Vasopressin: induced structural change in toad bladder luminal membrane. 1975.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
W A Kachadorian
Publications and source records attributed to W A Kachadorian.
Ca2+ dependence of alpha-adrenergic effects on the contractile properties and Ca2+ homeostasis of cardiac myocytes.
alpha-Adrenergic stimulation is known to enhance myocardial contractility. Adult rat left ventricular myocytes bathed in 1 mM [Ca2+] (Ca0) and electrically stimulated at 0.2 Hz responded to alpha-adrenergic stimulation with 50 microM phenylephrine and 1 microM propranolol with an increase in twitch amplitude to 177.1 +/- 25.6% of control (mean +/- SEM). In contrast, when cell Ca2+ loading was increased by bathing cells in 5 mM Ca0, alpha-adrenergic stimulation decreased twitch amplitude to 68.6 +/- 8.2% of control. Time-averaged cytosolic [Ca2+] of cells in 1.0 mM Ca0 is enhanced via an increase in the frequency of electrical stimulation. When myocytes were stimulated at 2 Hz in 1 mM Ca0, alpha-adrenergic stimulation did not increase twitch amplitude (103.8 +/- 12.4% of control). In myocytes loaded with the Ca2+ probe into-1, alpha-adrenergic effects during stimulation at 0.2 Hz (an increase in twitch amplitude in 1 mM Ca0 and a decrease in twitch amplitude in 5 mM Ca0) were associated with similar changes in the indo-1 transient. In 5 mM Ca0, spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR) occurred in the diastolic interval between twitches (2.9 +/- 1.4 spontaneous SR Ca2+ oscillations/min; n = 7); alpha-adrenergic stimulation abolished these oscillations in six of seven cells. Thus, an increase in the frequency of spontaneous diastolic SR Ca2+ release (i.e., Ca2+ overload) is not the mechanism for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. In experiments with unstimulated myocytes, we determined whether the effect of alpha-adrenergic stimulation on cell Ca2+ homeostasis and oscillatory SR Ca2+ release observed in 5 mM Ca0 occurs only during electrical stimulation, when voltage-dependent currents are operative, or also at rest. Unstimulated rat ventricular myocytes in 5 mM Cao exhibit oscillatory SR Ca2+ release; alpha-adrenergic stimulation decreased the frequency of these oscillations to 53.9 +/- 8.9% of control, and this effect was blocked by 1 microM prazosin. In unstimulated indo-1-loaded myocytes alpha-adrenergic stimulation decreased the resting indo-1 fluorescence ratio in 5 mM Ca0, whereas it had no effect in 1 mM Ca0. Additional experiments were aimed at defining a role for Ca(2+)-activated, phospholipid-dependent protein kinase C (PKC) for the negative inotropic effect of alpha-adrenergic stimulation in 5 mM Ca0. Short-term preexposure to 0.1 microM 4 beta-phrobol 12-myristate 13-acetate (PMA) has been shown to maximally activate PKC.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Effects of serosal hypertonicity on water permeability in toad urinary bladder.
We studied in toad urinary bladder the effects of serosal hypertonicity on tissue water permeability, granular cell luminal membrane water permeability, and granular cell luminal membrane particle aggregates and compared them with effects of antidiuretic hormone (ADH). In tissues challenged by a hypertonic (447 mosmol/kgH2O) serosal bath, luminal membrane aggregates were structurally similar to those caused by ADH. The tissue water permeability increase induced by serosal hypertonicity was much less than that caused by a maximally stimulating concentration of ADH on tissue in isotonic serosal baths with approximately the same transmural gradient. The difference is explained not only by a reduced incidence of luminal membrane aggregates but also by an increased resistance to water movement at a postluminal membrane site. Measurements of luminal membrane water permeability showed a close correlation with luminal membrane aggregate frequency, indicating that the calculated permeability of an individual aggregate was a constant. Thus the relation of luminal membrane aggregates to tissue osmotic permeability is modified by serosal hypertonicity. Morphological examination of these tissues suggested that luminal membrane aggregates may be less stable in the absence of hormone. This was evident by the proportionally greater number of structures interpreted as aggregates captured in the process of disassembly ("patches"). Membrane depressions containing intramembrane particles ("craters") were also observed. They corresponded in terms of frequency and size to coated pits as seen in thin sections.
Animals↗
Variability of cellular responsiveness to ADH stimulation in toad urinary bladder.
The hydrosmotic response of toad bladder to antidiuretic hormone (ADH) is quantitatively linked to the induced fusion of aggrephores with, and the appearance of aggregates of tightly packed intramembrane particles in the luminal membrane of granular cells. We used these morphological indexes of hormonally induced cell activation 1) to assess the variability of individual cell responsiveness to a maximally stimulating concentration of ADH and 2) to compare cell response patterns in paired tissues where the extent of whole tissue stimulation, as evidenced by transtissue water flow, was either maximal or submaximal. The results indicate that individual cell responsiveness within the same tissue to standardized maximal ADH treatment varies between two- and sevenfold, depending on the morphological endpoint measured. Furthermore, based on skewness in endpoint distribution, this variability appears to reflect inherent heterogeneity of granular cell reactivity to hormone. In relation to proportional tissue responses elicited by different stimulating concentrations of ADH, our observations of luminal membrane aggregate incidence suggest that the responding cells, whatever their sensitivity, participate in a graded, rather than "all-or-none," "on-off" manner.
Animals↗
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.
Animals↗
Water permeability and particle aggregates in ADH-, cAMP-, and forskolin-treated toad bladder.
Osmotic water flow was used to evaluate total tissue water permeability (Ptissue), and luminal membrane particle aggregates, presumed sites for transmembrane water movement, were quantified to assess luminal membrane water permeability, in bladders treated with maximally stimulating concentrations of antidiuretic hormone (ADH), adenosine 3',5'-cyclic monophosphate (cAMP), and forskolin. Aggregates were as numerous and occupied the same fractional area of the luminal membrane in response to cAMP treatment (10 mM) as treatment with ADH (20 mU/ml). Ptissue in cAMP-treated tissues, however, was only half of that induced by ADH (P less than 0.001). A similar disparity in the relationship between aggregates and Ptissue occurred for additional bladders treated with 50 microM forskolin, which is known to increase endogenous cAMP to levels much greater than caused by maximally stimulating concentrations of ADH. Although Ptissue achieved with forskolin was the same in paired bladders treated with ADH, aggregates were far more numerous (P less than 0.05) and occupied much more membrane area (P less than 0.05) with forskolin. These observations are consistent with the view that aggregate appearance in the luminal membrane is a function of intracellular cAMP. The finding that the hydrosmotic response of toad bladder to both cAMP and forskolin compared with ADH stimulation is reduced relative to measured changes in luminal membrane aggregates suggests that the effect of ADH in altering water permeability involves additional regulation via a non-cAMP-mediated mechanism. This latter event would appear to be by an effect of ADH on the permeability of a resistance at a postluminal membrane site and/or possibly on the permeability of aggregates in the luminal membrane.
Animals↗
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.
Animals↗
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)
Animals↗
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.
Animals↗
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.
Animals↗
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.
Animals↗
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.
Animals↗
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.
Animals↗
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.
Animals↗
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.
Animals↗
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.
Animals↗
Evidence for calmodulin dependence of water permeability in toad urinary bladder.
Explore the source record for details and available documents.
Animals↗
Relation of ADH effects to altered membrane fluidity in toad urinary bladder.
Membrane fluidity, urea permeability, and osmotic water permeability in toad urinary bladder are regularly enhanced by antidiuretic hormone (ADH). In addition, organized intramembranous particle aggregates, which correlate specifically with hormonally stimulated water permeability, are found in granular cell luminal membranes consequent to ADH stimulation. In this investigation ADH-stimulated changes in urea and osmotic water permeability and luminal membrane aggregates at room temperature (24.8 +/- 0.4 degrees C) and in the cold 10.6 +/- 0.2 degrees) were compared with corresponding changes in membrane fluidity, as assessed by n-butyramide permeability. Although a critical level of membrane fluidity is undoubtedly required, the occurrence of aggregates in the luminal membrane is independent of an accompanying hormonally induced change of membrane fluidity. ADH-stimulated osmotic water permeability in toad bladder is also independent of the coincident change in membrane fluidity, and as a process almost certainly involves membrane channels, not a solubility-diffusion process through membrane lipids. For ADH-stimulated transbladder urea movement, channels seem to be involved as well, and the change induced in membrane fluidity by ADH could be an underlying factor in their formation.
Animals↗