Fracture faces of osmotically disrupted zonulae occludentes.

Exposing the mucosal epithelium of the toad urinary bladder to 240 mM urea in Ringer's solution is known to cause a dramatic increase in the permeability of the zonulae occludentes and the appearance of distended, bubble-like compartments within these junctions. Examination of such osmotically disrupted junctions with the freeze-fracture technique reveals that these bubbles result from a distention of the compartments existing within the meshwork of interconnecting fibrils characteristic of the zonulae occludentes in this epithelium. Frequent discontinuities in the meshwork of fibrils are also found after osmotic disruption of the junction. These observations indicate the essential role of these fibrils in maintaining the characteristic properties of the zonula occludens as a site of cell-to-cell attachment and as a permeability seal.

The structure of the zonula occludens. A single fibril model based on freeze-fracture.

Replicas of freeze-fractured toad urinary bladder and gallbladder were analysed in an attempt to determine the fracturing properties and structure of the zonula occludens (tight junction). Chalcroft and Bullivant have proposed that the junction has a double set of fibrils with one set associated with each of the adjacent cell membranes. However, the fracturing pattern that is observed might also result from only a single set of fibrils which is shared by the adjacent membranes if fracturing occurred around either side of the fibrils. These two models predict quite different structures at regions of the junction where tranl sitions are made between face A and face B. The relative heights of face A and face B and the shape of the transition from face A to face B do not agree with that expected according to the two fibril model but agree exactly with that expected if only a single set of fibrils existed. Further evidence for the single fibril model is derived from fractures of the mucosa membrane which cross the junction to the membrane of the adjacent cell without deflection. Such fractures reveal a single ridge which appears to be identical to the juxtaluminal fibril of the junction. In addition, small ridges are occasionally found in place of the grooves on face B which, although not consistent with the double fibril model, is expected if the single fibril model were correct. Although alternative explanations might account for these observations, we believe that the simplest and most consistent explanation is that the zonula occludens fractures as would be expected of a single set of fibrils shared by adjacent cells.

Structural integrity of hepatocyte tight junctions.

The significance of discontinuities frequently found in freeze-fracture replicas of the tight junction was evaluated using complementary replicas of hepatocyte junctions from control and bile duct-ligated rats. An extensive analysis of complementary replicas using rotary platinum shadowing indicates that discontinuities in the protoplasmic (P) fracture face do not represent structural breaks in the tight-junctional network. In no case did P-face discontinuities correspond with interruptions in the groove network on the complementary extracellular (E) face. Quantitative analysis of replicas shows that P-face discontinuities result in part from "transfer" of material to the complementary E face (approximately 7% of the junctional length). However, many P-face discontinuities (7-30% of the junctional length) are matched only by a groove on the complementary E face. This finding demonstrates that a significant amount of material can be lost during freeze-fracture. An analysis of junctions from bile duct-ligated rats, which are known to have an increased paracellular permeability, shows comparable transfer and loss of material. However, the number of junctional elements and the tight-junction network density was significantly reduced by bile duct ligation. These observations indicate that discontinuities in tight-junctional elements result during the preparation of freeze-fracture replicas and are not physiologically important features of the junctional barrier. Variation in the number of elements provides the best explanation for observed differences in tight-junction permeability.

Modulation of cell membrane area in renal collecting tubules by corticosteroid hormones.

Isolated renal cortical collecting tubules obtained from rabbits treated chronically with desoxycorticosterone acetate (DOCA) have been found to possess elevated transepithelial potential differences and a greatly increased capacity for ion transport. Structural exmination of tubules from rabbits exposed to either DOCA or dexamethasone for 11--18 d reveals a marked increase in basolateral cell membrane area in these tubules. Morphometric analysis shows that this effect is specifically on the basolateral membrane area of only one of the two cell types found in this nephron segment. Increases of greater than 140% and 90% are found for the basolateral membrane area of the principal cells for DOCA and dexamethasone, respectively, but no change could be detected in the basolateral membrane area of the intercalated cells found in this nephron segment. No siginificant changes were found in luminal membrane area, cell number, or cell volume for either cell type. These observations demonstrate that significant changes in membrane area can occur in differentiated epithelia and suggest that this may be an important mechanism for modulating epithelial transport capacity.

Vasopressin: induced structural change in toad bladder luminal membrane.

Freeze-fracture electron microscopy demonstrates that vasopressin stimulation of isolated toad bladder alters the structure of the luminal membrane of granular cells. This alteration consists of an ordered aggression of intramembranous particles, and appears to be of functional significance, since the frequency of aggregation sites per area of membrane is closely correlated with vasopressin-induced osmotic water flow.

Transepithelial water flow regulates apical membrane retrieval in antidiuretic hormone-stimulated toad urinary bladder.

Antidiuretic hormone (ADH) increases the osmotic water permeability (Posm) of toad urinary bladder. This increase is believed to be produced by fusion of intracellular vesicles called aggrephores with the granular cell apical plasma membrane. Aggrephores contain intramembrane particle aggregates postulated to be water channels. ADH-stimulated Posm is decreased by osmotic gradient exposure, which is termed flux inhibition. We studied flux inhibition by exposing ADH-stimulated bladders to various osmotic gradients. Osmotic water flow was initially proportional to the applied osmotic gradient, but Posm decreased with time. Ultrastructural and quantitative studies of endocytosis demonstrate that apical membrane retrieval was a direct function of the transepithelial osmotic gradient. Posm remained unchanged when apical membrane retrieval was blocked by incubation of bladders at 2 degrees C, or under low water-flow conditions. These effects were reversed by increases in temperature or the applied osmotic gradient. We conclude that apical membrane retrieval causes the phenomenon of flux inhibition.

Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney.

Aldosterone stimulates sodium transport in the renal collecting duct by activating the epithelial sodium channel (ENaC). To investigate the basis of this effect, we have developed a novel set of rabbit polyclonal antibodies to the 3 subunits of ENaC and have determined the abundance and distribution of ENaC subunits in the principal cells of the rat renal collecting duct. Elevated circulating aldosterone (due to either dietary NaCl restriction or aldosterone infusion) markedly increased the abundance of alphaENaC protein without increasing the abundance of the beta and gamma subunits. Thus, alphaENaC is selectively induced by aldosterone. In addition, immunofluorescence immunolocalization showed a striking redistribution in ENaC labeling to the apical region of the collecting duct principal cells. Finally, aldosterone induced a shift in molecular weight of gammaENaC from 85 kDa to 70 kDa, consistent with physiological proteolytic clipping of the extracellular loop as postulated previously. Thus, at the protein level, the response of ENaC to aldosterone stimulation is heterogenous, with both quantitative and qualitative changes that can explain observed increases in ENaC-mediated sodium transport.

Relationship of aggregated intramembranous particles to water permeability in vasopressin-treated toad urinary bladder.

It has been previously demonstrated with freeze-fracture electron microscopy that vasopressin induces specific structural alterations of the luminal membrane of granular cells from toad urinary bladder in a dose-dependent fashion. These alterations consist of aggregated intramembranous particles and are observed both in the presence and absence of an osmotic gradient. We examined the effect of methohexital, a selective inhibitor of vasopressin-stimulated water flow, and the effect of phloretin, a selective inhibitor of urea permeability, on the structure of the granular cell luminal membrane. Methohexital treatment of the vasopressin-stimulated toad bladder reduced both the osmotic water flow and vasopressin-induced alterations of membrane structure to the same extent. Phloretin reduced urea permeability but not water flow or particle aggregation. Since neither agent affects vasopressin-stimulated sodium movement, these findings indicate that the phenomenon of particle aggregation is specifically related to vasopressin-induced water permeability and not to changes in urea or sodium permeability.

Rat renal arcade segment expresses vasopressin-regulated water channel and vasopressin V2 receptor.

The arcades are long, branched renal tubules which connect deep and mid-cortical nephrons to cortical collecting ducts in the renal cortex. Because they are inaccessible by standard physiological techniques, their functions are poorly understood. In this paper, we demonstrate that the arcades are a site of expression of two proteins, aquaporin-2 (the vasopressin-regulated water channel) and the V2 vasopressin receptor, that are important to regulated water transport in the kidney. Using a peptide-derived polyclonal antibody to aquaporin-2, quantitative ELISA in microdissected segments showed that aquaporin-2 is highly expressed in arcades and that the expression is increased in response to restriction of fluid intake. Immunocytochemistry revealed abundant aquaporin-2 labeling of structures in the cortical labyrinth in a pattern similar to that of the Na(+)-Ca2+ exchanger and kallikrein, marker proteins expressed in arcades but not in cortical collecting ducts. RT-PCR experiments demonstrated substantial aquaporin-2 and V2 receptor mRNA in microdissected arcades. In situ hybridization, using 35S-labeled antisense cRNA probes for the V2 receptor demonstrated strong labeling of both arcades and cortical collecting ducts. Thus, these results indicate that the arcades contain the specific proteins associated with vasopressin-regulated water transport, and may be a heretofore unrecognized site of free water absorption.

Water permeability of cochlear outer hair cells: characterization and relationship to electromotility.

The distinguishing feature of the mammalian outer hair cells (OHCs) is to elongate and shorten at acoustic frequencies, when their intracellular potential is changed. This "electromotility" or "electromechanics" depends critically on positive intracellular pressure (turgor), maintained by the inflow of water through yet uncharacterized water pathways. We measured the water volume flow, J(v), across the plasma membrane of isolated guinea pig and rat OHCs after osmotic challenges and estimated the osmotic water permeability coefficient, P(f), to be approximately 10(-2) cm/sec. This value is within the range reported for osmotic flow mediated by the water channel proteins, aquaporins. J(v) was inhibited by HgCl(2), which is known to block aquaporin-mediated water transport. P(f) values that were estimated for OHCs from neonatal rats were of the order of approximately 2 x 10(-3) cm/sec, equivalent to that of membranes lacking water channel proteins. In an immunofluorescence assay we showed that an anti-peptide antibody specific for aquaporins labels the lateral plasma membrane of the OHC in the region in which electromotility is generated. Using patch-clamp recording, we found that water influx into the OHC is regulated by intracellular voltage. We also found that the most pronounced increases of the electromotility-associated charge movement and of the expression of OHC water channels occur between postnatal days 8 and 12, preceding the onset of hearing function in the rat. Our data indicate that electromotility and water transport in OHCs may influence each other structurally and functionally.

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.

The evolution of granule fracture strength as a function of impeller tip speed and granule size for a novel reverse-phase wet granulation process.

The feasibility of a novel reverse-phase wet granulation process has been established previously and several potential advantages over the conventional process have been highlighted (Wade et al., 2014a,b,b). Due to fundamental differences in the growth mechanism and granule consolidation behaviour between the two processes the reverse-phase approach generally formed granules with a greater mass mean diameter and a lower intragranular porosity than those formed by the conventional granulation process under the same liquid saturation and impeller tip speed conditions. The lower intragranular porosity was hypothesised to result in an increase in the granule strength and subsequent decrease in tablet tensile strength. Consequently, the aim of this study was to compare the effect of impeller tip speed and granule size on the strength and compaction properties of granules prepared using both the reverse-phase and conventional granulation processes. For the conventional granulation process an increase in the impeller tip speed from 1.57 to 4.71 ms(-1) (200-600 RPM) resulted in an increase in the mean granule strength (p<0.05) for all granule size fractions and as the granule size fraction increased from 425-600 to 2000-3350 µm the mean fracture strength decreased (p<0.05). For the reverse-phase process an increase in impeller tip speed had no effect (p>0.05) on mean granule strength whereas, like the conventional process, an increase in granule size fraction from 425-600 to 2000-3350 µm resulted in a decrease (p<0.05) in the mean fracture strength. No correlation was found between mean granule fracture strength and the tablet tensile strength (p>0.05) for either granulation approach. These data support the rejection of the original hypothesis which stated that an increase in granule strength may result in a decrease in the tablet tensile strength. The similar tablet tensile strength observed between the conventional and reverse-phase granulation processes indicated that while mechanistic differences exist in the formation of the granules, which resulted in significant granule-scale fracture strength differences, the granule compaction properties at pharmaceutically relevant tableting pressures were unaffected.

Controlling granule size through breakage in a novel reverse-phase wet granulation process: the effect of impeller speed and binder liquid viscosity.

The feasibility of a novel reverse-phase wet granulation process has been established previously highlighting several potential advantages over the conventional wet granulation process and making recommendations for further development of the approach. The feasibility study showed that in the reverse-phase process granule formation proceeds via a controlled breakage mechanism. Consequently, the aim of the present study was to investigate the effect of impeller speeds and binder liquid viscosity on the size distribution and intragranular porosity of granules using this novel process. Impeller tip speed was found to have different effects on the granules produced by a conventional as opposed to a reverse-phase granulation process. For the conventional process, an increase in impeller speed from 1.57 to 3.14 ms(-1) had minimal effect on granule size distribution. However, a further increase in impeller tip speed to 3.93 and 4.71 ms(-1) resulted in a decrease in intragranular porosity and a corresponding increase in mean granule size. In contrast when the reverse-phase process was used, an increase in impeller speed from 1.57 to 4.71 ms(-1) resulted in increased granule breakage and a decrease in the mean granule size. This was postulated to be due to the fact that the granulation process begins with fully saturated pores. Under these conditions further consolidation of granules at increased impeller tip speeds is limited and rebound or breakage occurs. Based on these results and analysis of the modified capillary number the conventional process appears to be driven by viscous forces whereas the reverse-phase process appears to be driven by capillary forces. Additionally, in the reverse-phase process a critical impeller speed, represented by the equilibrium between centrifugal and gravitational forces, appears to represent the point above which breakage of large wet agglomerates and mechanical dispersion of binder liquid take place. In contrast the conventional process appears to be difficult to control due to variations in granule consolidation, which depends upon experimental variables. Such variations meant increased impeller tip speed both decreased and increased granule size. The reverse-phase process appears to offer simple control over granule porosity and size through manipulation of the impeller speed and further evaluation of the approach is warranted.

Role of nonacidic endosomes in recycling of ADH-sensitive water channel structures.

Toad urinary bladder epithelial cells respond to the hormone ADH by increasing the water permeability of their luminal membrane. This action is mediated by insertion into the apical membrane of specific water channels. In the absence of ADH these channels appear to be present in tubular cytoplasmic vesicles as morphologically distinctive intramembrane structures called particle aggregates. ADH induces these vesicles to fuse with the apical membrane, transferring their aggregate-water channels into the apical membrane. When ADH stimulation is removed (ADH reversal), aggregates and fluid-phase markers from the mucosal bath appear in water-permeable vesicles in the cytoplasm. We have examined the fate of fluid-phase markers and aggregates with time after ADH reversal. Although the fluid-phase markers horseradish peroxidase and colloidal gold are initially found predominantly in tubular vesicles near the apical surface, by 30 min the markers were found in perinuclear multivesicular bodies (MVBs) of heterogeneous size and shape. These MVBs appear to be nonacidic since they fail to accumulate DAMP. Acid phosphatase (AcPase) was undetectable in these structures. After 60 min, labeled MVBs tended to be smaller, and some of these structures displayed DAMP accumulation and AcPase activity. By evaluation of uncleaned replicas it was possible to localize recycled aggregate-water channels with respect to internalized fluid-phase markers. Thirty minutes after retrieval from the apical surface in tubular vesicles, aggregates could be localized to both the central body and tubular projections of labeled MVBs. At 60 min following reversal, most MVBs had a reduced number of aggregates compared with 30 min, and compact structures could be identified that contained markers but no detectable aggregates. These observations show that aggregates and fluid-phase markers enter a nonacidic endosomal compartment with an MVB morphology following ADH reversal. At extended times following reversal, labeled MVBS having lysosomal characteristics and labeled MVBs having no detectable aggregates can be found, suggesting that aggregates are sorted or degraded prior to this stage.

Visualization of endocytosed markers in freeze-fracture studies of toad urinary bladder.

Antidiuretic hormone (ADH) stimulation increases the apical membrane water permeability of granular cells in toad urinary bladder. This response correlates closely with the fusion of tubular cytoplasmic vesicles with the membrane and delivery of intramembrane particle (IMP) aggregates from the tubules (aggrephores) to the apical membrane. These aggregates are believed to be associated with the channels responsible for the water permeability increase. Removal of ADH triggers apical membrane endocytosis and disappearance of aggregates from the apical membrane. However, it has been unclear whether aggregate disappearance is due to disassembly of aggregates within the apical membrane or to their endocytic retrieval as intact structures. Using colloidal gold and horseradish peroxidase to follow endocytosis from the apical surface after ADH removal, we have directly observed in cross-fractured bladder cells the intramembrane structure of intracellular vesicles that contain these fluid-phase markers. Under these conditions, intact aggregates can be identified in the membrane of tubular endocytosed vesicles. This directly demonstrates that conditions which lower apical membrane water permeability cause the tubular aggrephores to "shuttle" intact aggregates from the apical membrane back into the cytoplasm. An additional population of vesicles with tracer are found which are spherical and display structural features of the apical membrane, as well as occasional aggregates. These vesicles may be responsible for retrieval of aggregates from the surface apical membrane.

Sex differences in negative emotional responses to chronic pain.

We proposed a sequential model of pain processing with pain intensity as stage 1, pain unpleasantness as stage 2, pain-related emotions (depression, anxiety, frustration, anger, fear) as stage 3, and overt behavioral expression of pain as stage 4. We tested hypotheses about relationships between sex and the first 3 stages of pain processing by conducting simultaneous regression analysis using LISREL-8 with data collected from 967 women and 680 men with chronic pain. We found the following results: (1) women reported higher pain-related frustration and fear; (2) frustration related most highly to pain intensity among women, as compared with anxiety and depression among men; (3) depression and frustration related most highly to usual and highest pain unpleasantness among women, as compared with frustration among men; and (4) contrary to expectations, pain-related emotions were more strongly related to pain for men. Consistent with the sequential model of pain processing, emotional response to pain was more closely related to pain unpleasantness than to pain intensity across sex. Anxiety and frustration were the emotions most highly related to pain. The current results highlight sex differences in the experience of chronic pain and the importance of assessing a range of emotions in patients with pain.

A prospective assessment of the Garren-Edwards Gastric Bubble and bariatric surgery in the treatment of morbid obesity.

Morbid obesity is a serious medical hazard, and effective alternatives to surgery have been unsuccessful. In 1985, the Garren-Edwards Gastric Bubble (GEGB) was offered as an adjunct to dietary and behavioral therapy for weight loss treatment. The safety and efficacy of the GEGB were compared with bariatric surgery, the current standard for the treatment of morbid obesity. Fifty-seven patients received GEGB and 77 underwent bariatric surgery. GEGB patients were divided into two groups: those who attended group therapy and those who did not. This study showed that bariatric surgery was far more effective in reducing excess body weight during a 12-month period compared with the GEGB plus group therapy and the GEGB alone. The morbidity from bariatric surgery was greater than in the GEGB-treated groups, while the cost for uncomplicated cases for a year's treatment was comparable. It is concluded that the GEGB does not offer an effective alternative to bariatric surgery in the treatment of morbid obesity.

Behavioral management of sleep disturbances secondary to chronic pain.

The clinical efficacy of a behavioral management program for treating insomnia secondary to chronic pain was evaluated within a multiple-baseline design across subjects. Treatment consisted of a combination of stimulus control and sleep restriction procedures. Daily sleep diaries and all-night polysomnographic (PSG) measures were used to document changes in sleep/wake patterns. The results showed that treatment was effective in improving sleep patterns in all three patients. A substantial decrease of time awake at night was obtained and this was reflected by reductions of sleep onset latency, wake time after sleep onset, and early morning awakenings. Sleep improvements were well maintained at follow-ups and were also paralleled by improved mood states. The findings indicate that behavioral procedures are effective for treating sleep disturbances associated with chronic pain conditions.

Feasibility assessment for a novel reverse-phase wet granulation process: the effect of liquid saturation and binder liquid viscosity.

A novel reverse-phase wet granulation process was developed and the feasibility of the process compared to a conventional wet granulation process. The reverse-phase granulation approach involves the immersion of the dry powder formulation into the binder liquid followed by controlled breakage to form granules. Conventional wisdom would warn against this approach due to the initial formation of a slurry or over-wetted powder formulation. However, a feasibility assessment of the novel approach was motivated by the potential advantages of eliminating traditional granule nucleation variables and reducing risk of uncontrolled granule growth. The effects of liquid saturation and binder liquid viscosity on the physical properties of granules formed using both the reverse-phase and conventional granulation processes were compared. Liquid saturation significantly affected the physical properties of granules prepared using both processes. At liquid saturation up to ∼1 the reverse-phase process typically resulted in larger, less porous granules than the conventional process. However, at a liquid saturation >1.1 the conventional process exhibited uncontrolled growth and significantly larger granule size as a result of decreased intragranular porosity. The response to liquid saturation was seen as a steady growth mechanism for the reverse-phase process compared to an induction growth mechanism for the conventional process, indicating potential robustness advantages of the reverse-phase approach. Despite institutional perceptions to the contrary, the reverse-phase process was shown to be feasible and merits further detailed investigation.