter vasopressin's effects on urea permeability or short-circuit current. Trifluoperazine also blocked stimulation of water flow by cyclic AMP and methylisobutylxanthine, implying a "postcyclic AMP" site of action. Consistent with these results, trifluoperazine did not decrease epithelial cyclic AMP content or the cyclic AMP-dependent protein kinase activity ratio. Assay of bladder epithelial supernate demonstrated calmodulin-like activity of 1.5 U/microgram protein. Morphologic studies of vasopressin-treated bladders revealed that trifluoperazine did not alter the volume density of cytoplasmic microtubules or significantly decrease the number of fusions between cytoplasmic, aggregate-containing, elongated vesicles and the luminal membrane. Nonetheless, the frequency of luminal membrane aggregates, structures that correlate well with luminal membrane water permeability, was decreased by greater than 50%. Thus, trifluoperazine appears to inhibit the movement of intramembranous particle aggregates from the fused intracellular membranes to the luminal membrane, perhaps by blocking an effect of calcium on microfilament function.

Relationship of aggregated intramembranous particles to water permeability in vasopressin-treated toad urinary bladder.

It has been previously demonstrated with freeze-fracture electron microscopy that vasopressin induces specific structural alterations of the luminal membrane of granular cells from toad urinary bladder in a dose-dependent fashion. These alterations consist of aggregated intramembranous particles and are observed both in the presence and absence of an osmotic gradient. We examined the effect of methohexital, a selective inhibitor of vasopressin-stimulated water flow, and the effect of phloretin, a selective inhibitor of urea permeability, on the structure of the granular cell luminal membrane. Methohexital treatment of the vasopressin-stimulated toad bladder reduced both the osmotic water flow and vasopressin-induced alterations of membrane structure to the same extent. Phloretin reduced urea permeability but not water flow or particle aggregation. Since neither agent affects vasopressin-stimulated sodium movement, these findings indicate that the phenomenon of particle aggregation is specifically related to vasopressin-induced water permeability and not to changes in urea or sodium permeability.

Regulation of ADH-stimulated water flow at a post-luminal barrier in toad bladder.

Recent studies show that ADH-stimulated water flow across toad bladder may be regulated at a site other than the luminal membrane. In these studies luminal membrane particle aggregate frequency has been used as a measure of luminal membrane water permeability. In fully stretched bladders the relationship between total tissue permeability and aggregate frequency is curvilinear, rather than linear. This implies a resistance in series with the luminal membrane that can become rate-limiting for water flow during ADH stimulation. The possibility that transtissue water movement is actually regulated at such a post-luminal membrane resistance is suggested by the finding that within 30 min following exposure to hormone, water flow becomes attenuated without any change in aggregate frequency. Supporting this possibility, recent data from follow-up studies suggest that the apparent water permeability per luminal membrane aggregate is not reduced with time. Finally, for bladders in which prostaglandin synthesis is inhibited (by naproxen), increases in both base-line water flow and water flow consequent to treatment with a submaximal dose of ADH (0.125 mU/ml), are much less than expected from simultaneously observed changes in luminal membrane aggregate frequency. In parallel experiments to these, moreover, direct measurements of luminal membrane water permeability from the rate of change of cell volume consequent to a transluminal membrane osmotic challenge, confirm that luminal membrane water permeability increases to the extent expected from changes in aggregate frequency. All of the data taken together argue for a post-luminal membrane barrier in toad bladder which regulates tissue permeability during ADH stimulation.

Barriers to water flow in vasopressin-treated toad urinary bladder.

Unstirred layers of water complicate the measurement of water permeability across epithelia. In the toad urinary bladder, the hormone vasopressin increases the osmotic water permeability of the granular epithelial cell's luminal membrane, and also leads to the appearance of aggregates of particles within this membrane. The aggregates appear to be markers for luminal membrane osmotic water permeability. This report analyzes the relationship between transbladder osmotic water flow and aggregate frequency, and demonstrates that flow across the bladder is significantly attenuated by unstirred layers of water or by structural barriers other than the luminal membrane when the luminal membrane is made permeable by vasopressin. This analysis in addition yields unique values for the permeabilities of both the luminal membrane and the barriers to water flow which lie in series with it.

Regulation of luminal membrane water permeability by water flow in toad urinary bladder.

Intramembranous particle aggregates (presumed sites for water flow) which appear in the luminal membrane consequent to ADH treatment are derived from cytoplasmic membrane structures (now termed "aggrephores") which fuse with the luminal membrane. We have previously shown that bladders stimulated in the absence of an osmotic gradient have about twice as many aggregates and about three times as many sites of aggrephore fusion as bladders stimulated with ADH in the presence of a 175 milliosmolal gradient. The present studies show that the frequency of fused aggrephores and luminal membrane aggregates can be modified as a consequence of alterations in transmembrane water flow initiated by changing the transbladder osmotic gradient during hormone stimulation. Bladders treated with ADH for 1 hr without a gradient and then for 1 hr with a gradient had approximately 1/3 as many aggregates and fusion sites as paired bladders treated for 2 hr without a gradient. Conversely, bladders treated with ADH for 1 hr with a gradient and then for 1 hr without a gradient had approximately 2x as many aggregates and fusion sites as bladders treated for 2 hr with a gradient. In other experiments we demonstrate that the time course of hormone washout is greatly accelerated if carried out in the presence of an osmotic gradient. In paired bladders that were first stimulated with ADH for 30 min in the absence of a gradient, aggregates and fusion sites as well as osmotic water permeability determined in fixed bladders, persisted at near maximum levels for 15 min of washout in the absence of a gradient.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of distension on ADH-induced osmotic water flow in toad urinary bladder.

We recently described a method by which the resistance to water flow of the luminal membrane of ADH-stimulated toad bladder can be quantitatively distinguished from that of barriers lying in series with it. This method requires estimated of both total bladder water permeability (assessed by transbladder osmotic water flow at constant gradient) and luminal membrane water permeability (assessed by quantitation of the frequency of ADH-induced luminal membrane particle aggregates). In the present study we examined the effect of bladder distension on transepithelial osmotic water flow before and during maximal ADH stimulation. Base-line water flow was unaffected by bladder distension, but hormonally stimulated flow increased systematically as bladders became more distended. Distension had no effect on the frequency of ADH-induced intramembranous particle aggregates. By comparing the relationships between aggregate frequency and hormonally induced water permeability in distended and undistended bladders, we found that distension appeared to enhance ADH-stimulated water flow by decreasing the resistance of the series permeability barrier while the apparent water permeability associated with each single luminal membrane aggregate was unaffected. In that bladder distension caused tissue thinning, the series resistance limiting ADH-stimulated water flow appears to be accounted for by deformable barriers within the bladder tissue itself, probably unstirred layers of water.

Cytochalasin B inhibition of toad bladder apical membrane responses to ADH.

The possible role of actin microfilaments in antidiuretic hormone (ADH)-induced increases in apical membrane water permeability was investigated in studies that evaluate inhibition by cytochalasin B of both permeability and membrane structural responses in the toad urinary bladder. Experiments were carried out in the absence of a transepithelial osmotic gradient to eliminate possible flow-induced distortions of the response. Measurements of osmotic water permeability after a brief tissue fixation with glutaraldehyde show that cytochalasin B reduces the permeability response to ADH by approximately one-third. Freeze-fracture electron microscopy indicates that the intramembrane particle aggregates, previously found to correlate closely with ADH-induced permeability, are reduced by about the same extent (28%) under these conditions. However, the frequency of apical membrane fusion events was not affected by cytochalasin B treatment. These results suggest that cytochalasin B treatment in the absence of an osmotic gradient alters the ADH-induced permeability through an effect on apical membrane aggregate frequency.

Effects of potassium-free media on ADH action in toad urinary bladder.

We studied the effects of potassium-free media on processes related to the hydro-osmotic response of toad bladder to ADH (20 mU/ml). Exposure of bladders to potassium-free media did not affect base-line osmotic water flow, but it promptly attenuated the level of osmotic water permeability induced by ADH. Both the frequency of hormonally induced intra(luminal)membrane particle aggregates (presumed sites for transmembrane water flow) and the number of luminal membrane fusion events (associated with aggregate delivery from the cytoplasm) were also reduced. Potassium-free media had no measurable effect either on cytoplasmic microtubule integrity or on mean aggregate size. Potassium repletion reversed the inhibitory effect of potassium-free media on ADH-related osmotic water permeability. For bladders fully stimulated with ADH in the presence of potassium, subsequent bathing media depletion of potassium led to an inhibition of ADH-related water flow and to reductions in membrane fusion sites and aggregates. We confirmed that the inhibitory effect of potassium-free media on ADH-induced osmotic water permeability results from serosal bathing medium potassium depletion alone and occurs at a post-cyclic AMP site. In addition, we found that ADH-stimulated water permeability was attenuated in bathing media containing a low potassium concentration (0.5 mM). The data are consistent with the view that potassium-free media or media containing low levels of potassium inhibit ADH-enhanced osmotic water permeability in toad bladder by interfering with the process of or leading to membrane fusion required for the delivery of water-conducting structures to the luminal membrane. In addition, some of our results imply that aggregates may turn over during sustained ADH stimulation.

Aggregate-carrying membranes during ADH stimulation and washout in toad bladder.

Aggregates of intramembrane particles are found in cytoplasmic structures that we now term "aggrephores." Antidiuretic hormone (ADH) causes aggrephores to fuse with the luminal membrane. Aggregates subsequently become dispersed in the membrane and behave as sites for water flow. In the present studies we examined further the behavior of aggrephores. 1) Bladders stimulated for 10, 30, or 60 min in the absence of an osmotic gradient had two to three times more fusions as those stimulated in the presence of a gradient, indicating that aggrephore fusion frequency is influenced by water flow. 2) ADH stimulation did not change the projected luminal surface area of granular cells. Thus fused aggrephores remain fixed while aggregates move from the aggrephore to flat areas of luminal membrane. 3) Horseradish peroxidase, present in the mucosal baths of bladders stimulated with and then washed of ADH, was found in the lumen of cytoplasmic aggrephores; aggrephores therefore detach from the luminal membrane during washout. The same bladders had about twice as many multivesicular body-type lysosomes as unstimulated bladders, suggesting that, after detachment, aggrephores may join or become lysosomes. 4) Colchicine did not affect the rate of disappearance of fusion sites during washout, whereas cytochalasin B slowed it, suggesting that aggrephore detachment depends of microfilaments, but not microtubules.

Effect of osmotic gradient on ADH-induced intramembranous particle aggregates in toad bladder.

Paired toad urinary bladders were prepared without or with an osmotic gradient (175 mosM) across them, stimulated for 2.5 (n = 6), 5 (n = 6), 30 (n = 6) or 60 (n = 6) min with ADH (20 mU/ml), and studied by freeze-fracture electron microscopy. Water permeability at these times was assessed in additional bladders (n = 6 for each case) after tissue fixation according to the technique of Eggena. After both 60 and 30 min of ADH stimulation, the presence of a gradient compared with the absence of one was associated with fewer aggregates (242 +/- 35 vs. 382 +/- 14 x 235 micron-2 at 60 min, P less than 0.01; 279 +/- 36 vs. 470 +/- 51 x 235 micron-2 at 30 min, P less than 0.01) and lower water permeability (8.4 +/- 1.1 vs. 18.8 +/- 1.8 microgram x min-1 x cm-1 x mosM-1 at 60 min, P less than 0.005; 9.2 +/- 1.0 vs. 22.0 +/- 2.1 microgram x min-1 x cm-2 x mosM-1 at 30 min, P less than 0.001). In addition, with a gradient both maximum water permeability and maximum aggregate frequency were reached nearly together; a similar correspondence occurred without a gradient. We conclude that in the presence of an osmotic gradient both the ADH-associated aggregates and the water permeability response to ADH are prevented from reaching the higher levels observed in bladders not exposed to a gradient.

Time-dependent attenuation of water flow in antidiuretic hormone-treated toad bladder.

Toad urinary bladders were subjected to sequential 30-min stimulation with antidiuretic hormone (ADH) followed by 30-min hormone washout over 4 h in the absence of a transmural osmotic gradient. Immediately thereafter, during test stimulation with hormone in the presence of a transmural gradient, transbladder water flow was profoundly inhibited, but intra(luminal)membrane particle aggregates, presumed markers of luminal membrane water permeability, were as numerous as in fully responsive controls. The protocol followed was designed to eliminate any distorting effect of prior water flow on cytoplasmic organization and to equalize, for both experimental and control tissues, the time of aggregate presence in the luminal membrane during final test stimulation. On the assumption that cytoplasmically stored and/or newly synthesized aggregates would be unaffected by the protocol followed, these observations appear to be consistent with the view that bladder refractoriness to prolonged ADH treatment may involve regulation of tissue water permeability at a resistance distal to the luminal membrane.

Regulation of water permeability in toad urinary bladder at two barriers.

The effects of prostaglandin synthesis inhibition by naproxen were studied in toad bladder. Luminal membrane water permeability was evaluated both by the frequency of intramembranous particle aggregates in granular cell luminal membrane and by direct assessment of the rate of change of cell volume during perfusion of an anisosmotic solution. Total tissue water permeability was assessed by transbladder osmotic water flow. Inhibition of prostaglandin synthesis caused luminal membrane water permeability to increase much more than expected from tissue permeability measurements. The addition of a very low dose of antidiuretic hormone (ADH) (0.125 mU/ml) during prostaglandin synthesis inhibition increased luminal membrane water permeability to the same level as maximal stimulation with ADH, while tissue water permeability failed to increase proportionately. The results imply the presence of a regulatable barrier to water movement across toad bladder that is distal to the luminal membrane and subject to control by either prostaglandins or ADH.

Possible roles for microtubules and microfilaments in ADH action on toad urinary bladder.

Intramembranous particle aggregates in the luminal membrane of toad bladder granular cells after vasopressin stimulation have been found to correlate closely and specifically with induced alterations of water permeability. Roles for microtubules and microfilaments in mediating the latter response have been proposed on the basis of studies involving colchicine and cytochalasin B, respectively. In the present investigation the effects of these agents on both initiating and sustaining vasopressin-induced osmotic water flow and the particle aggregation phenomenon were studied. The results indicate that during initiation the aggregation and water flow responses to vasopressin are each colchicine- and cytochalasin B-sensitive and that these sensitivities can be wholly additive. However, after full vasopressin stimulation is established, the same responses demonstrate sensitivity only to cytochalasin B, not to colchicine. The findings, therefore, suggest that microtubules and microfilaments may be independently necessary for the initiation of the aggregation and water flow responses to vasopressin, and that microfilaments, but not microtubules, are required for their maintenance.