Biomedical subjects

G M Roomans

Publications and source records attributed to G M Roomans.

At least 91 records · Page 5Linked to original sources

PubMed1996

In vitro systems and cultured cells as specimens for X-ray microanalysis.

In vitro systems and cultured cells are recognized as useful systems in many areas of biomedical research, including X-ray microanalysis. To be reliable, in an vitro system should have an elemental composition close to that of the tissue in situ, react in the same way to stimuli, and retain the in situ regulation of ion transport. In the present paper, four of the most commonly used in vitro systems will be reviewed: incubated tissue slices (liver and pancreas), isolated glands (submandibular gland acini, sweat glands), primary cell cultures (sweat glands, endometrium), and cell lines (the colon cancer cell line T84, immortalized sweat gland cells). Incubation of tissue slices of liver in Krebs-Ringers buffer caused a significant increase in Na and Cl and a decrease in K. Initially, these changes were also observed in the pancreas, but here the values gradually returned to normal. Isolated submandibular gland acini, and isolated sweat gland ducts and coils react in a similar way to stimulation as their in situ counterparts. In primary cultures of coil cells, however, part of the cell population acquires different ion transport characteristics. Technically simplest is the use of cell lines originating from cancer cells (e.g., the T84 cell line) and immortalized cell lines. X-ray microanalysis not only confirms data on ion transport obtained with other techniques, but adds the possibility to investigate the presence of subpopulations within a culture.

Animals↗

PubMed1996

A yohimbine-dependent, UK14,304 induced ion transient in HT29 cells studied by X-ray microanalysis.

The response of HT29 cells to alpha 2-adrenoceptor agonists was investigated by X-ray microanalysis. Treatment of the cells with 10 microM UK14,304, an alpha 2-agonist, resulted in a rise of intracellular Cl and Na, suggesting an antisecretory effect. When the cells were exposed to yohimbine or RX821002, both of which are alpha 2-antagonists, the former had no effect, whereas the latter produced a minor decrease of the cellular Na, Cl and K concentrations. However, when the cells were pretreated with yohimbine, UK14,304 induced a transient change in ion concentrations, which consisted of a rapid increase of intracellular Cl, Na and a decrease of K at 1 and 5 minutes. Concurrently, a marked increase in intracellular Ca was found. This transient change could also be induced by different doses of UK14,304 (100 nM or 10 microM) after preincubation with yohimbine (0.3 microM or 3 microM). In contrast, UK14,304 did not elicit a similar response after pretreatment of the HT29 cells with RX821002. Also, yohimbine did not affect the ion content of the cells after pretreatment with UK14,304. These results indicate that yohimbine may have a specific effect by which the cells are sensitized to the ion transient induced by UK14,304.

Brimonidine Tartrate↗

PubMed1996

Preparation of cultured smooth muscle cells from human myometrium for X-ray microanalysis.

Methodological aspects of the use of X-ray microanalysis in physiological and pharmacological experiments on cultured myometrial cells were investigated. Cultured human myometrial cells were grown from biopsies after detaching the fibroblasts. Of the cultured cells, 95-98% showed desmin-like immunoreactivity. Transmission electron microscopy showed that subcultured cells were different from myometrial cells in situ. The effects of washing the cells to remove external salt-rich medium were investigated. All solutions removed the external medium, resulting in lower concentrations of Na and Cl. In the cells washed with 0.3 M mannitol, most of the elemental concentrations were significantly lower than in their unwashed counterparts and those washed in the other solutions. In cells washed in either 0.15 M ammonium acetate or distilled water, no significant differences in P and K compared with their unwashed counterparts were found. There were also no significant differences between cells washed in ammonium acetate and in distilled water. In subsequent experiments ammonium acetate was used. Incubation of cells in standard Ringer's solution resulted in an increase in Na and Cl, and a decrease in K, concomitantly with an increase in Ca. Although Ringer's solution per se can elicit changes in diffusible elements in the cells, physiological and pharmacological effects of oxytocin could still be detected in Ringer's solution. However, effects of oxytocin were different when the experiment was done in culture medium, instead of in Ringer's solution.

Calcium↗

PubMed1995

Effects of K(+)-induced depolarization and purinergic receptor activation on elemental content in insulin-producing RINm5F-cells.

X-ray microanalysis was used to detect elemental changes in the insulin-producing tumor cell-line RINm5F. To improve discrimination between mobile ions and ions bound to macromolecules a new approach was employed, consisting of multivariate statistical analysis of correlations between the concentrations of Na, Mg, P, S, Cl, K, and Ca. RINm5F cells, cultured on Formvar-coated titanium grids, were stimulated with high K+ or ATP, that are both known to stimulate insulin release. The buffers used contained Ca2+ or one of the Ca(2+)-analogues Sr2+ and Ba2+, to represent Ca2+ uptake in response to stimulation. After stimulation the cells were shock-frozen and freeze-dried overnight. Incubation for 10-20 seconds in a Ca(2+)-containing buffer did not significantly affect elemental composition, whereas cellular Mg, P and K decreased in a Sr(2+)-containing buffer. Depolarization with high K+ concentration caused an increase in the cellular Na content, both in Ca(2+)- and Sr(2+)-containing buffers, but not in the buffer where Ca2+ had been replaced by Ba2+. X-ray microanalysis is useful for detection of elemental changes subsequent to stimulation of cultured cells. Moreover, multivariate statistical analysis strengthens the idea that stimulation of RINm5F cells causes redistribution of ions possibly due to changes in the state of binding of some elements to cellular proteins.

Adenosine Triphosphate↗

PubMed1995

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 Ca(2+)-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.

Adenosine Triphosphate↗

PubMed1995

Calcium-activated chloride fluxes in cultured NCL-SG3 sweat gland cells.

The dependence of chloride permeability of the human sweat gland cell line NCL-SG3 cell line on cytosolic free calcium ([Ca2+]i) was investigated. X-ray microanalysis, fura-2 fluorescence and patch clamp methodology were used. Carbachol and A23187 decreased cellular Cl and K for cells grown on permeable supports, but carbachol had no effect on cells grown on impermeable supports. In perforated patch experiments with impermeable supports, ATP and calcium ionophores increased the inward current (ic) whereas carbachol had no effect. ic was unaffected by cation channel blockers or removal of extracellular Na+ but was blocked by chloride channel blockers. Lowering bath Ca2+ decreased ic. On raising bath Ca2+ ic and [Ca2+]i responded with a transient rise which was not blocked by La3+ or D-600. La3+, but not D-600, blocked the entry of Mn2+. K(+)-depolarization and Bay-K-8644 had little effect on [Ca2+]i. The rise in [Ca2+]i may be mediated primarily via depletion operated Ca(2+)-channels. Irrespective of substrate NCL-SG3 cells have a chloride permeability which depends on [Ca2+]i.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1995

Dye-coupling between term pregnant human myometrial cells before labor: carboxyfluorescein versus lucifer yellow.

Term pregnant human myometrial cells in whole mounts were microinjected by pressure with the fluorescent probes Lucifer Yellow and carboxyfluorescein. Tissues obtained from acute and elective sections displayed weak dye-coupling when injected with Lucifer Yellow. Injection of carboxyfluorescein into cells from the elective sections resulted in a more extensive dye-coupling than that observed with Lucifer Yellow. These results indicate that term pregnant human myometrial cells are metabolically coupled before labor and carboxyfluorescein is superior to Lucifer Yellow in detecting the coupling.

Cell Communication↗

PubMed1995

Use of post mortem and in vitro tissue specimens for X-ray microanalysis.

Post mortem changes in the distribution of elements, as well as changes induced by dissection and incubation of tissue slices, were investigated by X-ray microanalysis of brain tissue, liver, pancreas and submandibular gland. Dissection itself causes minor changes in the intracellular ionic concentrations, but even a brief exposure of dissected tissue slices to a physiological buffer causes an increase in intracellular Na and Cl and a decrease in intracellular K concentration. The effect is most marked in brain tissue and least marked in submandibular gland slices. Incubation in fluid resembling the extracellular compartment in its ion composition results in a further increase of Na and Cl in brain tissue (cortex and hippocampus) and liver; in pancreas and submandibular gland, on the other hand, a stable situation throughout 2 h of incubation can be obtained. Incubation at lower temperature, and exchanging NaCl in the incubation medium for Na gluconate, has only relatively minor effects on the intracellular K/Na ratio. Exchanging the NaCl for K gluconate results in a high intracellular K/Na ratio throughout the incubation, but morphological evidence of tissue oedema was nevertheless observed. Physiological changes in intracellular ion content induced by cholinergic stimulation are similar in in vitro preparations as compared with stimulation in situ. The effect of dissection and brief incubation on the ionic composition of brain tissue is less pronounced 6 h after death than in a living, anaesthetized animal, but this is largely due to the post mortem changes that already have taken place.

Animals↗

PubMed1995

Morphometric analysis of gap junctions in nonpregnant and term pregnant human myometrium.

BACKGROUND AND METHODS: Some structures in the human myometrium, namely; caveolae, extracellular space and gap junctions were quantified in tissues from nonpregnant women, women who were not in labor, and women in spontaneous labor or oxytocin-induced labor, in order to reveal morphologic differences that would contribute to explaining the events leading to labor. Transmission electron micrographs taken from tissues from twenty-four pregnant and ten nonpregnant women were studied. RESULTS: Differences were observed in cell growth which was accompanied by enlargement of the extracellular space, and in gap junctions. Gap junctions were demonstrated in nonpregnant women, and even in one postmenopausal woman. Their frequency and size increased gradually between groups of pregnant women in the order of non-labor, oxytocin-induced labor and spontaneous labor. The increase in size of the junctions was less than the increase in number. CONCLUSIONS: Gap junctions are most frequently found in human myometrial cells in spontaneous labor, which strongly suggests a role for these structures in the termination of pregnancy. The effect of oxytocin on the appearance of gap junctions was negligible; hence, the contribution of oxytocin to establish myometrial contractile synchronicity is thought to be minor. Caveolae are probably not relevant for the initiation of labor as their distribution did not differ between the groups.

Adolescent↗

PubMed1995

In vitro effect of Hepes buffer on maintaining the number of gap junction plaques in human myometrium at term.

In human myometrium, at various stages of labour and in correlation with the concentration of progesterone and estradiol in maternal blood the formation of gap junctions has been described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated human myometrium at term to Hepes buffer, on the number of gap junction plaques. Biopsies of myometrium were obtained from 5 pregnant women at term: they had an elective caesarean operation in the 37th or 40th week of pregnancy. The biopsies were immersed immediately in Hepes buffer, trimmed under a stereo microscope into small strips and kept in warm (37 degrees C) oxygenated Hepes buffer supplemented with glucose (0.01 mM). Then some strips of myometrium were incubated at 37 degrees C for 10, 20, 40, 55, and 180 min. The number of gap junction plaques were counted: it decreased in strips of myometrium after 10, 20, 40, 55, and 180 min of in vitro experiment (P < 0.01 vs control). In some of our experiments, the decrease in quantity of gap junction plaques was very dramatic and far below 50% of their numbers found in control specimens but never reached the zero value.

Biopsy↗

PubMed1995

Human skin physiology studied by particle probe microanalysis.

Particle probe methods (electron probe and proton probe X-ray microanalysis) have been applied to investigate the distribution of elements and water over the different layers of the epidermis. For major elements, electron probe X-ray microanalysis (XRMA) provides the advantage of superior spatial resolution, but for trace element analysis the more sensitive proton probe (particle induced X-ray emission, PIXE) analysis has to be used. On a dry weight basis, the concentration of S is rather constant across the epidermis, whereas the concentrations of P, K, Cl and Na show gradients with high levels in stratum germinativum (basale) and stratum spinosum but low levels in the stratum granulosum and stratum corneum. Essentially, Fe and Zn are confined to the basal region in normal skin. The concentration of Ca, however, increased steadily from the basal region to the stratum corneum. The probe technique allows quantitative analysis of stratum-specific changes in elemental content in a variety of pathological conditions, e.g., changes induced by nickel, detergents and other chemicals, or in psoriatic skin. Of particular interest are findings of increased Fe and Zn in non-involved psoriatic skin. Since the different layers of the skin have different elemental concentrations and react differently under pathological conditions, the probe techniques are far superior to bulk chemical analysis in elucidating physiological and pathological processes in the skin.

Apoptosis↗

PubMed1994

Comparative study of freeze-substitution techniques for X-ray microanalysis of biological tissue.

A comparative study of some aspects of freeze-substitution as a preparative method for X-ray microanalysis of diffusible ions in biological specimens was carried out. Four substitution fluids were compared with each other and with cryosections. As criteria, elemental ratios as determined by X-ray microanalysis of thin sections of mouse pancreas were used. Freeze-substitution for 3 weeks in diethyl ether or for 2 days in tetrahydrofurane gave results comparable to those obtained in cryosections. Slow warming to room temperature after freeze-substitution in tetrahydrofurane gave slightly better results than fast warming. Freeze-substitution in acetone resulted in noticeable loss and redistribution of ions and freeze-substitution in methanol gave very poor results.

Animals↗

PubMed1994

Use of fluorescent europium chelates as labels in microscopy allows glutaraldehyde fixation and permanent mounting and leads to reduced autofluorescence and good long-term stability.

In the present study europium chelates were introduced as alternative fluorescent labels for microscopy and their effect on enhanced autofluorescence caused by the glutaraldehyde fixative was investigated. Glutaraldehyde fixation was used to stabilize the cells for a permanent mount after the immunocytochemical reaction. The europium signal in time-resolved fluorescence microscopy was shown to be free of autofluorescence when strong cross-linking fixation with glutaraldehyde was used and the signal-to-background ratio obtained was 2,400 or better. It was also shown that the europium signal was stable in daylight and at room temperature. Fluorescent europium chelate used in this experiment provides excellent contrast and long-term stability for the samples with glutaraldehyde fixation and permanent mounting.

Antibodies, Monoclonal↗

PubMed1994

Effect of extracellular K+ on hatching and outgrowth of mouse blastocysts in vitro.

The dependence of blastocyst development on the extracellular Na+/K+ ratio was investigated in an in vitro system. Hatching and outgrowth of mouse blastocysts was enhanced at Na+/K+ ratios between 3 and 10 compared to the ratio of about 25 typical for most culture media and serum. At a Na+/K+ ratio of 2, blastocyst hatching and outgrowth were inhibited. The requirement of blastocyst development for relatively high extracellular K+ concentrations agrees with the fact that K+ concentrations in oviduct and uterine secretions are higher than in serum. The findings can also be relevant in optimizing in vitro culturing techniques for blastocysts.

Animals↗

PubMed1994

Morphometric analysis of gap junction density in human myometrium at term.

In the human myometrium, the formation of gap junctions in the various stages of labor and, in correlation with the concentration of progesterone in the maternal blood, has not been described adequately. The accepted hypothesis that raised concentrations of progesterone in maternal blood in animals prevent the formation of gap junctions was scrutinized in human myometrium. Myometrial tissue was examined by electron microscopy for the presence of gap junctions and analyzed morphometrically in 17 women who had an elective or emergency cesarean operation at the 38th or 39th week of pregnancy. The concentrations of progesterone and estradiol in the maternal and cord blood was measured by means of radioimmunoassay. The frequency and area of gap junctions varied in relation to the presence or absence of labor. In the myometrium of women in labor, gap junctions were most frequent (5.2/1000 microns of cell membrane), their area largest (0.32 microns 2/1000 microns of cell membrane) and their fractional area greatest (0.20%). When the women were not in genuine labor or when contractions were weak, the percentage fractional areas of gap junctions were low (0.020-0.090) and the gap junctions very small. In the pre- and active phases of labor, the concentration of progesterone in maternal blood was significantly higher (p < 0.025 and 0.03 respectively) than in women who were not in labor. Hence, despite high concentrations of progesterone in the maternal and cord blood at term, the myometrium presented numerous gap junctions of relatively large area.

Adult↗

PubMed1994

Direct intracellular injections for studying human myometrial gap junctions prior to labor.

Direct intracellular microinjection of a fluorescent dye (Lucifer Yellow) was performed on ex-situ muscle strips from term pregnant women not in labor. The aim was to characterize the intracellular distribution of LY to obtain criteria for an intracellular injection in whole mounts and ultimately to study the gap junctional communication between myometrial cells in those tissues. Fifteen injections performed on biopsies from ten cases showed a well-bordered fusiform shape and were considered to be intracellular. The intercellular spread of the dye into an adjacent cell was observed in three injections (three cases). The average cell dimension was 284 +/- 95 microns for length and 5 +/- 1.5 microns for width (n = 17). The intracellular injection was confirmed by light microscopy of cross sections. It is concluded that limited coupling exists between myometrial cells of women prior to labor.

Cell Communication↗

PubMed1994

Use of in vitro systems for X-ray microanalysis.

The use of X-ray microanalysis in human pathology may require the use of cryoprepared tissue. Often it is impossible to carry out freezing of the tissue in an optimal way, and in addition, it is difficult to carry out experiments in living patients. The use of in vitro systems and cell cultures allows separation of the process of tissue removal and the freezing procedure, and also makes testing of pharmacological or toxic substances possible. In experiments with animal tissue it was shown that incubation in a physiological buffer induced significant changes in the concentrations of Na, K, and Cl. In general, the concentrations of Na and Cl increased, those of K decreased. Prolonged incubation of brain tissue (cortex and hippocampus) and of liver resulted in further changes of the cellular ion contents in the same direction. Incubation of pancreas and submandibular gland resulted in a limited reversal of the changes induced by dissection. The submandibular gland in vitro showed the same response to cholinergic stimulation as the gland in situ. The use of cell cultures for X-ray microanalysis is briefly reviewed and illustrated by an example of analysis of an immortalized sweat gland cell line. It was shown that these cells respond to stimulation by cAMP with loss of Cl and that this response was unaffected by the type of substrate the cells were grown on.

Animals↗

PubMed1994

Changes of ion and water content of mouse intestinal cells after pilocarpine and isoproterenol stimulation.

Cytoplasmic monovalent ion and water contents in morphologically defined mice jejunal cells were measured by X-ray microanalysis in order to gain insight into the cell-type specificity of intestinal electrolyte transport mechanisms. Ion and water contents were measured independently. It was found that in some cases net fluxes of ions and water do not correspond to the assumption of constant osmotic activity of cytoplasmic Na and K ions. Stimulation of secretion with the cholinergic secretagogue pilocarpine resulted in efflux of Cl- from an influx of K+ into crypt enterocytes containing small secretion granula (crypt A cells). No significant changes in ion concentrations were found in crypt enterocytes without secretion granula (crypt B cells). Crypt A cells were more likely to be stimulated by pilocarpine than crypt B cells, and the basolateral K+ efflux pathway in crypt A cells appeared to be rate-limiting. In villus enterocytes, pilocarpine stimulated Cl- efflux. Isoproterenol caused marked changes in the cytoplasmic Cl content of all epithelial cells. These changes were reversed by inhibition of adenylate cyclase by alloxan, with the sole exception of Cl increase in villus absorptive cells. The results are consistent with an cAMP-mediated stimulated secretion in crypt epithelial cells and a predominantly cAMP-independent stimulation of absorption in villus cells. The results obtained suggest a transcellular route of Cl absorption in the mouse jejunum.

Animals↗