Epithelial Cell Volume Research Articles

Stereology and ultrastructure of the seminal vesicle of C57/BL/6J mice following chronic alcohol ingestion

Excessive alcohol consumption causes metabolic changes and pathologic alterations in testes and accessory sex organ in different animal species. The aim of the present study was to evaluate the macroscopic, histologic and ultrastructural alterations provoked by chronic ingestion of different ethanol concentrations over increasing periods of time on the secretory epithelium of the seminal vesicle of C57/BL/6J mice in using stereological methods. Sixty male adult mice were divided into three experimental groups: Control, Alcoholic 25% and Alcoholic 35%, respectively, receiving tap water and tap water containing ethanol diluted to 25 and 35° Gay Lussac. All mice were fed with the same solid diet. After 150 and 250 days of treatment the animals were sacrificed and the seminal vesicles were collected and processed for light and transmission electron microscopy. The cellular, cytoplasmic and nuclear volumes and the area density of autophagic and secretory vacuoles were measured. The histologic alterations observed in the alcoholic mice consisted of a reduction in epithelial size and cell volume, with maintenance of the same nuclear and cytoplasmic ratio as verified in the control groups. The ultrastructural alterations were: increased density of dense body area, decreased density of secretory granule area, and dilated rough endoplasmic reticulum and Golgi cisternae. We conclude that chronic ethanol ingestion causes depleting morphologic alterations in the epithelial cells of the seminal vesicle and negatively affects the secretory process of this gland.

Expression, localization, and regulation of aquaporin-1 to -3 in rat urothelia.

Although mammalian urothelia are generally considered impermeable to constituents of urine, in vivo studies in several species indicate urothelial transport of water and solutes under certain conditions. This study investigates the expression, localization, and regulation of aquaporin (AQP)-1, -2, and -3 in ureteral and bladder tissues in 48-h dehydrated and water-loaded female Wistar rats. Immunoblots of homogenates of whole ureter and bladder identified characteristic approximately 28- and 35- to 44-kDa bands for AQP-1, -2, and -3. AQP-1 was localized to capillary and arteriole endothelial cells, whereas AQP-2 and -3 circumferentially lined the epithelial cell membranes except for the apical membrane of the epithelial cells adjacent to the lumens of both ureter and bladder. AQP-2 was also present in epithelial cell cytoplasm. Dehydration resulted in 160-200% increases of AQP-3 signal and 24-49% increases of AQP-2 signal but no change in AQP-1 signal on immunoblots of homogenates of ureters and bladders. AQPs in genitourinary tract urothelia likely play a role in the regulation of epithelial cell volume and osmolality and may play a role in bulk water movement across urothelia.

CFTR contributes to cAMP-mediated inhibition of Na+/H+ exchange in murine jejunum

In small intestinal epithelium of wild-type (WT) mice, increases in intracellular cAMP result in stimulation of CFTR-mediated anion secretion and inhibition of electroneutral Na + absorption (via Na+/H ÷ exchange). However, several reports indicate that anion secretion and inhibition of Na t absorption do not occur following cAMP treatment of the intestine in both cystic fibrosis (CF) patients and CFTR knockout (CFTR(-)) mice. To better understand the role of CFTR during cAMP inhibition of intestinal Na ÷ absorption, we investigated: (1) whether the anion secretory function of CFTR is required for complete inhibition of Na + absorption; and (2) whether electroneutral Na ÷ absorption in the OF intestine can be normally regulated by other physiological mechanisms, e.g. extracellular hyperosmolarity. ~Na flux studies were performed on WT and CFTR(-) murine jejunum mounted in Ussing chambers (voltage-clamped) and bathed in physiological Ringers solution. Under basal conditions (+ vehicle), wr jejunum exhibited net Na ÷ absorption at a rate of 6.1 _+ 1.3/~Eq/cm2.hr (n =6). Basal Na ÷ absorption could be completely inhibited by treatment with 100/~M 5-(N-EthyI-N-isopropyl} amiloride (EIPA), a specific inhibitor of Na÷/H + exchanger activity. Treatment of the W'i jejunum with 10/~M forskolin/100 p,M IBMX to increase intracellular cAMP also completely inhibited net Na ÷ flux in WT mice (0.4 _+ 0.5 ,~Eq/cm2.hr, n=6), but did not affect net Na + absorption in the CFTR(-) jejunum (basal = 4.8 _+ 0.9 vs. treated = 4.5 _+ 0.8/~Eq/em2.hr; n=6#). To examine the role of CFTR-mediated anion secretion, Wl jejuna were simultaneously treated with forskolin/IBMX and humetanide (100/~M serosal) to inhibit CFTR-mediated Clsecretion. Bumetanide treatment recovered 46% of net Na ÷ absorption (2.2 _+ 0.4 ~Eq/cm2.hr, n = 6). To examine whether intestinal Na + absorption can be inhibited in the absence of CFTR, net Na* absorption was measured after addition of 300 mM mannitol to the bath solutions of WT and CFTR(-) jejunua. Extracellular hyperosmolarity completely inhibited Ha + absorption in both WT (-0.2 _+ 0.7/~Eq/cm2.hr, n=6) and CFTR(-) intestine (0.1 _+ 0.2 p.Eq/cm2.hr, n =9). In conclusion, functional CFTR is required for complete inhibition of intestinal Na ÷ absorption during cAMP stimulation but not during extracellular hyperosmolarity. These findings suggest the hypothesis that complete cAMP inhibition of intestinal Na ÷ absorption may require changes in epithelial cell volume resulting from CFrR-mediated anion secretion. Supported by NIH and AHA.

Gross anatomical and histomorphometric characteristics of the oviduct and uterus during the pubertal transition in sheep.

Gross anatomical and histometric characteristics of oviducts and uteri were evaluated during the pubertal transition in ewe lambs. Lambs were hysterectomized prepubertally (PP; n = 10) or 26 +/- 2 h after onset of first (FE; n = 13) or third estrus (TE; n = 12), or estrus in mature pluriparous ewes (ME; n = 4). Gross characteristics included diameters, lengths, and weights of oviducts; diameters of uterine cornua; and weights of uteri. Histometric characteristics of ampullae, isthmi, and uterine cornua included epithelial cell volume, total luminal surface area, and total number of cells per ampulla or isthmus and area of the muscularis. Side of the reproductive tract did not affect (P > 0.10) any gross or histometric characteristic. Gross uterine characteristics increased (P < 0.05) dramatically between PP ewes and FE ewes, and then increased (P < 0.05) to a lesser degree between FE and TE ewes, and again (P < 0.05) between TE and ME ewes. Histological characteristics of uteri followed a similar developmental pattern: large changes (P < 0.05) between PP and FE ewes, slight increases between FE and TE ewes, and no change (P > 0.10) between TE and ME ewes. Oviductal weight and length showed little change (P > 0.10) at puberty or by third estrus, but they were greater (P < 0.05) in ME ewes than in the other groups. The diameters of ampullae increased (P < 0.05) at puberty, showed no change between FE and TE ewes, and were greatest (P < 0.05) in mature ewes. These changes seemed to be due to increases (P < 0.05) in mucosal cell height and muscularis area at puberty, and to muscularis area and submucosal thickness between TE and ME ewes. The surface area of the ampullae increased (P < 0.05) at puberty or by third estrus in a linear manner between PP, FE, TE, and ME ewes. The diameters of isthmi did not change (P > 0.10) at puberty but increased (P < 0.05) between FE and TE, and were greatest (P < 0.05) in ME ewes. The histological characteristics of isthmi followed the pattern observed in the ampullae. However, the surface area of the isthmi showed no change (P > 0.10) at puberty or by third estrus and was largest (P < 0.05) in ME ewes. Results of this study indicate that gross morphological characteristics of uteri and oviducts increase at puberty with either slight increases or changes after two estrous cycles. This relationship is also reflected by histological characteristics of these organs during the pubertal transition of sheep.

Osmotic water permeabilities of cultured, well-differentiated normal and cystic fibrosis airway epithelia.

Current hypotheses describing the function of normal airway surface liquid (ASL) in lung defense are divergent. One theory predicts that normal airways regulate ASL volume by modulating the flow of isosmotic fluid across the epithelium, whereas an alternative theory predicts that ASL is normally hyposmotic. These hypotheses predict different values for the osmotic water permeability (P(f)) of airway epithelia. We measured P(f) of cultures of normal and cystic fibrosis (CF) airway epithelia that, like the native tissue, contain columnar cells facing the lumen and basal cells that face a basement membrane. Xz laser scanning confocal microscopy recorded changes in epithelial height and transepithelial volume flow in response to anisosmotic challenges. With luminal hyperosmotic challenges, transepithelial and apical membrane P(f) are relatively high for both normal and CF airway epithelia, consistent with an isosmotic ASL. Simultaneous measurements of epithelial cell volume and transepithelial water flow revealed that airway columnar epithelial cells behave as osmometers whose volume is controlled by luminal osmolality. Basal cell volume did not change in these experiments. When the serosal side of the epithelium was challenged with hyperosmotic solutions, the basal cells shrank, whereas the lumen-facing columnar cells did not. We conclude that (a) normal and CF airway epithelia have relatively high water permeabilities, consistent with the isosmotic ASL theory, and the capacity to restore water on airway surfaces lost by evaporation, and (b) the columnar cell basolateral membrane and tight junctions limit transepithelial water flow in this tissue.

Neuregulin-1 and human epidermal growth factor receptors 2 and 3 play a role in human lung development in vitro.

The human epidermal growth factor receptor (HER) family consists of four distinct receptors: HER1 (epidermal growth factor receptor), HER2, HER3, and HER4. Their specific activating ligands are collectively known as neuregulins (NRG). We hypothesized that one member of the NRG family, NRG-1, and the HER family would play a role in fetal lung development. To test this hypothesis, we defined NRG-1 and HER gene expression in mid-trimester human fetal lung tissue. HER2 and HER3 messenger RNA and protein were detected in the fetal lung, but HER4 expression was not detected. Immunohistochemical staining of fetal lung tissue localized HER2 and HER3 protein to the developing lung epithelium. NRG-1 expression was not found in freshly isolated human fetal lung, but it was observed in fetal lung explants after 2 d of explant culture. Immunohistochemistry of cultured human fetal lung explants revealed that NRG-1 protein was also expressed in pulmonary epithelial cells. Exposing human fetal lung to recombinant NRG-1 activated the HER receptor complex as measured by approximately 4-fold increases in receptor phosphotyrosine content. In addition, NRG-1 increased explant epithelial cell volume density approximately 2-fold (P < 0. 03); increased epithelial cell proliferation approximately 2-fold, as determined by bromodeoxyuridine labeling (P = 0.002); and reduced surfactant protein-A (SP-A) levels by 53% (P < 0.05). These data are consistent with an autocrine regulatory process mediated by NRG-1 activation of HER2/HER3 heterodimers expressed on developing human fetal lung epithelial cells. Receptor activation results in increased lung epithelial cell proliferation and volume density, and decreased SP-A production, a marker of type II pneumocyte differentiation.

Selective cloning of cell surface proteins involved in organ development: epithelial glycoprotein is involved in normal epithelial differentiation.

Coordinating the activities of neighboring cells during development in multicellular organisms requires complex cellular interactions involving secreted, cell surface, and extracellular matrix components. Although most cloning efforts have concentrated on secreted molecules, recent work has emphasized the importance of membrane-bound molecules during development. To identify developmental genes, we raised antibodies to normal embryonic pancreatic epithelial cell surface proteins. These antibodies were characterized and used to clone the genes coding for the proteins by a panning strategy. Using this approach, we cloned the rat homologue of the mouse epithelial glycoprotein (EGP). Our immunohistochemistry data, describing the expression of EGP during rat development, as well as our in vitro data, looking at the effect of the anti-EGP antibody and the extracellular domain of EGP on embryonic pancreatic epithelial cell number and volume, strongly suggest a role for EGP during pancreatic development.

Postnatal time frame for renal vulnerability to enalapril in rats.

Angiotensin-converting enzyme inhibition or angiotensin II type 1 receptor blockade in neonatal, but not in weaned, rats induces irreversible renal histologic abnormalities and an impaired urinary concentrating ability. The aim of the present study was to define the postnatal time frame when the rat kidney is vulnerable to an interruption of the renin-angiotensin system. Male Wistar rats received daily injections of enalapril (10 mg/kg, intraperitoneally) during different age intervals within 3 to 24 d,of age. Fluid handling and urinary concentrating ability, renal function under pentobarbital anesthesia, and kidney histology using stereologic techniques were evaluated in adult rats. Enalapril treatment within 3 to 13 d after birth induced abnormalities in renal function and morphology long-term, whereas treatment initiated at 14 d of age did not. The main histologic alterations were papillary atrophy, and a reduction in the volume of tubular epithelial cells in association with an increase in the proportion of interstitium, throughout the cortex and outer medulla. Functionally, the predominant defect was an impairment in urinary concentrating ability, which correlated with the degree of papillary atrophy. In conclusion, the vulnerable age interval for the induction of irreversible renal abnormalities by enalapril was the first 13 d after birth in the rat. This postnatal time span coincides with the completion of nephrogenesis and a period of marked tubular growth and differentiation, suggesting a pivotal role for angiotensin II in these processes.

Morphometric Analysis of the Epithelium of Mammary Fibroadenomas During the Proliferative and Secretory Phases of the Menstrual Cycle.

Fibroadenoma of the breast is a benign neoplasia with a mixture of glandular and mesenchymal elements of high incidence and is mainly present in young women. This aspect and the presence of estrogen and progesterone receptors reinforce its hormone dependency. Recent studies have demonstrated that the proliferative activity of the mammary epithelium is significantly increased during the luteal phase. In order to assess whether fibroadenomas behave like the normal breast under the cyclic action of hormones we counted the number of mitoses and calculated the nuclear volume of epithelial cells in both the proliferative and luteal phases. We selected 20 women with clinically diagnosed fibroadenomas, 10 in the proliferative phase and 10 in the secretory phase. Our results showed no significant differences in mitotic index or nuclear volume between the proliferative and the luteal phases of fibroadenomas. We therefore conclude that the epithelium of fibroadenomas behaves like a true neoplasia whose regulation may occur via paracrine mechanisms, which in turn would explain its self-limited growth.

Protective Effect of a Bioflavonoid Proanthocyanidin-BP1 in Glycerol-Induced Acute Renal Failure in the Rat: Renal Stereological Study

Renal ischemia as well as oxygen metaholites ploy an important role in renal Injury during myoglobinuric acute renal failure (ARF). On the other hand, flavonoids. a diverse group of constituents naturally occurring in plants, have a strong antioxidative activity, and have been implicated in vascular relaxation. In this study the protective effect of a new bioflavonoid proanthocvanidin-BP1 (BP1), extracted from seeds of grapes, was evaluated in glycerol-induced ARF in rats. Stereological methods were used to quantify changes in renal morphology associated with ARF. Volume density of tubular lumen and intratubular cast formations, nuclear parameters (area, diameter, volume) of epithelial cells in the cortical proximal tubules, and glomerular parameters (surface area, diameter, volume, perimeter) were estimated on kidney sections of rats treated either with 50% glycerol (8 ml/kg i.m.) alone. BP1 (20 mg/kg i.p.) in addition to glycerol, or BP1 alone. It was noted that the volume density of tubular lumen and cast formations were significantly lower (p < 0.001) in kidneys of the rats treated with BP1 in addition to glycerol, compared with those treated with glycerol alone. There were no significant differences in glomerular and nuclear parameters between glycerol treated, and BP1 in addition to glycerol treated rats. Renal function was significantly improved in rats treated with BP1 in addition to glycerol. The results suggest that BP1 is a protective agent in glycerol model of ARF. This effect is probably due to the antioxidative activity of BP1 and reduced toxicity of myoglobin in renal tissue. Moreover, it is possible that the ability of BP1 to protect the kidney is dependent upon renal vascular relaxation. The potential beneficial effects of bioflavonoid-BP1 demonstrated in experimental ARF could be considered in therapy of myoglobinuric ARF.

The determination of the cellular volume of avian, porcine and bovine phagocytes and bovine mammary epithelial cells and its relationship to uptake of tilmicosin.

In order to compare the intracellular concentration of antimicrobial agents in phagocytic and nonphagocytic cells, the knowledge of their cell volume is essential. For the first time, the determination of the avian, porcine, and bovine polymorphonuclear neutrophils (PMN), monocyte-derived macrophages, macrophages, and bovine mammary epithelial cell volume was performed using [3H]-water and [14C]-carboxyinulin. The comparison of all the cells showed that the PMN have a size range between 3.58 and 4.04 microL per mg of protein, and are smaller than the monocyte-derived macrophages and mammary epithelial cells (4.32-5.01 microL per mg of protein). The macrophages show the largest size (5.84-6.57 microL per mg of protein). The cellular uptake of tilmicosin in these cells was then determined. The examination of the intracellular/extracellular concentration ratios (Ci/Ce) after 4 h of incubation with 10 mg/mL of [14C]-labelled tilmicosin revealed that tilmicosin was well accumulated and showed a ratio of 137, 169 and 193 in avian PMN, porcine PMN, and bovine alveolar macrophages, respectively. The cellular uptake data also demonstrated that tilmicosin accumulated in nonphagocytic bovine mammary epithelial cells. The importance of the use of the appropriate species and cell type specific cell volume values for calculations was exemplified by calculating the Ci/Ce of tilmicosin using cell volume data found in the literature for human and mouse cells. The subsequent comparison of these data with the Ci/Ce calculated with the actual cell volume appropriate for the species tested revealed an under evaluation of 3-13% in monocyte-macrophages, an over evaluation of 7-18%, 16-31% and 69% in PMN, macrophages, and epithelial cells, respectively. This study highlights the importance of the proper cell volume in order to determine the Ci/Ce. Moreover, the cell volumes determined here for avian, porcine and bovine cells should facilitate further in vitro and in vivo cellular studies by veterinary researchers.

Cold inhibition of cell volume regulation during the freezing of insect malpighian tubules.

Cells in freeze-tolerant tissues must survive substantial shrinkage during exposure to the hyperosmolarity that results as solutes are excluded from extracellular ice. We investigated the possibility that this hyperosmotic shock elicits an acute regulatory volume increase (RVI) by monitoring the response of epithelial cell volume in the Malpighian tubules of the New Zealand alpine weta (Hemideina maori) during exposure to low temperature/hyperosmolarity (mimicking freezing conditions) or during an actual freeze/thaw cycle. The cross-sectional area of cells in isolated Malpighian tubules was measured using differential interference contrast microscopy. At 20 degrees C, cells held in saline containing 400 mmol1-1 glucose exhibit an RVI in response to hyperosmotic shock. Cross-sectional area decreased by 30% immediately after a change from iso-osmotic (0.7 osmol1-1) to hyper-osmotic saline (2.1 osmol1-1, equal to the osmotic shock encountered during freezing to -4 degrees C) and then returned to 21% below the control value 30 min after the exposure. Although substantial cellular function of Malpighian tubules was retained at low temperature (the rate of fluid secretion by isolated tubules at 4 degrees C was 72% of that measured at 20 degrees C), no RVI was observed at 0% degrees C; cross-sectional area was 39% below the control value immediately after the hyperosmotic exposure and 36% below the control value 30 min after hyperosmotic exposure. Dibutyryl cyclic AMP potentiated the RVI observed at 20 degrees C, but failed to elicit an RVI at 0 degrees C. A substantial RVI was also absent when the saline contained trehalose rather than glucose, regardless of whether the tubules were held at 20 degrees C or 0 degrees C. The cross-sectional area of cells in saline containing glucose remained at approximately 30% below the control value during an entire 30 min period of actual freezing to -4 degrees C, suggesting that an acute volume regulatory response was in fact inhibited during mild freezing. The inhibition of an acute RVI during mild freezing may serve to avoid the energetic expenditure associated with volume regulation at a time when the normal defence of cell volume appears to be unnecessary.

The effect of fasting and refeeding on thyroid follicule structure and thyroid hormone levels in young and old rats

In order to assess morphological and functional plasticity of the thyroid gland in aging the effects of fasting and refeeding on the thyroid morphology and thyroid hormone serum levels were compared with morphometry and RIA in young and old rats. Young (4 months) and old (24 months) male Wistar rats were fasted for 40 h and sampled, or fasted and fed thereafter for 4 or 24 h. In control (fed) old animals the thyroid follicules were larger, the follicular epithelium was smaller and colloid resorption was smaller than in young rats. ‘Thyroid activation index’ (epithelial volume/colloid volume ratio) was almost twice lower in the thyroids of control old rats. As the result of fasting, height, surface area and volume of epithelial follicular cells decreased in the thyroids of fasted young rats but not in old ones. On the contrary, in thyroids of fasted old rats the dimensions of epithelial cells did not change and thyroid colloid resorption was increased. After 24 h of refeeding, thyroid morphology in both young and old rats did not differ significantly as compared with control animals. Upon fasting, serum levels of thyroxine (T4) and triiodothyronine (T3) decreased by 28 and 38% in young and by 35 and 46% in old rats, respectively. However, T4 and T3 serum concentrations did not differ significantly between age groups in both fed and fasted states. During refeeding the increase in serum thyroxine concentration was delayed in old rats as compared with young ones. The results of morphological, morphometric and hormonal investigations indicate the existence of age-related changes in the structure and function of thyroid follicular cells.

Effects of chronic hypertonic stress on regulatory volume increase and Na-K-2Cl cotransporter expression in cultured corneal epithelial cells.

The optical and barrier properties of the corneal epithelium, which are essential for normal vision, depend on the maintenance of epithelial membrane integrity and tight junctional continuity between neighboring cells. Changes in tear film osmolality can occur in daily living (e.g., wind exposure, or swimming in fresh or salt water) and lead to alterations in epithelial cell volume that could affect epithelial topography and tight junction continuity. If these changes are uncompensated, they could compromise the corneal epithelial optical and barrier function. In some other cells, compensation includes reswelling to their isotonic volume through an appropriate regulatory volume increase (RVI). Frequently, this response is dependent on the activity of the bumetanide-sensitive, ouabaininsensitive Na-K-2Cl cotransporter.1

A novel approach to resolve cellular volume responses to an anisotonic challenge.

The ability of cells to regulate their volume in response to a change in external osmolality can be essential to the maintenance of their normal function. In the case of corneal epithelial cells, changes in tear film osmolarity can occur in daily living because of potential exposure to the drying effect of wind, fresh or salt water, etc. Such changes can lead to alterations in epithelial cell volume that affect cell function. For some cells, the inability to undergo volume regulation contributes to rapid cell death.1 KeywordsRegulatory Volume DecreaseEhrlich Ascites Tumor CellRapid Cell DeathOsmotic ResponseHypotonic ChallengeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Three-dimensional estimation of the glandular volume, and of the number and volume of epithelial cells in two glands from the antral mucosa of five healthy volunteers.

Specimens of antral mucosa were taken from five healthy volunteers and processed for microscopic evaluation. Consecutive 50-micron-thick sections were cut to estimate the volume of two antral glands. Two glands in each section were followed throughout by projecting their profiles from two microscopes mounted in parallel. The glandular volume was estimated using Cavalieri's principle. The mean volume of epithelial cells was estimated by systematic random sampling of the sections with an optical disector of known sample volume. The total number of cells per gland was calculated after determination of the volume of the glands and the mean volume of the epithelial cells. The mean volume of the antral glands was 13.6 +/- 1.67.10(6) microns3, whilst the mean volume of the epithelial cells was 1256 +/- 240 microns3. The total number of epithelial cells per gland was 11216 +/- 1104. In conclusion, using stereological methods, the total number of cells as well as the cell volume and the volume of antral glands can be determined in routine biopsy specimens of human stomach provided that the total depth of the mucosa is present in the section.

Activation of Na+/H+ Exchange Is Required for Regulatory Volume Decrease after Modest “Physiological” Volume Increases in Jejunal Villus Epithelial Cells

Epithelial cell volume increases that occur because of the uptake of Na+-cotransported solutes or hypotonic dilution are followed by a regulatory volume decrease (RVD) due to the activation of K+ and Cl- channels. We studied the relationship of Na+/H+ exchange (NHE) to this RVD in suspended guinea pig jejunal villus cells, using electronic sizing to measure cell volume changes and fluorescent spectroscopy of cells loaded with 2', 7'-bis(carboxyethyl)-5()-carboxyfluorescein to monitor intracellular pH (pHi). When the volume increase achieved by these cells during Na+ solute absorption was duplicated by a modest 5-7% hypotonic dilution, their pHi first acidified and then alkalinized. This alkalinization was blocked by 5-(N-methyl-N-isobutyl) amiloride (MIA; 1 microM), an inhibitor of NHE. The RVD subsequent to 5-7% hypotonic dilution was prevented by Na+-free medium and by amiloride and non-amiloride derivatives. The order of potency of these inhibitors was as follows: MIA > 5-(N,N-dimethyl) amiloride > cimetidine > clonidine, in keeping with the pattern attributable to NHE-1 as the isoform of NHE responsible for increase in pHi after modest volume increases. A substantial 30% hypotonic dilution caused acidification, and RVD following this larger volume increase was not affected by MIA. To assess the effect of hypotonicity on the activity of NHE, we measured the rate of MIA-sensitive pHi recovery from an acid load (dpHi/dt) in 5 and 30% hypotonic media. pHi recovery was faster in 5% hypotonic medium compared with isotonic medium and slowest in 30% hypotonic medium, which suggested that the activity of NHE was stimulated in the slightly hypotonic medium, but inhibited in the 30% hypotonic medium. To determine the role of activated NHE in RVD after a modest volume increase, cells were hypotonically diluted 7% in MIA to prevent RVD and then alkalinized by NH4Cl or acidified by propionic acid addition. Only after alkalinization was there complete volume regulation. We conclude that in Na+-absorbing enterocytes, the NHE-1 isoform of Na+/H+ exchange is stimulated by volume increases that duplicate the "physiological" volume increase occurring when these cells absorb Na+-cotransported solutes. The subsequent alkalinization of pHi is a required determinant of the osmolyte loss that underlies this distinct volume regulatory mechanism.

Mucociliary clearance during and after isocapnic hyperventilation with dry air in the presence of frusemide

We have previously shown that mucociliary clearance (MCC) decreased during and increased after isocapnic hyperventilation (ISH) with dry air, both in asthmatic and healthy subjects. Inhaled frusemide, an inhibitor of the Na+/K+/2Cl- and NaCl co-transporters on the basolateral membrane of the epithelial cell, prevents the airway narrowing provoked by ISH with dry air. The co-transport system controls epithelial cell volume and chloride secretion and, thus, frusemide has the potential to modify the rate of recovery of periciliary fluid volume during and after ISH with dry air, and hence affect MCC. Frusemide also blocks mediator release from mast cells, which may also modify the increase in MCC after ISH. Eleven asthmatic and 11 healthy subjects inhaled frusemide (35.7 +/- 0.44 mg) or its vehicle, from a Fisoneb ultrasonic nebulizer 30 min before ISH with dry air, on two separate occasions. MCC was measured using 99mTc-sulphur colloid and a gamma camera. Frusemide, compared to its vehicle, did not affect MCC during or 45 min after ISH. However, in the presence of frusemide, the onset of the increase of MCC after ISH was significantly delayed for approximately 10 min in the whole right lung (p < 0.002) and central region (p < 0.01) in the asthmatic but not in the healthy subjects. These findings could be explained by frusemide delaying the recovery of the periciliary fluid volume after ISH with dry air and/or interfering with the stimulus that causes the increase in MCC in the asthmatic subjects after ISH.

Influence of Prepubertal Dietary Regimen on Mammary Growth of Holstein Heifers

One hundred-sixteen Holstein heifers (mean BW, 175kg) were randomly assigned to diets of alfalfa silage or corn silage and were fed to gain approximately 725 or 950 g/d in order to study the influence of prepubertal diet and rate of gain on mammary growth and milk production. Blood was collected before puberty for hormone determination, and 8 heifers per group were killed at puberty for evaluation of tissue variables. Serum growth hormone was reduced, and IGF-I was increased, in the group of heifers reared at a high rate of gain on the corn silage diet. Accompanying the decline in growth hormone, total mammary parenchymal DNA and RNA was reduced in heifers reared at a high rate of gain on the corn silage diet. Mammary parenchyma in heifers of the latter group contained a greater volume of adipocytes and a lower volume of epithelial cells than did mammary parenchyma in heifers of other groups. Data are consistent with previous investigations that showed a deleterious effect of prepubertal rapid weight gain on mammogenesis when accompanied by excess body fat deposition. However, this effect did not cause a decline in subsequent milk production.

Regulatory volume decrease in a renal distal tubular cell line (A6) I. Role of K+ and Cl−

Changes in volume of A6 epithelial cells were monitored by recording cell thickness (Tc). The response of Tc to a reduction of the basolateral osmolality from 260 to 140 mosmol/kg was recorded while transepithelial Na+ transport was inhibited by 20 microM amiloride. With Cl--containing bathing media, this osmotic challenge elicited a rapid rise in Tc followed by a regulatory volume decrease (RVD). Substitution of SO4(2-) or gluconate for Cl- markedly reduced the RVD, whereas cells completely maintained their ability to regulate their volume after replacing Cl- by NO3(-). A conductive pathway for Cl- excretion is suggested, which is insensitive to NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid], an inhibitor of some types of Cl- channels. Ba2+ (5 or 20 mM) reduced the RVD. A more pronounced inhibition of the RVD was obtained with 500 microM quinine, a potent blocker of volume-activated K+ channels. K+-induced depolarization of the basolateral membranes of tissues incubated with SO4(2-)-containing solutions completely abolished the RVD. Noise analysis in the presence of Ba2+ showed the activation of an apical K+ conductive pathway. These results demonstrate that cell volume regulation is controlled by processes involving Cl- and K+ excretion through conductive pathways.