Regulation of epithelial Na+ permeability by protein kinase C is tissue specific.

Protein kinase C (PKC) is a major regulator of a broad range of cellular functions. Activation of PKC has been reported to stimulate Na+ transport across frog skin epithelium by increasing the apical Na+ permeability. This positive natriferic response has not been observed with other epithelial preparations, and could reflect the specific experimental conditions of different laboratories, or species or organ specificity of the response to PKC. In the present study, measurements were conducted with skins and urinary bladders from the same animals of two different species. The PKC activator TPA uniformly increased the transepithelial Na+ transport (measured as amiloride-sensitive short-circuit current, ISC, across skins from Rana temporaria and Bufo marinus, and inhibited ISC across bladders from the same animals. Inhibitors of PKC (staurosporine, H-7 and chelerythrine) partially blocked the TPA-induced stimulation of ISC across frog skin. The specificity of the PKC response by amphibian skin could have reflected an induction of moulting, similar to that observed with aldosterone. However, light micrographs of paired areas of frog skin revealed no evidence of the putative moulting. Separation of stratum corneum from the underlying stratum granulosum could be detected following application of aldosterone. We conclude that the effect of PKC on epithelial Na+ channels is organ, and not species specific. The stimulation of Na+ permeability in amphibian skin does not arise from sloughing of the stratum corneum. These observations are consistent with the hypothesis that the natriferic action arises from the calcium-independent isozyme of PKC previously detected in frog skin.

Selective stimulation of epithelial cells in colonic crypts: relation to active chloride secretion.

Stimulation of Cl secretion by prostaglandin E2 (PGE2) was measured as the short-circuit current (Isc) across isolated epithelium of the rabbit distal colon. Cellular morphology of columnar and goblet cells during secretion was monitored using light and electron microscopy. Stimulation by PGE2 altered epithelial cell morphology only by a reduction of vacuolar space in the apical pole of crypt columnar cells, consistent with release of vacuole contents. Imaging of isolated crypts using differential interference microscopy confirmed the release of material from columnar cells during the onset of secretion. Inhibition of Cl secretion with the loop diuretic bumetanide did not block vacuole release. The actin filament-disrupting agent, cytochalasin, reduced the PGE2-stimulated Isc by 40% and blocked emptying of the vacuolar space. These electrical and morphological results indicate that the process of active ion secretion is associated with release of the macromolecular contents from apical vacuoles through a mechanism involving the cytoskeleton. In addition, this relationship supports the concept that vacuolated columnar cells of the crypts of Lieberkühn are the cell type that secretes Cl in response to PGE2.

Sulfate transport in apical membrane vesicles isolated from tracheal epithelium.

Sulfate uptake in apical membrane vesicles isolated from bovine tracheal epithelium is shown to occur into an osmotically sensitive intravesicular space, via a carrier-mediated system. This conclusion is based on three lines of evidence: 1) saturation kinetics; 2) substrate specificity; and 3) inhibition by the anion transport inhibitors SITS and DIDS. The affinity of the transport system is highest in low ionic strength media (apparent Km = 0.13 mM) and decreases in the presence of gluconate (apparent Km = 0.68 mM). Chloride appears to cis-inhibit sulfate uptake and to trans-stimulate sulfate efflux. Cis-inhibition and trans-stimulation studies with a variety of anions indicate that this exchange system may be shared by HCO3-, S2O3(2-), SeO4(2-), and MoO4(2-) but not by H2PO4- or HAsO4(2-). Studies indicate that protons may play two distinct roles in sulfate transport in this system. 1) Their possible modifier role is suggested by the fact that protons affect SO2-4 transport in an uncompetitive manner. 2) The possibility that the proton gradient may act as an energy source for a secondary active transport is indicated by the fact that the imposition of a proton gradient stimulates a transient movement of sulfate in to the tracheal apical membrane vesicle, against its concentration gradient, causing an "overshoot" phenomenon. Our studies show that the carrier-mediated system can function in the absence of chloride. The overshoot observed in the presence of a proton gradient (OH- gradient) indicates that under those conditions the mechanism of transport may be a SO4(2-)-OH- exchange. The fact that chloride cis-inhibits and trans-stimulates SO4(2-) transport indicates that SO2-4 uptake may also occur via a SO4(2-)-Cl- exchange. Studies carried out so far do not enable us to conclude unequivocally whether the tracheal apical membrane system displays two distinct carrier activities (SO4(2-)-Cl-; SO4(2-)-OH-) or one anion exchanger, which like the erythrocyte anion exchanger, may interact with SO4(2-), Cl-, and H+. The fact that the anion transport inhibitors DIDS and SITS inhibit SO4(2-) transport in the presence or absence of chloride suggests that the latter possibility may be the case.

Cell volume regulation in rabbit proximal straight tubule perfused in vitro.

Volume regulation in the perfused proximal nephron of the rabbit was examined quantitatively with a computer-based method for estimating cell volume from differential interference-contrast microscopic images of isolated nephron segments. Following a hyperosmotic challenge (290-390 mosmol), the cells shrank as simple osmometers without a subsequent regulatory volume increase. Conversely, cell swelling induced by a hyposmotic challenge (290-190 mosmol) was completely reversed with a triphasic time course in which a rapid (less than 2 min) initial volume decline was followed by secondary swelling and shrinking phases. A similar regulatory volume decrease was observed following isosmotic cell swelling that was induced by exposure to 290 mosmol, urea-containing solutions. In addition, the cells partially reversed isosmotic swelling that was induced by the luminal replacement of a relatively impermeant cation (i.e., choline) with Na+ and a concomitant increase in luminal solute entry. Our results support two conclusions. First, there exist quantitative differences between the volume regulatory behaviors of perfused and nonperfused proximal tubules, the latter of which exhibit an incomplete and monotonic reversal of hyposmotic cell swelling (M. Dellasega and J. Grantham, Am. J. Physiol. 224: 1288-1294, 1973). Second, the primary physiological role of cell volume regulation in the proximal nephron may be to minimize isosmotic cell swelling associated with acute imbalances in the rates of cell solute entry and exit.

Regulatory volume decrease in perfused proximal nephron: evidence for a dumping of cell K+.

We utilized the microscopic and morphometric procedures described in the preceding paper to examine the role of a swelling-activated dumping of K-salt in the reversal of hyposmotic cell swelling in the perfused proximal nephron. The rate of the regulatory volume decrease that follows cell swelling in dilute solutions was reduced by two maneuvers that attenuate the K+ chemical potential difference across the basolateral membrane; inhibiting the Na+-K+ pump (e.g., with ouabain) and raising the peritubular K+ concentration. The rate of the regulatory volume decrease was also inhibited by peritubular quinine, which blocks K channels and volume regulation for a number of mammalian cells. Additionally, exposure to hyposmotic solutions resulted in a sustained and quinine-sensitive increase in the apparent permeability of the basolateral membrane to K+ salt, which was monitored qualitatively as the rate of cell volume change that was induced by a perturbation in the peritubular K+ concentration. The simplest interpretation of these results is that the reversal of hyposmotic cell swelling in the proximal nephron is referable at least in part to a swelling-activated loss of K-salt and water from the cells.

Intracellular solute gradients during osmotic water flow: an electron-microprobe analysis.

In an attempt to quantify possible intracellular water activity gradients during ADH-induced osmotic water flow, we employed energy dispersive X-ray microanalysis to thin, freeze-dried cryosections obtained from fresh, shock-frozen tissue of the toad urinary bladder. The sum of all detectable small ions (Na + K + Cl) in the cellular water space was taken as an index of the intracellular osmolarity. Presuming that all ions are osmotically active, they comprise about 90% of the cellular solutes. When the cells were exposed to dilute serosal medium, the reduction in the sum of the ions agreed well with the expected reduction in osmolarity. After inducing water flow by addition of ADH and dilution of the mucosal medium, all epithelial cells showed a fall in osmolarity. The change was more pronounced in granular cells than in basal or mitochondria-rich cells, consistent with the notion that granular cells represent the main transport pathway. Most significantly, intracellular osmolarity gradients, largely caused by an uneven distribution of K and Na, were detectable in granular cells. The gradients were not observed after ADH or mucosal dilution alone, or when the direction of transepithelial water flow was reversed. We conclude from these results that there is a significant cytoplasmic resistance to water flow which may lead to intracellular gradients of water activity. Concentration gradients of diffusible cations can be explained by a flow-induced Donnan-type distribution of fixed negative charges. With regard to transepithelial Na transport, the data suggest that ADH stimulates transport by increasing the Na permeability of the apical membranes of granular cells specifically.

Analysis of structural changes during hypotonic swelling in Ehrlich ascites tumor cells.

Studies were undertaken to quantify structural changes associated with swelling of Ehrlich ascites tumor cells in hypotonic medium. Cells transferred from isotonic (294 mosmol/kg H2O) to hypotonic (98 mosmol/kg H2O) medium swelled rapidly. Subsequently, approximately 40% of the water initially gained was lost, a phenomenon referred to as volume-regulatory decrease (VRD). During the initial rapid cell swelling, blister-like protrusions or blebs were formed on the cell surface. These blebs were examined by routine light microscopy, differential interference-contrast (DIC) microscopy, and scanning and transmission electron microscopy. Microscopic observations and the distribution of ATPase antibodies indicated that the blebs were formed from plasma membrane. During VRD, the blebs coalesced to form a smooth but expanded membrane surface that appeared to be separated from the original cytoplasm by a layer of less dense ground substance. Computer-assisted morphometry from digitized DIC images of the initial swelling phase indicated that all of the volume gained was sequestered in the blebs. We suggest that bleb formation may allow increases in cell volume without disruption of cytoplasmic organization and may be a protective response to a variety of stressful stimuli. The subsequent VRD is accompanied by reduction of this expanded compartment.

Microscopic investigation of structure and function in living epithelial tissues.

In this paper we shall illustrate the utility of direct microscopic methods for studying living epithelia. Beginning with an exposition on the available strategies for visualization of unstained biological materials, the rationale that leads to the choice of differential interference-contrast optics for examination of epithelia is illustrated. Findings from toad urinary bladder, Necturus gallbladder, and rabbit cortical collecting tubule are reviewed. Emphasis on renal structures is provided with a report on work in progress on proximal tubule volume regulation and on structural examination of the isolated perfused macula densa. Conclusions are drawn with respect to the advantages and shortcomings of discussed optical methods and with respect to the choice of model epithelia.

Functional analysis of tight junction organization.

The functional basis of tight junction design has been examined from the point of view that this rate-limiting barrier to paracellular transport is a multicompartment system. Review of the osmotic sensitivity of these structures points to the need for this sort of analysis for meaningful correlation of structure and function under a range of conditions. A similar conclusion is drawn with respect to results from voltage-clamping protocols where reversal of spontaneous transmural potential difference elicits parallel changes in both structure and function in much the same way as does reversal of naturally occurring osmotic gradients. In each case, it becomes necessary to regard the junction as a functionally polarized structure to account for observations of its rectifying properties. Lastly, the details of experimentally-induced junction deformation are examined in light of current theories of its organization; arguments are presented in favor of the view that the primary components of intramembranous organization (as viewed with freeze-fracture techniques) are lipidic rather than proteinaceous.

Myocardial cell volume and coronary resistance during diminished coronary perfusion.

The present study was undertaken to determine whether diminished coronary blood flow without abrupt reperfusion results in myocardial cell swelling and, if so, whether the increment in tissue water is related to an increase in coronary vascular resistance. In 19 anesthetized open-chest dogs on right heart bypass with controlled coronary perfusion a decrease in coronary flow resulted in an increase in intracellular water and a progressive increase in coronary resistance. In 15 additional dogs, 180 min of ischemia produced by partially occluding the circumflex coronary artery resulted in significant increases in myocardial water content and progressive decreases in regional myocardial blood flow (microsphere technique). A significant correlation was found between the progressive decreases in myocardial flow and the increases in myocardial water (r = -0.82, P less than 0.001). In five experiments, hypertonic mannitol prevented water accumulation and progressive decreases in blood flow in the ischemic tissue. Thus myocardial ischemia produced by a decrease in antegrade coronary perfusion results in myocardial cell swelling and an associated progressive perfusion deficit.

Morphologic response of the rabbit cortical collecting tubule to peritubular hypotonicity: quantitative examination with differential interference contrast microscopy.

The isolated and perfused cortical collecting tubule of the rabbit was examined by differential interference contrast microscopy in order to characterize the morphologic response of this nephron segment to peritubular hypotonicity. Computer-assisted, morphometric procedures were developed to obtain measurements of cell volume and lateral intercellular space geometry from interference contrast images of perfused nephron segments. Following dilution of the bath from 290 to 190 mOsm in the absence of antidiuretic hormone (T = 25 degrees C), the cells swelled rapidly to a new steady-state volume which was maintained for at least 20 to 30 min and which was about 90% of that predicted for ideal osmometric behavior. The increase in cell volume was accomplished entirely by bulging of the cells into the lumen; lateral space width and outside tubule diameter were unaffected by peritubular hypotonicity. In addition, the swelling of the cells was associated with an apparent swelling of intracellular organelles, e.g., nuclei and mitochondria. Our results indicate that cells of the mammalian collecting tubule swell without the capacity for significant volume regulation at 25 degrees C and without the cytoplasmic vacuolation and dilation of the lateral intercellular spaces observed following the onset of antidiuretic hormone-dependent volume reabsorption (E. Ganote , J. Grantham , H. Moses, M. Burg and J. Orloff , J. Cell Biol. 36:355, 1968).

Quantitative analysis of the structural events associated with antidiuretic hormone-induced volume reabsorption in the rabbit cortical collecting tubule.

We quantitatively examined the influence of antidiuretic hormone (ADH)-dependent volume reabsorption on the morphology of the rabbit cortical collecting tubule. Estimates of cell volume and the geometry of the lateral intercellular spaces were extracted from differential interference contrast images of perfused nephron segments using the morphometric procedures described in the preceding paper (K.L. Kirk , D.R. DiBona and J.A. Schafer, J. Membrane Biol. 79:53-64, 1984). The results indicate that ADH addition in the presence, but not absence, of a lumen-to-bath osmotic gradient (130 to 290 mOsm) stimulated transepithelial volume flow and simultaneously increased the volumes of both the cells (+28%) and the lateral intercellular spaces (+78%). In addition, the formation of cytoplasmic vacuoles could be observed during the latter stages of the swelling response, and vacuole formation continued well after new steady-state values for transepithelial water flow and cell volume had been reached. Two main conclusions can be drawn from these results. First, the cytoplasmic vacuoles comprise a slowly filling compartment that lies in parallel to the transepithelial pathway for ADH-stimulated volume reabsorption. Second, from the magnitude of the cell volume increase, we estimate that the hydraulic conductivities of the opposing cell membranes are nearly equal during maximal ADH stimulation.

Bioenergetics of Na+ transport across frog skin: chemical and electrical measurements.

Enzymatically prepared split frog skins consisted purely of epithelial cells. Electrical parameters and the cell contents of ATP, ADP, phosphocreatine (PCr), creatine, inorganic phosphate, protein, and water were measured in skins maintained at room temperature. Studies were conducted under base-line conditions, 15 and 60 min after adding vasopressin, and 30 min after adding amiloride. Intracellular ionic activities and concentrations were obtained from previous results. The data demonstrated that 1) the base-line concentration ratio of PCr/ATP was 0.53 +/- 0.03; 2) the average molar free energy of hydrolysis of intracellular ATP was approximately 15.0 kcal X mol-1 under control conditions, changing by less than or equal to 3% with changes in transport; and 3) the free energy of extruding 3 mol of Na+ and accumulating 2 mol of K+ was approximately 9.8 kcal X mol-1 under base-line conditions; the difference between the molar free energies of ATP hydrolysis and of transport work remained large, despite large changes in transepithelial transport. The simplest conclusion is that the Na+ pump of frog skin operates far from equilibrium.

Cytoplasmic involvement in ADH-mediated osmosis across toad urinary bladder.

Several lines of investigation have suggested that antidiuretic hormone (ADH) may have direct effects on the cytoskeletal organization of granular epithelial cells in the toad urinary bladder. To some extent, these effects are in concert with the well-established action of ADH on the hydraulic permeability of the mucosal plasma membrane, but it appears that other conformational adjustments (largely cytoplasmic) may be of comparable importance. The thrust of this review is that the hormone brings about a general restructuring of the granular cells so that the epithelium as a whole may function efficiently as an osmotic pathway. Details of cytoskeletal changes are far from clear as yet, but interference with or modulation of these particular effects infer that cytoplasmic organization is the seat of feedback control of osmotic flow rate, the basis for viability in the presence of dramatic cytosolic dilution and a major factor in the observed disparity in osmotic and diffusional permeability coefficients. In the interest of stimulating new thoughts and experiments in this area, a number of preliminary findings have been freely cited.

Microscopic methods for analysis of function in isolated renal tubules.

Using differential interference-contrast and fluorescence microscopy we have performed longitudinal observations of living, isolated and perfused nephron segments. With interference-contrast images we have made morphometric measurements (epithelial volume) and have compared those data to simultaneously measured physiologic parameters (delta psi T, Jv). Fluorescence images have allowed us to characterize qualitatively the distribution of a dye probe (acridine orange) within the epithelium and to follow changes in distribution in response to experimental maneuvers. In addition, preliminary results indicate that it should be possible to obtain quantitative information on the intracellular distribution of fluorescent probes, such as acridine orange, which have strong emission intensities. This should aid in the characterization of the cellular profiles of physiologic parameters such as pH or membrane potential. Collectively, these optical and physiological techniques should allow a meaningful analysis of structure and function in renal epithelia where a broad spectrum of transport processes can be examined.

Partial characterization of insulin receptors from rat myocytes.

Isolated myocytes were prepared from the adult rat heart and characterized for viability. The myocytes were exposed to 125I-insulin, and the 125I-insulin-receptor complex was extracted with 1% Triton X-102 and then applied to a DEAE-Sephacel column. When the chromatography were applied to a Sepharose CL-6B column, a 140,000-dalton complex with high specific radioactivity was found. Alternatively, when myocyte insulin receptors were first extracted with 1% Triton X-102 without prior exposure to 125I-insulin and then applied to a DEAE-Sephacel column, three peak protein fractions were obtained. They were treated separately with 125I-insulin and the 125I-insulin-protein complexes were covalently cross-linked with disuccinimidyl suberate. The cross-linked samples were applied to a Sepharose CL-6B column and the radioactive protein fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first and second peak samples from the ion exchange chromatography yielded complexes of low specific radioactivity, which appear to be formed by nonspecific random binding of 125I-insulin to the solubilized membrane proteins. In contrast, Sepharose CL-6B gel filtration of the cross-linked sample from the third peak fractions gave a major highly radioactive 125I-insulin-receptor complex with a molecular weight of 370,000 and a minor complex of low radioactivity with a molecular weight of 140,000. Upon sodium dodecyl sulfate-gel electrophoresis, the 370,000-dalton complex was dissociated to 130,000- and 82,000-dalton components and the 140,000-dalton complex was dissociated to a 47,000-dalton component.

Microfilaments and the hydrosmotic action of vasopressin in toad urinary bladder.

The role of cytoskeletal elements in the antidiuretic hormone (ADH)-induced hydrosmotic response of the toad bladder has been investigated with the microfilament and microtubule disrupting agents, cytochalasin B and colchicine. In the presence of a transmural osmotic gradient, addition of ADH resulted in a measured osmotic permeability coefficient (Pf) of 190 +/- 12 micrometers/s; preincubation with cytochalasin B (2 x 10(-5) M) reduced this value to 138 +/- 6. However, ADH-induced diffusional permeability to tritiated water (PD) was enhanced by 17% with cytochalasin B. The toxin caused a pronounced vacuolation in granular cells only when both ADH and an osmotic gradient were present. This led to experiments with brief glutaraldehyde fixation after hormonal stimulation but before osmotic gradient imposition. Subsequent osmotic flow measurements yielded Pf values of 180 +/- 18 and 210 +/- 7 micrometers/s for ADH and ADH + cytochalasin B, respectively. With gradient imposition only after tissue fixation, no structural alterations were found. We conclude that cytochalasin B inhibition is through disruption of the intracellular portion of the pathway for osmotic flows. Comparable experimentation with colchicine indicated that its interference is primarily with ADH action on the mucosal membrane itself. These results may bear on the significance of particle aggregation in the mucosal membrane, which has been thought to indicate ADH-induced water channels but which is reported to be precluded by cytochalasin B treatment.

Extracellular Ca2+ and the effect of antidiuretic hormone on the water permeability of the toad urinary bladder: an example of flow-induced alteration of flow.

The extracellular Ca2+ requirement for antidiuretic hormone (ADH) stimulation of water permeability in the toad urinary bladder has been critically examine. The polarity of the tissue was maintained with 1 mM Ca2+ in the mucosal bathing medium and a serosal bath nominally free of Ca2+. Under these conditions, ADH-induced osmotic water flow was inhibited by more than 60% while enhancement of the diffusional permeability to water was unaffected. Structural studies revealed that low serosal Ca2+ led to parallel alterations in epithelial architecture that amounted to a significant distortion of the osmotic water pathway. Prevention of these alterations, or restoration of normal cell-cell contact showed that the reduction of serosal Ca2+ did not restrict hormonal action per se, but that it resulted in a weakening of cell-cell junctions such that intercellular space distension during water flow occurred to a point where the geometric conditions for maintenance of osmotic flow were compromised. We conclude that extracellular Ca2+ is not a requirement for the molecular aspects of ADH action but that, in its absence, a direct measurement of ADH-induced osmotic flow proves to be an inaccurate index of the hormone-generated changes in epithelial transport characteristics. Under certain conditions the ADH-effect on the tissue's hydraulic permeability is probably best assessed by measurement of the diffusional permeability to water; although accuracy in this determination is difficult, it is not as strongly dependent on tissue geometry.

Structural responses to voltage-clamping in the toad urinary bladder. I. The principal role of granular cells in the active transport of sodium.

The structural consequences of clamping the transepithelial potential difference across the toad's urinary bladder have been examined. Reducing the potential to zero (short-circuiting) produced no apparent changes in the morphology of any of the four cell types which comprise the epithelium. Computer assisted, morphometric analysis of quick frozen specimens revealed no measurable difference in granular cell volume between open-and short-circuited preparations. However, when the open-circuit potential was quantitatively reversed (serosa negative with respect to mucosa), some of the preparations showed a marked increase in granular cell volume. To examine this more systematically twelve preparations were voltage-clamped at 50 mV (serosa negative); eight of the twelve revealed prominent granular cell swelling relative to control, short-circuited preparations. Only in this group of eight had the external circuit current fallen substantially during the clamping interval. Mitochondria-rich cells were not affected detectably. Application of the diuretic amiloride prior to clamping at reversed potential prevented granular cell swelling in every case. Goblet cells which were often affected by the - 50 mV clamp were not protected by the diuretic. Granular cell swelling thus appeared to be dependent on sodium entry at the mucosal surface. We also observed that, after voltage reversal, the apical "tight" junctions of the bladders were blistered as they are with hypertonic mucosal media. This blistering was associated with an increase in passive ionic permeability and was not prevented by application of amiloride. This finding is consistent with the evidence that the junction is a complex barrier with asymetric, and hence, rectifying properties for intrinsic ionic conductance as well as hydraulic permeability. These findings, together with others from the literature, lead to the conclusion that the granular cells constitute the principal, if not sole, elements for active sodium transport across toad urinary bladder and that they swell when sodium entry exceeds the transport capacity of the pump at the basal-lateral surface.