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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Vasopressin and collecting duct intramembranous particle clusters: a dose-response relationship.

Vasopressin increases the permeability of collecting ducts to water. Administration of this hormone is also associated with an increase in intramembranous particle clusters in rat collecting duct luminal membrane (CDLM) as revealed by freeze-fracture electron microscopy. To determine whether this morphologic alteration of CDLM is quantitatively related to the dose of vasopressin, anesthetized Brattleboro homozygous rats were given the hormone at different doses.

Effect of hydrazine on transport on toad urinary bladder.

Vasopressin stimulates osmotic water flow and urea permeability in the toad urinary bladder via separate cAMP-responsive mechanisms. Hydrazine (10--20 MM), added to the bladder's serosal bath, reversibly enhanced the effect of both low and saturating levels of vasopressin on osmotic water flow, without increasing urea permeability. A small increase in basal water flow was also observed.

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.

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.

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.