Ion transport in primary cultures from human sweat gland coils studied with X-ray microanalysis

Chloride secretion in primary cultures of cells originating from the secretory coil of human sweat glands was investigated by electron probe X-ray microanalysis. The total intracellular Cl concentration was lowered by muscarinic agonists (carbachol and acetylcholine), as well as by the calcium ionophore A23187. The muscarinic agonists also lowered the cellular K concentration. Cl^- secretion induced by these agonists could be inhibited by the chloride channel blocker NPPB. After cAMP stimulation, the frequency distribution of the Cl concentration changed from Gaussian to bimodal, indicating that cAMP induces Cl^- secretion only from a subpopulation of the cells. Also ATP stimulated Cl^- secretion, indicating the presence of purinergic receptors. The results suggest that some of the cells in addition to Ca²⁺ -regulated Cl^- channels also possess cAMP-activated Cl^- channels. Hence, the primary cultures still possess the Cl^- transport mechanisms known to be present in intact glands. It can, however, not be excluded that some coil cells have acquired ductal characteristics during culture.     

Changes in elemental content during apoptotic cell death studied by electron probe X-ray microanalysis

Recent data suggest that changes in ionic content, primarily potassium, play a pivotal role in the progression of apoptosis. However, the changes in total element content, i.e., sodium (Na), magnesium (Mg), phosphorous (P), chlorine (Cl), potassium (K), and calcium (Ca), during apoptosis have not been evaluated. Electron probe X-ray microanalysis (EPXMA) was used to measure total element content in U937 cells before and after the induction of apoptosis. As an experimental model we used U937 cells irradiated with ultraviolet (UV) light. Apoptosis was evaluated with phase-contrast microscopy, with scanning and transmission electron microscopy, and with the fluorescent dye bisbenzimide (Hoechst 33342). Plasma membrane permeability as a measure of cell death was determined by trypan blue dye exclusion. To investigate element content with EPXMA, cells were cryoprepared, i.e., cryofixed and freeze-dried, and analyzed as whole cells using a scanning electron microscope. We found that the UV irradiation induced rapid (within 2 h) morphological changes associated with apoptosis, such as plasma membrane blebbing, condensation of the chromatin, and the formation of membrane-bound apoptotic bodies. At this time, 95% of the apoptotic cells excluded trypan blue dye. EPXMA results demonstrated that UV light-irradiated apoptotic cells (cells with membrane-bound apoptotic bodies) had a lower Cl content (P < 0.001) and K content (P < 0.001) and a higher Na content (P < 0.001) in comparison with nonirradiated control cells. Also, P and Ca content was higher in apoptotic cells than in control cells, but this difference did not reach statistical significance. No differences were found in Mg. These data indicated that morphological changes characteristic of apoptotic cell death are related with significant changes in sodium, chlorine, and potassium content. In addition, we demonstrated that these changes in elemental composition were not associated with loss of cell membrane integrity.     

Ion transport by rabbit colon

Summary Descending rabbit colon, stripped ofmuscularis externa, absorbs Na and Cl under short-circuit conditions and exhibits a residual ion flux, consistent with HCO₃ secretion, whose magnitude is approximately equal to the rate of active Cl absorption. Net K transport was not observed under short-circuit conditions. The results of ion replacement studies and of treatment with ouabain or amiloride suggest that the short-circuit currentI _sc is determined solely by the rate of active Na transport and that the net movements of Cl and HCO₃ are mediated by a Na-independent, electrically-neutral, anion exchange process. Cyclic AMP stimulates an electrogenic Cl secretion, abolishes HCO₃ secretion but does not affect the rate of Na absorption under short-circuit conditions. Studies of the effect of transepithelial potential difference on the serosa-to-mucosa fluxesJ _sm ⁱ of Na, K and Cl suggest thatJ _sm ^Na ,J _sm ^K and one-third ofJ _sm ^Cl may be attributed to ionic diffusion. The permeabilities of the passive conductance pathway(s) are such thatP _KP _NaP _Cl=1.00.070.11. Electrolyte transport byin vitro rabbit colon closely resembles that reported fromin vivo studies of mammalian colon and thus may serve as a useful model for the further study of colonic ion transport mechanisms.     

Ion compartmentation in Porphyra umbilicalis determined by electron-probe X-ray microanalysis

The ion composition of cell compartments in the intertidal red alga Porphyra umbilicalis adapted for two weeks in 3.5 x artificial seawater was determined by X-ray microanalysis of unfixed, frozen, bulk specimens. A procedure is described for the calculation of ion concentrations in the main cell compartment, cytoplasm, vacuoles and plastid. The results indicate high K⁺ and low Na⁺ concentrations in cytoplasm and plastid. Sodium ions are preferentially localized in vacuoles. Both, vacuoles and plastid contain high Cl^- concentrations.     

Ion compartmentalization in frog oocytes as demonstrated by X-ray microanalysis

The distributions of K, Na, Mg and Ca within frog ovarian and oviductal oocytes were studied by electron probe wavelength dispersive X-ray microanalysis. An important heterogeneity could be found both in nuclear and jelly coated oocytes. The highest K, Mg and, to a lesser extent, Na concentrations were found in the pigmented area of the peripheral cytoplasm. There is a certain correlation between the distribution of K and Mg. The concentration of K (but not of Na) in the nucleus was higher than that in the non-pigmented cytoplasm. The distribution of Ca was rather uniform. The high amounts of K, Na and S determined in the oocyte jelly coat seem to have become accumulated by ion-exchange mechanism. Oocyte pigment granules are believed to be the site of ion compartmentalization and to play a role in regulation of intracellular ionic composition.     

Subcellular distribution of zinc in rat prostate studied by X-ray microanalysis. I. Normal prostate

Synopsis Zinc is distributed subcellularly throughout the lateral prostate of the rat in both the stromal and epithelial elements. The connective tissue appears to be a major store of zinc. Within the epithelium, the highest concentrations of the element are found in the lysosomes, nucleoli, nuclear chromatin, secretory granules and luminal secretion. Histochemical studies indicate that the metal is bound relatively tightly within the nucleoli (associated with RNA) and in the secretory products of the cytoplasm. Changes in tissue zinc concentration, observed by other workers, following changes in various external stimuli, may not necessarily be reflected by proportionate changes in epithelial concentrations. The role of zinc in the epithelium is considered to be at least two-fold: firstly, for incorporation into vital cellular mechanisms necessary for cell maintenance and, secondly, for involvement in secretory products. It is also possible that the metal participates in the physiology of the sub-epithelial stroma.     

Post-mortem elemental redistribution in rat studied by X-ray microanalysis and electron microscopy

Summary Post-mortem elemental redistribution in various tissues from rat was studied by means of electron probe X-ray microanalysis, and correlated with morphological changes in these tissues. Pancreas, liver and cardiac muscle were removed from the animal either immediately, or after some hours after death. Elemental distribution at the cellular level was studied by X-ray microanalysis of thick cryosections. Calcium redistribution at the subcellular level was studied using tissue fixed with glutaraldehyde/oxalate. In all tissues, post-mortem redistribution of electrolytes had taken place within 2 h. The cellular concentrations of Na, Cl and Ca increased markedly, those of Mg and K decreased; no significant changes were found in the concentrations of P and S. The number of oxalate precipitates (indicating the presence of calcium) increased both in the mitochondria and in the cytoplasm and endoplasmic reticulum, reaching a maximum at 2 h. Morphological changes included mitochondrial swelling and vesiculation of the endoplasmic reticulum. Since the post-mortem ion shifts are similar to those encountered in some diseases and types of cell injury, great care has to be taken in the interpretation of X-ray microanalytical results from autopsy material.     

Mucosubstances of rabbit granulocytes studied by means of electron-microscopic radioautography and X-ray microanalysis.

Rabbit bone marrow cells have been studied by means of light- and electron-microscopic radioautography and X-ray microanalysis. Carrier free sulfuric acid was used as the radioactive precursor in this experiment. Immature granulocytes showed more active incorporation than mature ones. Silver grains were observed in the Golgi apparatus and granules in three kinds of granulocytes. Electron-microscopically, immature granules showed the incorporation of inorganic sulfate, while mature ones did not. Sulfur was detected in all kinds of granules of the three granulocyte types by X-ray microanalysis. It is concluded that incorporated inorganic sulfur may be utilized for the synthesis of acid glycosaminoglycans and the sulfur detected by X-ray microanalysis may be that contained in the acid glycosaminoglycan. The sulfur detected in the specific granules of the heterophil probably derives from proteins or polypeptides incorporating sulfur containing amino acids.     

X-ray microanalysis of cAMP-induced ion transport in cystic fibrosis fibroblasts.

cAMP-induced ion transport in normal and cystic fibrosis (CF) fibroblasts was investigated by X-ray microanalysis. Stimulation with cAMP causes an increase in cellular Na content and a decrease in cellular Cl and K content. No significant difference in response between CF and normal cells was noted. In this respect, fibroblasts differ from epithelial cells, where cAMP-induced Cl- efflux blocked in CF patients. Isoproterenol produced similar changes in Na and K content as cAMP, but did not effect Cl content.     

X-ray microanalysis of etoposide-induced apoptosis in the PC-3 prostatic cancer cell line.

Apoptosis comprises a critical intracellular defense mechanism against tumourigenic growth. We have been interested in the relationship between morphological changes and intracellular concentration of several cations after etoposide-induced apoptosis in androgen-independent prostate cancer cells. SEM and X-ray microanalysis were performed on freeze-dried PC3 cells after etoposide treatment, and correlated with the morphological features observed after examination by light and fluorescence microscopy. Cell viability assays were also performed. A significant decrease in intracellular Cl(-) and K(+)and a progressive increase in Mg(2+) and Na(+) were observed, with parallel changes in cellular volume as cells passed through three morphological stages of apoptosis. The use of EPXRMA made it possible to evaluate alterations in element composition in prostate cancer cell apoptosis and may be a helpful tool for further studies on apoptosis in prostate cancer.     

Ion transport by rabbit colon. I. Active and passive components.

Descending rabbit colon, stripped of muscularis externa, absorbs Na and Cl under short-circuit conditions and exhibits a residual ion flux, consistent with HCO3 secretion, whose magnitude is approximately equal to the rate of active Cl absorption. Net K transport was not observed under short-circuit conditions. The results of ion replacement studies and of treatment with ouabain or amiloride suggest that the short-circuit current ISC is determined solely by the rate of active Na transport and that the net movements of Cl and HCO3 are mediated by a Na-independent, electrically-neutral, anion exchange process. Cyclic AMP stimulates an electrogenic Cl secretion, abolishes HCO3 secretion but does not affect the rate of Na absorption under short-circuit conditions. Studies of the effect of transepithelial potential difference on the serosa-to-mucosa fluxes Jism of Na, K and Cl suggest that JNasm,JIsm and one-third of JCl-sm may be attributed to ionic diffusion. The permeabilities of the passive conductance pathway(s) are such that Pk:PNa:PCl= 1.0:0.07:0.11. Electrolyte transport by in vitro rabbit colon closely resembles that reported from in vivo studies of mammalian colon and thus may serve as a useful model for the further study of colonic ion transport mechanisms.     

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.     

Mucosubstances of rabbit granulocytes studied by means of electron-microscopic radioautography and X-ray microanalysis

Summary Rabbit bone marrow cells have been studied by means of light-and electron-microscopic radioautography and X-ray microanalysis. Carrier free sulfuric acid was used as the radioactive precursor in this experiment. Immature granulocytes showed more active incorporation than mature ones. Silver grains were observed in the Golgi apparatus and granules in three kinds of granulocytes. Electron-microscopically, immature granules showed the incorporation of inorganic sulfate, while mature ones did not. Sulfur was detected in all kinds of granules of the three granulocyte types by X-ray microanalysis. It is concluded that incorporated inorganic sulfur may be utilized for the synthesis of acid glycosaminoglycans and the sulfur detected by X-ray microanalysis may be that contained in the acid glycosaminoglycan. The sulfur detected in the specific granules of the heterophil probably derives from proteins or polypeptides incorporating sulfur containing amino acids.     

Carbonic anhydrase supports electrolyte transport in Drosophila Malpighian tubules. Evidence by X-ray microanalysis of cryosections

Electron probe X-ray microanalytical studies on the role of carbonic anhydrase in electrolyte transport in the cells of Drosophila Malpighian tubules indicate that carbonic anhydrase delivers protons and bicarbonate ions to ion transport systems in the cell membrane. After injection and after feeding acetazolamide or hydrochlorothiazide, known inhibitors of carbonic anhydrase, the contents of potassium, magnesium and chloride in the apical cytoplasm and in the cytoplasm close to the basal plasma membrane decreased. We explain our measurements by the hypothesis of a basal Mg-H-antiport system in parallel with Cl-HCO(3)-antiport, inhibitable by DIDS. Zinc is supposed to enters cells and intracellular Zn storage vacuoles by a negatively charged Zn-anion-complex in exchange for HCO(3)(-) ions. This antiport is inhibitable by SITS. The content of the Zn storage vacuoles is acid, as shown by red fluorescence after incubation of Malpighian tubules with acridine orange. Red fluorescence is absent after preincubation in a medium containing an inhibitor of carbonic anhydrase. Carbonic anhydrase was demonstrated cytochemically in the Golgi-ER complex, Golgi vesicles and intercellular space. We suppose that carbonic anhydrase is synthesized and stored in the Golgi-ER-complex from where it is released into the tubule lumen.     

Study of maturation of membrane transport function in red blood cells by X-ray microanalysis

Summary Red blood cells of certain species of animals, such as dogs and cats, contain low potassium and high sodium, whereas the erythropoietic stem cells giving rise to these cells are of high potassium type. This paper examines the sequence of membrane transport changes during erythropoiesis by analyzing the K, Na and Fe in single bone marrow cells, reticulocytes and mature red blood cells with X-ray microanalysis. The relationship between K/Na ratios and Fe/(K+Na) ratios were examined by X-ray microanalysis. The K/Na ratios give a measure of the membrane cation transport function. The Fe/(K+Na), which is analogous to hemoglobin concentration, gives an index of maturation stage. The relationships between K/Na and Fe/(K+Na) in the marrow cells of normal adult dog and those of a phenylhydrazine-injected dog with accelerated erythropoiesis show that the modification of cation composition occurs after the initiation of hemoglobin synthesis but before its completion. Similar relationships in the reticulocytes obtained from phenylhydrazine-injected dogs as well as from newborn dogs show a consistent decrease in K/Na with increased Hb, indicating a drastic change in cation composition during the maturation of the reticulocytes. Therefore the modification in membrane transport function must have occurred before or during the formation of reticulocytes.     

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.     

Chloride secretion in the submandibular gland of adult and early postnatal rats studied by X-ray microanalysis

Submandibular acinar cells of 1-day-old, 7-day-old, and adult rats were analyzed with X-ray microanalysis after stimulation with carbachol for different time periods (2–7 min). In unstimulated animals, marked differences in elemental content between compartments could be observed: secretory granules had a higher Ca and lower P and K content than other cell compartments. Comparison between different age groups showed significant differences for Ca, which increased with age in all compartments; Mg increased with age in the secretory granules and the apical cytoplasm. Only the glands from adult animals showed a significant effect of cholinergic stimulation: a transient decrease in Cl and K. The Cl concentration in the secretory granules decreased to 60% of the control value, which suggests that the granules release Cl upon stimulation. In young animals, no or little change in elemental distribution was observed after stimulation. This may indicate that Cl-secretion mechanisms are much less prominent in young animals. The ultrastructure of submandibular secretory granules depends on the preparation method: condensed and electrondense in freeze-substituted unfixed tissue, decondensed and more translucent in aldehyde-fixed tissue. This may indicate that the granules can transport water, and swell during the process of aldehyde fixation.     

The formation of type-I concretions in Drosophila Malpighian tubules studied by electron microscopy and X-ray microanalysis

There are two types of concretions in Drosophila Malpighian tubules: Type-I concretions originate in the distal segments of the anterior tubules, type-II concretions in the adjacent transitional segment between the apical microvilli. Type-I concretions are formed with the aid of carbonic anhydrase within intracellular vesicles, which migrate to the apical cell membrane where they are discharged into the lumen by exocytosis. The carbonic anhydrase inhibitors acetazolamide or hydrochlorothiazide prevent the formation of concretions by interruption of bicarbonate supply. In addition, the formation of concretions can be reduced by feeding with sodium cellulose phosphate.