Abdul J. Mia

Aquaporins (Water Channels) Role in Vasopressin-Activated Water Transport

Abstract The discovery of water channels (aquaporins) was a breakthrough in research on water transport. Aquaporins are a family of intrinsic membrane proteins that function as water-selective channels (except aquaporin-3 and aquaporin-7, which are permeable to urea and glycerol as well) in the plasma membranes of many cells. Aquaporin-0 (MIP26) functions to maintain fluid balance in the lens. Aquaporin-1 is involved in water reabsorption in the kidneys proximal tubules and the thin descending Henles loop, aqueous humor formation in eye, cerebrospinal fluid formation in brain, and airway hydration in lung. Aquaporin-2 is the only water channel that is activated by vasopressin to enhance water reabsorption in the kidney collecting duct. Aquaporin-3 also contributes to water reabsorption in the kidney collecting duct but is unresponsive to vasopressin. It also appears that aquaporin-3 may contribute to cornea transparency. Aquaporin-4 is involved in cerebrospinal fluid transport in brain, water transport in the kidney collecting duct, aqueous humor transport in the eye, and airway hydration in the lung. Aquaporin-5 apparently is coupled to fluid secretion in exocrine tissues. Although the exact function of aquaporin-6 is not known due to its uncertain localization, its restricted presence in the kidney may suggest a potential role in water transport. Aquaporin-7 appears to play a role in the cryopreservation of the sperm whereas aquaporin-8 is responsible for the secretion of pancreatic juice. The major focus of this review is a discussion of aquaporins in renal epithelia, and particularly the mechanisms associated with vasopressin-mediated water transport involving aquaporin-2 and the signal transduction pathways linked to vasopressin action.

Morphometric analysis of epithelial cells of frog urinary bladder. I. Effect of antidiuretic hormone, calcium ionophore (A23187) and PGE2

Changes in epithelial cell morphology, especially at the apical plasma membrane, are frequently cited as initial evidence for antidiuretic hormone (ADH)-induced increase in membrane permeability. The effects of ADH and agents that alter and modify calcium and prostaglandin concentrations on the morphology and cytology of the epithelial cells of frog (Rana pipiens) urinary bladder are presented using the techniques of transmission and scanning electron microscopy. It was found that, like ADH, calcium ionophore, A23187, produce intense microvilli formation, microfilament mobilization and an increase in the density of granules and membrane associated vesicles, suggesting a prominent role of calcium in these processes. Moreover, our results suggest that these membrane and cytosolic transformations may be mediated in part through prostaglandin formation, as exogenous PGE2 mimicked these effects, and indomethacin, a prostaglandin synthesis inhibitor, attenuated ionophores effect on luminal cytomorphology. However, unlike ADH, prostaglandins and ionophore inhibit hormonal-induced increase in transepithelial water flow. These results suggest that other components more distal to the luminal membrane, perhaps the basolateral membrane, may be rate-limiting for transepithelial water flow and possibly are regulated by either changes in calcium concentrations or prostaglandins.

Is protein kinase C alpha (PKCα) involved in vasopressin-induced effects on LLC-PK1 pig kidney cells?

The involvement of protein kinase C (PKC) in vasopressin‐induced effects on renal water reabsorption is still unresolved. Activation of PKC can be detected by its translocation from the cytosol (C) to the plasma membrane (PM). In LLC‐PK1 cells, the redistribution of PKCα, a predominant isoform of PKC detected, was studied utilizing western blotting after stimulation with vasopressin. Vasopressin (100 mU/ml) failed to induce a translocation of PKCα from the C to the PM. By contrast, phorbol myristate acetate (PMA, 200 nM), a potent activator of PKC, induced a relocalization of PKCα from the C to the PM. After 2 hours of treatment of cells with PMA, PKCα was predominantly detected in the PM and absent from the C. These results suggest that the signal transduction pathway of vasopressin in LLC‐PK1 cells does not involve PKCα activation and translocation.

Modulation of cytoskeletal organization and cytosolic granule distribution by verapamil in amphibian urinary epithelia

The present study examines the role of calcium in modulating epithelial cytomorphology by using verapamil, a calcium antagonist, and considering its effects on cytosolic granule distribution and exocytosis in toad urinary bladder. The effect of verapamil on the detection and distribution of microfilaments in toad urinary bladder using immunogold labeling techniques in toad urinary bladder epithelial cells was also examined. Verapamil, which inhibits antidiuretic hormone (ADH)-mediated water flow, increased the number, size and distribution of dense calcium-containing secretory granules in bladder epithelial cells. This calcium antagonist prevented granule exocytosis, such that, six-times the number of granules were present in verapamil-treated tissues. The normal cytomorphological changes that accompany the actions of ADH were attenuated by verapamil, including ADH-induction of microvilli. ADH increased the number of actin microfilaments as determined using protein A-gold by immunolabeling, whereas, verapamil treatment was unremarkable as compared to control. The results suggest that calcium may play a prominent role in mediating granule exocytosis and membrane fusion events that normally accompany hormone action.

Surface membrane remodeling following removal of vasopressin in toad urinary bladder

Vasopressin (ADH) increases transepithelial water flow in renal epithelia by a process that involves the insertion of water channels into the apical membrane. The objective of the present study was to examine membrane surface remodeling under conditions that promote the recovery of water channels. Hemibladders were set up as sacs with an imposed osmotic gradient. The control sacs received no hormone treatment, whereas the other sacs were stimulated with 100 mU/ml ADH for 10 or 15 min to induce exocytosis and enhanced water flow. ADH was then washed from the tissues with fresh buffer rinses to abolish the hormone actions. These tissues were then allowed to recover for 15, 30 and 60 min. During this time water channels are recovered intracellularly by a process of endocytosis. This time period was called the retrieval period. At specified time intervals, tissues were fixed and processed for SEM or embedded in epon for ultrathin sectioning for TEM studies. Control tissues, regardless of the length of time, showed little or no sign of surface remodeling that was indicative of endocytosis during pre- or post-buffer washes, whereas the ADH-treated tissues showed a time-dependent remodeling of the apical membrane during activation and following removal of the hormone during the retrieval period. At the 10 min retrieval period, greater than 47% of the granular cells showed extensive surface remodeling. By 30 and 60 min posthormone treatment during recovery, fewer than 23% of granular cells showed signs of surface membrane changes. During retrieval the apical membrane undergoes a transition with a loss of both microridges and microvilli prior to membrane restoration. These observations suggest that apical membrane remodeling is crucial for the restoration of membrane permeability following hormone activation and termination.

Morphometric analysis of epithelial cells of frog urinary bladder, II. Effect of ADH, calcium ionophore (A23187) and verapamil on isolated dissociated cells.

Isolated frog urinary bladder epithelial cells, upon dissociation lose their polarity and develop microridges and occasional microvilli in a global fashion. These cells, when exposed only to isotonic Ringers solution manifest a membrane conformation with smooth discontinuous microridges, a cytoplasm with numerous free ribosomes, rough ER, thin Golgi cisternae, mitochondria, small vacuoles, electron-dense granules, few microtubules, and numerous microfilaments and intermediate filaments with an apparent random distribution, the dissociated cells, when treated with ADH or calcium ionophore (A23187), have the appearance of numerous elongated microvilli over the entire cell surface. The cytoplasm, under these conditions, is occupied by large vacuoles with a distribution of long profiles of aggrephores and associated vesicles. The peripheral cytoplasm as well as the cavities of the elongated microvilli of these cells contain large concentrations of microfilaments often showing a strong axial orientation to the long axis of the microvilli. Many of these filamentous elements appear in contact with the apical membrane of these microvilli with an alignment with the external glycocalyx. There is an indication that these morphocytological changes as revealed by SEM and TEM studies, correlated with a redistribution and realignment of microfilaments and possibly microtubules as detected by fluorescent microscopy using immunofluorescent antibody labeling for actin and tubulin. Cells treated with verapamil, a calcium antagonist, presented dwarf and stout microvilli with little detectable alterations in the cytoplasmic compositions from that of non-hormonal treated cells. Verapamil prevented ADH induction of microvilli, with the membrane, under these conditions, appearing as compact microridges. The results indicate that calcium ionophore, like ADH, produces intense formation of microvilli in dissociated cells, mobilization and realignment of microfilaments, microtubules, increase in the density of vesicles, aggrephores and possibly secretory granules, whereas the calcium antagonist, verapamil, opposes these actions. The results suggests a prominent role of calcium in the morphological changes induced by ADH.

The ATP-depleting reagent iodoacetamide induces the degradation of protein kinase C alpha (PKCα) in LLC-PK1 pig kidney cells

The alkylating reagent iodoacetamide, a potent inhibitor of sulfhydryl proteases, was found to stimulate the selective degradation of protein kinase C alpha (PKC alpha) isoform (80 KDa). Treatment of LLC-PK1 cells with iodoacetamide (0.5-15 mM) for 30-90 minutes at room temperature allowed by western blotting on total cell homogenate, revealed the appearance of an 50 KDa band that was still recognized with the antibody. However, iodoacetamide (15 mM) resulted in the total disappearance of the 80 KDa protein. Serine protease inhibitors, metalloprotease inhibitors and leupeptin failed to prevent the degradation of PKC alpha. The degradation persisted at 4 degrees C and in the absence of Ca2+. Iodoacetamide had no direct effect on purified PKC alpha. PKC activities in iodoacetamide-treated cells were also inhibited. In conclusion, the degradation of PKC alpha is a novel phenomenon. The degradation process could not be prevented by known protease inhibitors or in the absence of Ca2+ or by incubation at 4 degrees C and appears to involve interactions with unknown cellular intermediates.

Adaptive changes of H+ secreting cells in the epidermis of the leopard frog RANA PIPIENS

1. Mitochondria-rich (MR) cells in the integument of the southern leopard frog, Rana pipiens, berlandieri, were stained with AgNO3 under a variety of environmental and metabolic treatment conditions known to increase H+ excretion rates across the skin. In this tissue AgNO3 proved to be a good stain for discriminating the MR cell populations from the granular cells. 2. High salinity adapted southern frogs showed no change in the MR cell population. The inability of the MR cell number to significantly increase suggested that the increased H+ excretion rates previously seen in these animals were not due to increased MR cell proliferation. 3. The MR cell population was found to increase in the NaNO3 adapted frogs, demonstrating the contribution of altered extracellular Cl- concentrations on the regulation of MR cell density. 4. Animals that were placed in chronic metabolic acidosis or pre-treated with ibuprofen demonstrated an increased MR cell population. The current observations are consistent with previous findings that these treatment regimes increase H+ excretion, suggesting that one of the cellular adaptive mechanisms responsible for increasing H+ excretion involves increasing the MR cell density. 5. The results further suggest that prostaglandins may play a role in regulating H+ excretion in MR cells, and that either changes in intracellular pH or prostaglandin formation regulates cell proliferation.

Effect of Temperature on Apical Membrane Remodeling in ADH-Stimulated Toad Urinary Bladders

Abstract Pretreatment and removal of vasopressin (ADH) in toad urinary bladder renal model tissues induces endocytosis at 25°C. The objective of the current study is to determine if apical membrane remodeling, as well as transepithelial water flow, can be affected by lowering the temperature to 15°C. Control toad urinary bladders in the presence of an osmotic gradient at either 25°C or 15°C when visualized by scanning electron microscopy (SEM) show a typical apical membrane surface with no apparent surface differences. ADH-treated tissues following 15-min stimulation at 25°C or 15°C revealed a propagation of apical microvilli on their surface membranes. After 15 min following removal of ADH, bladder tissues at 25°C or 15°C showed surface invaginations involving over 44% and 80% of granular cells, respectively. The rate of water flow in tissues at 15°C remained elevated compared to tissues held at 25°C. This was consistent with the observation that ADH-stimulated tissues following washout at 15°C still had marked apical membrane surface involvement. However, at 30 min and 60 min postwashout, ADH-stimulated tissues at 15°C recovered considerably, with a reduction in the number of shallow apical membrane invaginations involving fewer than 33% and 20% of granular cells respectively. This may indicate that the membrane undergoes continuous remodeling even at cold temperature conditions but with a different half-time. Control bladder tissues subjected to transmission electron microscopy (TEM) reveal a dense cytoplasmic profile with a scattered distribution of secretory granules, rough ER cisternae, mitochondria, and little or no vacuolation. In contrast, ADH-stimulated bladder tissues displayed a vacuolated cytoplasm, expanded rough ER cisternae, and ruffled basolateral membranes. These observations suggest that the apical membrane undergoes considerable reorganization following cessation of hormone action and that lowering the temperature reduces the rate of membrane remodeling and thus may provide a means to monitor the processes of endocytosis and the mechanisms responsible for water channel retrieval.