A morphometric study of 24-hour variations in subcellular structures of the rat pancreatic acinar cell

Summary Subcellular structures of pancreatic acinar cells were examined at six evenly spaced time points in the 24-h period (light cycle: 06.00 h–18.00 h) in four Wistar male rats at each time point. At each sampling point, the area and circumference of acinar cell bodies and the area, number and circumference of their cytoplasmic organelles were measured using a semiautomatic computer system for morphometry and a point-counting method.The area, number and circumference-area ratio of the cytoplasmic organelles were subject to strong circadian variations, and the cellular area and circumference exhibited weak circadian variations. Variation pattern of the cytoplasmic organelles suggested an intracellular route for secretory proteins during a 24-h span. From the results it was possible to divide the 24-h period into three stages. 1. The resting or protein synthetic stage (00.00 h to 08.00h): the area of the rough surfaced endoplasmic reticulum (rER) was strongly increased, and that of zymogen granules was clearly decreased. 2. The granule accumulation stage (08.00h to 16.00h): the area of the rER was markedly decreased; that of zymogen granules was increased. 3. The secretion stage (16.00 h to 00.00): as a result of the release of zymogen granules from the acinar cell, the area of zymogen granules decreased, and that of the rER increased. The relationship between the area of the rER and zymogen granules varied in a reciprocal manner. Other cytoplasmic organelles, namely the Golgi complex, condensing vacuoles, mitochondria and lysosomes also varied prominently during the 24-h span, corresponding to variations in the rER and zymogen granules.     

Single-channel recordings from the apical membrane of the toad urinary bladder epithelial cell

Summary The patch-clamp technique for the recording of single-channel currents was used to investigate the activity of ion channels in the intact epithelium of the toad urinary bladder. High resistance seals were obtained from the apical membrane of tightly stretched tissue. Single-channel recordings revealed the activity of a variety of ion channels that could be classified in 4 groups according to their mean ion conductances, ranging from 5 to 59 pS. In particular, we observed highly selective, amiloridesensitive Na channels with a mean conductance of 4.8 pS, channels with a similar conductance that were not Na-selective and channels with mean conductance values of 17–58 pS that were mostly seen after stimulation of the tissue with vasopressin or cAMP. When inside-out patches from the apical membrane were exposed to 110mm fluoride, large conductances (86–490 pS) appeared.     

A morphometric study of developing pancreatic acinar cells of rats during prenatal life

Summary The pancreatic acinar cells of rat embryos obtained at 15, 17, 19 and 21 days of gestation have been examined using fine-structural and morphometric techniques.Morphometric analysis demonstrates significant variations in the average volume of the cell, nucleus and cytoplasm, and the volume, surface and numerical densities of various cytoplasmic organelles during fetal life. In particular, the volume and surface densities of rER exhibit maximal values at 19 days of gestation, suggesting that secretory proteins are produced most actively at this time. Further-more, membrane continuity between the nuclear envelope and rER is frequently discernible in acinar cells, indicating that at this stage the rER is mainly derived from the nuclear envelope. Zymogen granules first appear at 17 days of gesstation. By 21 days, they occupy the greater part of the cytoplasm of the acinar cells, no polarity being seen in their distribution pattern. No direct evidence for the secretion of zymogen granules has been observed during fetal life.It therefore appears that membrane transport involved with intracellular movement of newly synthesized proteins from rER via the Golgi complex to zymogen granules occurs in one direction and lacks regulation.     

Measurement of the composition of epithelial cells from the toad urinary bladder

Summary Two methods are described by which epithelial cells from toad urinary bladders can be obtained for analysis of their intracellular water and electrolyte contents. In the first, a method similar to that described in 1968 by J. T. Gatzy and W. O. Berndt, sheets of epithelial cells are scraped from bladders after incubation in sodium Ringer's and collagenase (400 mg/liter). The scraped cells were incubated under various conditions and their composition subsequently determined. Oxygen consumption was also measured. In the second method, epithelial cells were scraped from hemibladders removed from chambers. These cells were then analyzed without further incubation. The morphology of epithelial cells obtained by each method is illustrated. Both methods yield similar results and evidence is provided that the derived intracellular values obtained truly reflect the composition of the epithelial cells.     

Intramembrane particle structures in epithelial cells of the toad urinary bladder: a quantitative freeze-fracture study

Freeze-fracture electron microscopy reveals intramembrane particle arrays in basal membranes of granular epithelial cells as well as both upper and lower plasma membranes of the underlying basal cells in the toad urinary bladder. These particle arrays are morphologically indistinguishable from the luminal membrane aggregates which are known to be associated with antidiuretic hormone (ADH)-stimulated water transport. In both granular and basal cells particle arrays are frequently located in and/or around the openings of vesicular and/or tubular structures fused to the plasma membranes, suggesting that they may be transferred from the cytoplasm by membrane fusion. Quantification of cytoplasmic aggrephores in control granular cells shows that they can be numerous and as close to the basolateral membrane as they are with the luminal membrane, to which they are known to fuse and deliver aggregates upon ADH stimulation. Aggrephore-like tubules were also found in the basal cells. Particle array densities were quantified for 6 pairs of control and ADH-stimulated hemibladders. At least 1440 microns 2 area of plasma membrane for each membrane domain was examined. Results indicate that the presence of these particle arrays in granular and basal cell membranes is highly variable and that exposure to ADH does not cause a statistically significant increase in their frequency.     

Electron microprobe analysis of the different epithelial cells of toad urinary bladder

Summary The electrolyte composition of toad urinary bladder epithelial cells has been measured using the technique of electron microprobe analysis. Portions of hemi-bladders, which had been mounted in chambers and bathed with a variety of media, were layered with albumin solution on their mucosal surfaces and immediately shock-frozen in liquid propane at –180°C. From the frozen material 1–2m thick cryosections were cut and promptly freeze-dried for 12 hr at –80°C and 10^–6 Torr. Electron microprobe analysis using a scanning electron microscope, an energy dispersive X-ray detector, and a computer programme, to distinguish between characteristic and uncharacteristic radiations, allowed quantification of cellular ionic concentrations per kg tissue wet wt by comparison of the intensities of the emitted radiations from the cells and from the albumin layer. Granular, mitochondrial-rich, and basal cells, and the basal portions of goblet cells, showed a similar composition, being high in K (about 110mm/kg wet wt) and low in Na (about 13mm/kg wet wt). The apical portions of goblet cells were higher in Ca and S and lower in P and K, presumably reflecting the composition of the mucus within them. With Na-Ringer's as the mucosal medium, cells gained Na and lost K, when their serosal surfaces were exposed to ouabain, 10^–2 m. Replacement of mucosal Na by choline virtually prevented these ouabain-induced changes. Cellular ion contents were unchanged when Na in the serosal medium was replaced by choline. No differences in Na and K concentrations were detected between nuclei and cytoplasm. These results provide independent support for the hypothesis that the cellular Na transport pool in toad bladder epithelial cells derives exclusively from the mucosal medium and that no important recycling of Na occurs from the serosal medium to the cells.     

A comparative study in morphometric grading of transitional cell carcinoma of the urinary bladder

To overcome the considerable observer inconsistency in the histologic grading of transitional cell carcinomas, the value of four different morphometric grading methods was investigated in 61 tumors of the bladder. Only two methods showed satisfactory reproducibility. Both methods, one based on random nuclear sampling and the other on selective nuclear sampling, showed an increase in the mean and standard deviation of the nuclear area with higher tumor grades (P less than .00001). Morphometric classification of the learning set (44 cases) was in agreement with the unequivocally assessed histologic grade in 35 cases (79.5%) using random sampling and in 38 cases (86.4%) using selective sampling. By reducing the grading classes to "low" (grades 1 and 2) and "high" (grade 3) and by introducing a classification probability threshold (0.80), an accurate morphometric classification was achieved in 38 cases (86.4%) using random sampling and in 41 cases (93.2%) using selective sampling. Of the 17 cases with histologic grading discrepancies, all 10 low-grade tumors (with discrepancies of grade 1 versus grade 2) were correctly classified as low-grade carcinomas by both of the morphometric methods; in the remaining 7 cases, with low-versus-high discrepancies (grade 2 versus grade 3), the selective method yielded better correlation with the tumor stage and clinical follow-up. It is concluded that morphometric classification is an acceptable alternative for histologic grading by pathologists, provided that the reproducibility of the method is confirmed. Although both random and selective sampling yielded satisfactory classifications, the selective method gave more reliable results as confirmed by the clinical behavior.     

Effects of voltage clamping on epithelial cell composition in toad urinary bladder studied with X-ray microanalysis

Toad urinary bladder epithelial cells were incubated in Na Ringer's with the serosal surface of the epithelium clamped at either +50 mV, O mV (short-circuited) or –50 mV with respect to the mucosal surface. Following incubation, portions of tissue were coated with an external albumin standard and rapidly frozen. Cryosections were freeze-dried and cell composition determined by x-ray microanalysis. Cell water and ion contents were unaffected when tissues were short-circuited rather than clamped close to their open-circuit potential difference (+50 mV). Incubation with vasopressin at +50 mV, and under short-circuit conditions, caused Na uptake without cell swelling or gain in Cl. Clamping at –50 mV resulted in uptake of water and ions, with considerable variation from cell to cell. These variations in cell composition were exacerbated by vasopressin. The greater the increase in water content, the greater the rise in cell Cl. However, there was no consistent pattern to the associated changes in cation contents. Most cells gained some Na. In some cells, this gain was accompanied by an increase in K. In others, the gain of Na was predominant and cell K content actually fell. At –50 mV with ouabain, many of the cells also gained water. As was found in our earlier study with ouabain under short circuit conditions (Bowler et al., 1991), there was considerable variation in the extent of the Na gain and K loss; some cells were largely depleted of K while in others the K content remained relatively normal. These results indicate differences between granular cells in the availabilities in the plasma membranes of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control.This work was supported by a grant from the Health Research Council of New Zealand. Purchase of the equipment was made possible through grants from the Medical Research Council of New Zealand, the Medical Distribution Committee of the Lottery Board, the University Grants Committee, the Telford Trust, the New Zealand Neurological Foundation and the National Heart Foundation. We are grateful for the excellent technical assistance of Ms. S. Zellhuber-McMillan.     

An ultrastructural study of periderm granules in the regenerating feather of the jungle fowl

Summary Periderm granules in the support cells of regenerating feathers of mature male Jungle Fowls were studied ultrastructurally and histochemically. Histochemical results showed the absence of carbohydrate and lipid, and the presence of protein in the periderm granules. The periderm granules were measured at successive levels of feather regeneration. The mean size of the periderm granules increased significantly as the regenerating feather matured, and this observation was suggestive of a storage function, perhaps of surplus or waste protein. The cells in which the periderm granules are found also contain glycogen. There are numerous desmosomal junctions on their interdigitating plasma membranes. These transient cells may collect waste, provide nutrition, and serve as a protective barrier for the definitive cells of the regenerating feather.     

Effects of potassium-free media and ouabain on epithelial cell composition in toad urinary bladder studied with X-ray microanalysis

Summary The technique of X-ray microanalysis was used to study the composition of toad urinary bladder epithelial cells incubated in Na Ringer's and K-free medium, with and without ouabain. Following incubation under short-circuit conditions, portions of tissue were coated with an external albumin standard and plunge-frozen. Cryosections were freeze-dried and analyzed. In Na Ringer's, granular and basal cells, and also the basal portion of the goblet cells, had similar water and ion compositions. In contrast, mitochondria-rich cells contained less Cl and Na. On average, the granular cells and a subpopulation of the basal cells lost K and gained Na after ouabain and in K-free medium alone. However, there was considerable variation from cell to cell in the responses, indicating differences between cells in the availabilities of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control. In contrast, the compositions of both the low Cl, mitochondria-rich cells and a sub-population of the basal cells were little affected by the different incubation conditions. This is consistent with a comparatively low Na permeability of these cells. The results also indicate that (i) much, if not all, of the K in the dominant cell type, the granular cells, is potentially exchangeable with serosal medium Na, and (ii) Na is accumulated from the serosal medium under K-free conditions. They also provide information about the role of the (Na–K)-ATPase in the maintenance of cellular K in K-free medium, being consistent with other evidence that removal of serosal medium K inhibits transepithelial Na transport by decreasing Na entry to the cells from the mucosal medium, rather than solely by inhibiting the basolateral membrane (Na–K)-ATPase.     

Fluorescent measurements of intracellular free calcium in isolated toad urinary bladder epithelial cells

Summary Sodium-calcium exchange has been suggested to play a pivotal role in the regulation of cytosolic free calcium (Ca _f ) by epithelial cells. Using isolated epithelial cells from the toad urinary bladder, Ca _f has been measured using the intracellular Casensitive fluorescent dyes Fura 2 and Quin. 2. Dye loading did not alter cell viability as assessed by measurements of ATP and ADP content or cell oxygen consumption. When basal Ca _f was examined over a wide range of cell dye content (from 0.04 to 180 nmol dye/mg protein) an inverse relationship was observed. At low dye content, Ca _f was 300–380 nM and, as dye content was increased, Ca _f progressively fell to 60 nM. Using low dye content cells, in which minimal alteration in Ca steady state would be expected, the role for plasma membrane Na–Ca exchange was examined using either medium sodium substitution or ouabain. While medium sodium substitution increased Ca _f , prolonged treatment with ouabain had no effect on Ca _f despite a clear increase in cell sodium content. The lack of effect of ouabain suggests that Na–Ca exchange-mediated Ca efflux plays a minimal role in the regulation of basal Ca _f . However, exchange-mediated Ca efflux may play a role in Ca _f regulation when cytosolic calcium is elevated.     

Electrical characteristics of the apical and basal-lateral membranes in the turtle bladder epithelial cell layer

(1) The active transport of Na⁺ across the turtle bladder epithelial cell layer consists of a passive entry step through a Na⁺-selective path in the apical membrane and an active extrusion step through Na⁺ pump-containing path in the basal-lateral membrane together with some back-leakage through the paracellular spaces and tight junctions between the epithelial cells. This hypothesis has now been verified qualitatively and to some extent, quantitatively by the use of an intracellularly-located microelectrode in conjunction with a conventional assembly of extracellularly-located macroelectrodes mainly in short-circuited bladders bathed by Na⁺-rich Ringer media. Under these conditions, the intracellular potential (V_sc) averaged 38.4 mV with the cell electronegative; the fractional resistance of the apical membrane ($\text{?R}{}_{\mathrm{<mtext>a</mtext>}}$) averaged 0.55; while the concomitant transepithelial parameters, short circuiting current (I_sc) and electrical conductance (G_t), average 68.6 μA/cm² and 0.98 mS/cm², respectively. (2) The relation between these parameters and the transepithelial flow of Na⁺ (orI_sc) is evoked by blocking Na⁺ entry into the cell (by the mucosal addition of amiloride or removal of mucosal Na⁺). Amiloride-induced blockade of the Na⁺ entry step results in a rapid hyperpolarization of the cell interior during which V_sc = —79.1 mV and $\text{?R}{}_{\mathrm{<mtext>a</mtext>}}\text{= 0.92}$. I_sc and G_t (equivalent to the shunt conductance under these conditions) averaged 5 μA/cm² and 0.35 mS/cm², respectively. The entire process is reversible on re-admission of Na⁺ entry into the cell. (3) A slow depolarization of the cell interior in the period of blocked transapical Na⁺ entry is opposite to that expected from an electroneutral Na⁺-K⁺ exchanging pump; but instead is the predictable response of an electrogenic Na⁺ pump in parallel with a passive K⁺-selective conductance in the basal-lateral membrane. (4) The electrogenicity concept is substantiated after pretreatment of the bladder with serosal ouabain, which changes the response of V_sc to amiloride (from the aforementioned biphasic response) to a step-function response, attributable mainly to the development of a slowly dissipating K⁺ diffusion potential across the basallateral membrane. (5) Under open-circuit conditions, the electronegativity of cell to mucosa (V_a) is a linear inverse function of the electropositivity of serosa to mucosa (V_t). For V_t ? 100 mV, V_a is positive; and for V_t between ?30 and 90 mV, V_a is negative.     

Electron-microscopic study of the development of the periarteriolar zone in splenic white pulp of rats

Summary To obtain more information concerning the origin of interdigitating cells, the postnatal development and morphology of the periarteriolar lymphatic sheath in splenic white pulp of rats was investigated by light- and electron-microscopy. Special attention was paid to the ontogeny of interdigitating cells. The spleens of the animals were studied in the age range from 1 h to 28 days after birth.The splenic white pulp of neonatal rats consists only of a few reticuloblasts, which are concentrically arranged around central arterioles. After 21 h an increase in promonocytes and monocytes was noted. Between the fifth and seventh postnatal day monocytogenic cells with a light and almost translucent cytoplasm appear, which display long cytoplasmic projections between the adjacent cells. Neighbouring lymphocytes often insert finger-like processes into the invaginated cellular membrane of these transitional forms. This intimate cellular contact is supported by zonulae occludentes. These cells represent transitional forms between monocytes and interdigitating cells.From seven days of age onwards typical interdigitating cells were present as in adult animals. After the differentiation into an inner and outer periarteriolar lymphatic sheath, the T-cell-dependent area of splenic white pulp has attained its adult appearance and further changes are not to be expected.On the basis of these findings, it is highly probable that interdigitating cells develop via transformation of monocytes.This investigation was supported by the Deutsche Forschungsgemeinschaft (^*He 537)     

Fura-2 handling in a polarized epithelial barrier: the toad urinary bladder.

Toad bladders sacs were placed inside quartz cuvettes. When fura-2 AM was added to the mucosal compartment, low temperature (4 degrees C) almost completely blocked the transepithelial transfer of fluorescence observed at 20 degrees C (20 degrees C = 371 +/- 56, 4 degrees C = 29 +/- 29 fluorescence intensity in arbitrary units (FIAU), excitation at 340 nm, emission at 510 nm). Simultaneously, fluorescence accumulation inside the tissue was significantly higher (20 degrees C = 25 +/- 5, 4 degrees C = 91 +/- 24% increase on basal levels (%IBL)). When fura-2 AM was added to the serosal side, low temperature also reduced the serosal to mucosal transfer (20 degrees C = 149 +/- 36, 4 degrees C = 61 +/- 35 FIAU). Nevertheless, in this situation tissue accumulation, that was significantly higher that the one observed when fura-2 AM was added to the mucosal side, was reduced at low temperature (20 degrees C = 300 +/- 30, 4 degrees C = 48 +/- 7 %IBL). Spectral analysis of the mucosal and serosal compartments indicated that free fura-2 was transferred from the intracellular to the serosal compartment, but not to the mucosal one. These results indicate that fura-2 appears as a useful tool to evaluate the cellular distribution and traffic of polycyclic charged and non-charged molecules.     

Isolation and characterization of granules of the toad bladder

Summary The electron-dense granules that lie just below the apical plasma membrane of granular epithelial cells of toad urinary bladder contribute glycoproteins to that apical membrane. Also, exocytosis of granules (and tubules) elicited by antidiuretic hormone potentially doubles that apical surface, during the same period the transport changes characteristic of the hormonal response occur.Granules separated from other membrane systems of the cells provide the material to assess the importance of the granules as glycocalyx precursors and in hormone action. We used isosmotic media to effect preliminary separations by differential centrifugation. Then granules were isolated by centrifugation on self-forming gradients of Percoll of decreasing hypertonicity.We find qualitative and quantitative changes in protein composition and enzymic activities in the isolated fractions. The primary criterion for granule purification was electron microscopic morphology. In addition, polypeptide species found in the granule fraction are limited in number and quantity. The granules are enzymically and morphologically not lysosomal in nature. Granules may provide the glycoproteins of the apical glycocalyx but they differ from the isolated plasma membrane fraction enzymically, in protein composition and in proportion of esterified cholesterol.We conclude that the granules are not average plasma membrane precursors. Their role in the membrane properties of the toad urinary bladder may now be evaluated by characterizing permeability and other properties of the isolated organelles.     

Nocodazole inhibition of the vasopressin-induced water permeability increase in toad urinary bladder

Nocodazole is a synthetic antitumor drug that binds rapidly to tubulin. When this drug is applied to toad bladder prior to vasopressin stimulation it inhibits the vasopressin response. A maximum inhibition (68%) is reached with a dose level of 10 μ/ml applied one-half hour prior to vasopressin stimulation (20 mU/ml). This compares with an inhibition of 50% seen with a 3-h exposure of the tissue to colchicine (0.1 mM) prior to stimulation with vasopressin. Application of nocodazole (1 μ/ml) 3 min after hormonal stimulation shows no inhibition of the response at one-half hour past stimulation. These data support the view that microtubules are involved in the vasopressin-induced increase in water permeability in toad bladder and also indicate that this involvement is limited to the period prior to or directly after stimulation.     

Some effects of ouabain on cellular ions and water in epithelial cells of toad urinary bladder

The Journal of Membrane Biology - Transepithelial sodium transport was virtually abolished when toad urinary hemibladders, mounted in chambers and short-circuited, were exposed on their serosal...     

Electron-microscopic studies on the relationship between the frequency of parathyroid storage granules and serum calcium levels in the rat

Summary In the rat parathyroid, the mean number of storage granules (NSG) per chief cell has been electron-microscopically studied and correlated with the mean serum calcium level (SCL). In animals given 4% CaCl₂ plus vitamin D₂ for 3 days, SCL is significantly elevated and NSG is increased. When these animals are injected with 2% EDTA, SCL is lowered to 8 mg/dl, but NSG is not affected; in those injected with 4% EDTA, however, SCL declines to a minimum (5.8 mg/dl) after 30 min, and NSG is also decreased. Control SCL are 8.9 mg/dl. These results indicate that storage granules may not be released until SCL is depressed to a certain level.In rats 3 weeks after castration, the chief cells show hyperplastic changes and SCL is at a low concentration (8.0 mg/dl). NSG, however, remains almost within control limits. Castrated animals injected with 4% EDTA show a hypocalcemia and a decrease in NSG, but NSG gradually recovers over a period of 6h. These data suggest that storage granules can be produced even under lower calcium concentrations. It is concluded that storage granules may be constantly produced and stored, and are released only as an emergency supply of hormone.