Effects of colchicine and cytochalasin B on hypertonicity-induced changes in toad urinary bladder

Summary Coincident with an increase in the water permeability of toad urinary bladder induced by serosal hypertonicity, a transformation of the ridge-like surface structures of the granular cells into individual microvillous structures occurs. This study was initiated to establish whether the transformation is mediated by the cytoskeletal network and, thus, can be prevented by disruption of microtubulemicrofilament function with colchicine or cytochalasin B (CB). Scanning electron microscopy revealed the characteristic branching ridges on granular cells of control bladder incubated with colchicine or CB. In contrast, transformation of ridges to discrete microvilli was observed in experimental bladders exposed to serosal hypertonicity alone or in combination with either colchicine or CB. These results suggest that the mechanism underlying hypertonicity-induced surface changes which are associated with increased water permeability does not involve either microtubules or microfilaments.     

Cellular changes in the toad urinary bladder in response to metabolic acidosis

Summary The urinary bladder ofBufo marinus excretes H⁺ and NH ₄ ⁺ , and the H⁺ excretion is increased when the animal is placed in metabolic acidosis. The mitochondriarich (MR) cells mediate the H⁺ excretion by the bladder. The purpose of this study was to determine if there is a change in MR cells of the bladder during metabolic acidosis. Bladders from normal toads and from toads that had been placed in metabolic acidosis were used. The bladders were mounted between plastic chambers and H⁺ excretion measured. The bladder was then fixed and prepared for scanning (SEM) and transmission (TEM) electron micrograph studies. SEM's at low magnification were used to count the various cell types and the TEM's were used to confirm the different cell types. Fields were randomly selected and a total of 2500 cells counted in each group. The bladders from toads in metabolic acidosis had a consistently higher ratio of MR cells to granular cells than did the normal bladders. These results indicate that during metabolic acidosis there is an increased number of MR cells in the bladder, and this increases the bladder's capacity to excrete H⁺.     

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 NH₄Cl induced metabolic acidosis, respiratory acidosis, and a group in NaHCO₃ 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.     

Evaluation by capacitance measurements of antidiuretic hormone induced membrane area changes in toad bladder

A technique for estimating effective transepithelial capacitance in vitro was used to investigate changes in epithelial cell membrane area in response to antidiuretic hormone (ADH) exposure in toad bladder. The results indicate that transepithelial capacitance increases by about 30% within 30 min after serosal ADH addition and decreases with ADH removal. This capacitance change is not blocked by amiloride and occurs whether or not there is a transepithelial osmotic gradient. It is blocked by methohexital, a drug which specifically inhibits the hydro-osmotic response of toad bladder to ADH. We conclude that the hydro-osmotic response of toad bladder to ADH is accompanied by addition of membrane to the plasmalemma of epithelial cells. This new membrane may contain channels that are permeable to water. Stimulation of Na⁺ transport by ADH is not related to membrane area changes, but appears to reflect activation of Na⁺ channels already present in the cell membrane before ADH challenge.     

Kinetics of cyclic AMP in toad bladder

Tissue distributions of cyclic [³H]AMP and [¹⁴C]inulin in toad bladder were determined and their kinetics analyzed. We found that both the epithelial and the other cells of the toad bladder handcle cyclic AMP similarly. Moreover, we found that the distribution of cyclic AMP did not differ from that of inulin, an extracellular marker. Kinetic analysis suggests that the rate of coefficient of cyclic AMP metabolism is much larger than the exchange rate coefficient, which explains why distribution of both cyclic AMP and inulin are similar.     

Stretch-induced excitation and action potential changes of single cardiac cells

Mechanoelectric coupling (MEC) has been studied extensively in the heart at the tissue and organ levels, but to only a limited extent in single cells because of the technical challenges. New results are presented in which MEC was studied in 57 single frog ventricular myocytes that were held on both ends by glass holding pipettes. Axial stretch was applied either by displacement of the pipettes, or by a glass fiber around which the cell was wrapped, that was displaced in a pulsatile or sinusoidal fashion. Electrical activity of the cell was monitored either by active contraction, by intracellular action potentials, or by focal extracellular potentials. Of more than 350 stretches applied to 57 cells with amplitudes ranging from 3% to 35%, only 4 cases of mechanically induced stimulation were observed. In 252 stretches applied to 32 cells in which action potential duration (APD) was measured, no change >20% was observed, except in 3 cells in which APD increased by >100%, and in 2 cells with extended triggered activity. Thus, in contrast to studies in intact tissue, single frog ventricular myocytes are generally insensitive to direct axial stretch. However, robust mechanosensitive responses were observed in 7 of 57 (12%) cells. The results of other single cell studies are reviewed, and the significance of differences in tissue-level and single cell results is discussed.     

Ouabain-dependent potassium-potassium exchange in the toad bladder

Summary Recent results from this laboratory have indicated the existence of two potassium compartments in the isolated toad bladder. Only one of these, containing less than 10% of total intracellular potassium, appears to be related to the sodium transport system, since potassium influx at the serosal border of this compartment is coupled to the sodium efflux which occurs there. Ouabain, which specifically inhibits serosal sodium exit, has no effect on potassium fluxes and compartment sizes in bladders mounted in normal (2.5mm K) Ringer's solution. However, in the presence of this inhibitor, removal of serosal potassium results in a significant decrease in the rate coefficient for potassium efflux into the serosal medium, while an increase in serosal potassium results in a significant rise in this parameter, which appears to saturate at approximately 5mm K. This sensitivity to serosal potassium is seen neither in the absence of ouabain nor when the sodium pump is inactivated by removal of sodium from the mucosal medium. Furosemide, which also inhibits the sodium transport system, both inhibits potassium transport parameters in normal Ringer's and abolishes the potassium-sensitive potassium efflux seen in the presence of ouabain. Thus, the Na–K pump appears to operate as a K–K exchanger when the sodium system is inhibited by ouabain; this K–K exchange mechanism is inhibited by furosemide. One explanation for these results is that ouabain effects an alteration in the affinities of the transport system for sodium and potassium.     

Microfilament-rich cells in the toad bladder epithelium

Summary Basal cells of the bladder epithelium ofBufo marinus have been found heterogenous and consist of microfilament-rich cells (MFR-cell) and undifferentiated cells (Un-cell). The MFR-cell, which represents approximately 20% of the epithelial cell population, lies between the epithelial layer lining the urinary space and the basement membrane; it extends under several epithelial cells by processes of varying widths and lengths which contact, via desmosomes, other MFR-cells, as well as cells in the superficial layer, i.e., granular and mitochondria-rich cells. The cytoplasm of MFR-cell is filled with intermediate filaments arranged in bundles which run parallel to the plane of the epithelium and no dense granules, typical of granular cells, have been detected. Strong immunofluorescence for actin is associated with cells which occupy the same basal position as MFR-cells. Undifferentiated cells have no contact via desmosomes with adjacent cells and their cytoplasm is filled with free ribosomes; they lack bundles of intermediate filaments and posses no specialized organelles.After a 4-hr pulse of³H-thymidine, 1.5% of epithelial cells incorporate thymidine into nuclear DNA, out of which 3/4 are basally 1/4 are apically located. Identification of cell types by electron microscopy reveals that 10% of undifferentiated basal cells are labeled, whereas less than 0.1% of granular cells and no MFR-cells incorporate³H-thymidine into DNA. When dissociated from the epithelium and separated by isopycnic centrifugation, MFR-cells possess a mean buoyant density of approximately 1.025, cosediment with mitochondria-rich cells and exhibit a strong immunofluorescence for actin. The function of MFR-cells remains unknown; however, they may play a role in cell coupling and responses to hormonal and physical factors.