Biomedical subjects

E Gruenstein

Publications and source records attributed to E Gruenstein.

At least 19 recordsLinked to original sources

PubMed1992

Histamine H1 receptors in UC-11MG astrocytes and their regulation of cytoplasmic Ca2+.

Experiments were carried out on UC-11MG human astrocytoma cells, a continuous cell line that expresses a broad range of the biochemical and electrophysiological properties found in well-differentiated astrocytes. Because of a number of recent reports that astrocytes may express receptors for a variety of neuro-active substances, we measured the effects of 12 different neurotransmitters on intracellular free Ca2+ (Ca2+i) in UC-11MG cells. Of these neurotransmitters only histamine was found to have a significant effect. Further characterization of the nature of the histamine response showed that UC-11MG cells express mepyramine-sensitive H1 receptors the activation of which causes both mobilization of Ca2+ from intracellular stores and entry of Ca2+ from the extracellular solution. No evidence was found for the presence of H2 receptors. The Ca2+i response was maximal at 300 microM histamine and was attenuated by increasing cell density. We suggest that this neurotransmitter may play a role in astrocytic function in the human CNS.

Astrocytes↗

PubMed1992

Histamine stimulates glycogenolysis in human astrocytoma cells by increasing intracellular free calcium.

Astrocytes from a variety of sources, including the human UC-11MG astrocytoma line, express receptors for histamine on their plasma membranes, but the function of these receptors is largely unknown. Here we report studies on the effect of histamine on newly synthesized glycogen in the human astrocytoma-derived cell line, UC-11MG. We have found [3H]glycogen hydrolysis with a EC50 of 2 microM and a maximum effect of 30% at 300 microM histamine. The glycogenolytic effect of histamine was completely blocked by the H1 receptor antagonist, mepyramine, and was insensitive to the H2 receptor antagonist, cimetidine. Histamine-induced glycogenolysis was significantly reduced in the absence of extracellular Ca2+ and the residual response could be accounted for by Ca2+ released from intracellular stores. The Ca2+ ionophore, ionomycin, induced a similar concentration-dependent increase in both intracellular Ca2+ concentration and in glycogenolysis. These results suggest that one function of astrocytic histamine receptors in vivo may be the stimulation of glucose release from astrocytes, and that this process is mediated by increased intracellular free Ca2+. The glycogenolytic effect of histamine and other neurotransmitters in different systems, and the possible implication of astrocytic glycogenolysis in the pathophysiology of ischemia are discussed.

Astrocytoma↗

PubMed1992

Intracellular acidification is associated with enhanced morphological transformation in Syrian hamster embryo cells.

A series of studies has indicated that the frequency of morphological transformation induced by chemical carcinogens in early passage Syrian hamster embryo (SHE) cells is significantly higher when these cells are cultured in medium of reduced bicarbonate concentration and pH (6.70) compared with cells cultured in medium of higher pH. It has also been shown that intercellular gap junctional communication is decreased in these cells when they are cultured at pH 6.70 compared with medium of higher pH. The purpose of the studies reported here was to characterize the effect of changing extracellular pH on intracellular pH in SHE cells. The frequency of morphological transformation induced by benzo(a)pyrene was established at various extracellular pHs and compared with intracellular pH values. Cells cultured in medium of pH ranging from 6.70 to 7.35 were loaded with the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, and either the steady-state intracellular pH values or the kinetics of change in intracellular pH following refeeding of the cultures with medium of pH ranging from pH 6.70 to pH 7.35 was monitored via image analysis techniques. Results from these studies indicate that, at culture medium pH above 6.95, SHE cells were relatively insensitive to changes in extracellular pH, maintaining an intracellular pH of 7.30 to 7.35 in medium containing 0% serum or pH 7.05 to 7.10 in medium containing 20% fetal bovine serum. At extracellular pHs below 6.95, intracellular pH decreased and, in the presence of serum, equilibrated with extracellular pH. The decrease in intracellular pH was closely associated with an increase in benzo(a)pyrene-induced morphological transformation frequency observed in parallel studies. These results indicate that SHE cells have active intracellular pH regulatory activities and suggest that intracellular acidification plays a role in the increased frequency of transformation observed in SHE cells cultured under acidic conditions.

Animals↗

PubMed1992

Association of cytoplasmic free Ca2+ gradients with subcellular organelles.

Previous investigations have identified gradients of intracellular free (Ca2+)i (Ca2+i) in the cytoplasm of human fibroblasts. In this study we have compared the spatial distribution of these gradients with the subcellular distribution of cytoplasmic organelles. Using the Ca(2+)-sensitive dye fura-2 and organelle-specific fluorescent dyes, we have found that the highest Ca2+ concentrations are found in the perinuclear cytoplasm and that these regions co-localize with the Golgi apparatus. The area occupied by the endoplasmic reticulum, which includes the Golgi region plus an adjacent area, is also significantly elevated above the average cellular (Ca2+)i. Most mitochondria are located in regions different from those with the highest (Ca2+)i. A variety of phenomena which could have given rise to artifactual (Ca2+)i gradients have been ruled out, including compartmentalization of fura-2 in subcellular organelles, incomplete hydrolysis of fura-2AM esters, and the presence of pH gradients which might change the Ca2+ binding characteristics of fura-2. The existence of gradients in (Ca2+)i between ER and Golgi containing regions of the cytoplasm supports the hypothesis (Sambrook: Cell 61:197-199, 1990) that the traffic of membrane bound vesicles from ER to Golgi is directed by local variations in (Ca2+)i.

Calcium↗

PubMed1991

Inhibition of lactate-induced swelling by dichloroacetate in human astrocytoma cells.

High levels of tissue lactate exacerbate tissue damage that results from cerebral ischemia and reperfusion injury that follows. Post-ischemic treatment with dichloroacetate (DCA) facilitates a decrease in lactate in the central nervous system (CNS) of animals during reperfusion following experimental ischemia, thus it may help to ameliorate ischemic cell damage. It has been suggested that the lactate lowering effect is mediated through a stimulatory effect of DCA on pyruvate dehydrogenase (PDHC) activity. We have studied such a hypothesis in a human astrocytoma derived cell line, UC-11MG. Under conditions resembling those of the ischemic tissue (i.e. high lactate and low pH) these cells accumulate lactate, driven by the inwardly directed proton gradient, and swell as a consequence of the osmotic effect of intracellular lactate. We have demonstrated that DCA increases PDHC activity and also reduces lactate-induced swelling. However, we also found that these two effects could be uncoupled and that the ability of DCA to prevent swelling is still present in the absence of any stimulation of PDHC. We also demonstrated that DCA competitively inhibits the uptake of lactate (Ki = 1.9 mM) and increases the efflux of lactate in a trans-acting manner that suggests the presence of a lactate-DCA exchange. We present a mechanism by which reduction in the rate of lactate uptake could account for the observed inhibition of swelling. This effect of DCA on lactate transport indicates another possible mechanism of action for DCA in facilitating the decrease in lactate observed in vivo during reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Astrocytoma↗

PubMed1990

Analysis of dystrophin in fast- and slow-twitch skeletal muscles from mdx and dy2J mice at different ages.

Muscles from mdx, control, and dy2J/dy2J mice at different ages were analyzed for dystrophin in an attempt to relate the chronology of the protein expression with the final phenotypes in regenerated, normal, and dystrophic muscle, respectively. Immunostaining and gold staining of electrophoresis gels were carried out in the investigation. At 5, 25, and 219 days of age, control muscles exhibited dystrophin bands in both the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles. Muscles from the mdx mice at comparable ages (8, 28, and 217 days) never exhibited bands for dystrophin, although titin, nebulin, myosin, and other protein bands were present at intensities comparable to those in control muscles. The dystrophin band was present in both the EDL and SOL from dy2J/dy2J dystrophic mice. As indicated by the present study, the dystrophin deficiency from mdx tissue is not transient. This suggests that dystrophin is not necessary for the success of mdx muscle regeneration.

Age Factors↗

PubMed1990

Intracellular Ca2+ measurement with Indo-1 in substrate-attached cells: advantages and special considerations.

The dual emission, Ca2+ sensitive fluorescent dye, Indo-1, offers several potential advantages over its dual excitation analogue, Fura-2. Most notable among these advantages are increased speed of measurement using dual wavelength photometry and the absence of a requirement for special quartz optics. Despite these potential advantages, only a tiny fraction of the microscopic studies of intracellular free calcium ([Ca2+]i) on substrate-attached cells has employed Indo-1. Among the reasons for the infrequent use of Indo-1 are the fact that it exhibits somewhat different spectral properties in the cytosol than it does in extracellular buffers, and the notion that it is much more sensitive to photobleaching than Fura-2. We report here that under our experimental conditions, Indo-1 photobleaching is small and does not noticeably affect the measurement of free Ca2+, even after 30 minutes of continuous illumination. We also report a new method for creating in situ standard curves that is easy, reproducible, and yields values for [Ca2+]i that are identical to those obtained with Fura-2. In addition, we have found that Indo-1 is less subject than Fura-2 to compartmentalization within subcellular organelles. These results provide baseline data to take advantage of the significant improvement afforded by Indo-1 in the measurement of rapid [Ca2+]i responses and the avoidance of compartmentalization artifacts during experiments of long duration.

Calcium↗

PubMed1990

An alkaline pH-activated Cl(-)-anion exchanger regulates pH homeostasis in fibroblasts.

Alkalinization of the cytoplasm in human fibroblasts activates Cl- efflux. The pathway of Cl- efflux is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or by removal of Cl- from the extracellular medium, but it is insensitive to the Cl(-)-cation cotransport inhibitor bumetanide. These data indicate that the alkali-stimulated Cl- pathway is a Cl(-)-anion exchanger. One function of this pathway appears to involve intracellular pH homeostasis. Thus, after alkalinization of the cytoplasm, recovery of the pH to basal levels is impaired by DIDS or by removal of HCO3- from the extracellular medium, but it is unaffected by the Na(+)-H+ exchange inhibitor amiloride. Reports from a number of laboratories, including our own, have described a biphasic efflux of Cl- in which kinetics suggested the presence of two subcellular Cl- compartments. The data presented in this study provide a mechanism to explain this kinetic behavior. The biphasic Cl- efflux kinetics are the result of a transient alkalinization of the cytoplasm associated with a change in PCO2 as cells are transferred from the incubator to the bench top for the efflux assay.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1990

Histamine receptors in human fibroblasts: inositol phosphates, Ca2+, and cell growth.

Histamine stimulated inositol phosphate formation by human skin fibroblasts. The effect of histamine was reduced but still readily apparent in the absence of extracellular Ca2+. Histamine caused a transient increase in intracellular free Ca2+ as detected by indo-1 and fura-2 fluorescence studies on cell populations and on individual cells. Similar increases were observed in the absence of extracellular Ca2+, indicating that the effect was primarily due to mobilization of Ca2+ from intracellular stores, presumably by inositol trisphosphate (IP3). The effects of histamine on phosphoinositide metabolism and intracellular Ca2+ were inhibited by pretreatment of the cells with phorbol esters, suggesting that the histamine receptor in fibroblasts is subject to feedback regulation by protein kinase C. Histamine inhibited the incorporation of [3H]-thymidine into DNA. The effects of histamine on inositol phosphate formation, intracellular Ca2+, and thymidine incorporation were blocked by the H1 receptor antagonist mepyramine. Our results indicate that human skin fibroblasts have H1 receptors coupled to the formation of inositol phosphates and mobilization of intracellular Ca2+. We suggest that this H1 receptor also mediates a block of the cell cycle and that histamine may play a physiological role in the regulation of fibroblast proliferation.

Aminopyridines↗

PubMed1989

Electrophysiological and biochemical characterization of a continuous human astrocytoma cell line with many properties of well-differentiated astrocytes.

Astrocytes comprise about 25% of the cellular volume of the brain, and their main function is to maintain homeostasis of the neuronal environment. These cells are commonly identified on the basis of their membrane electrical properties and the presence of specific proteins. We have characterized the human astrocytoma cell line designated UC-11MG and have shown these cells have many of the traits of differentiated astrocytes. Many of the UC-11MG cells have a large resting membrane potential, averaging -74 mV. The slope of the Em vs log [K]o cuve was 58.5 mV per decade [K]o. The cells were inexcitable when exposed to brief depolarizing current pulses. The astrocytoma traits are virtually identical to those previously reported for normal astrocytes. The astrocytoma cells also express glutamine synthetase activity which is considered specific to astrocytes among brain cells. Previous work had also demonstrated the presence of other astrocyte markers glial fibrillary acidic protein and S-100 protein in the UC-11MG cells. The steady-state ion transport properties of Na+, Cl-, and K+ were also characterized in these cells, and the rates of efflux were found to be similar to those in other astrocytes, with the major difference being the presence of a second kinetic compartment in the UC-11MG cells. From this work, we conclude that the UC-11MG cell line displays prominent features associated with differentiated astrocytes, and may provide an excellent model system for the study of human astrocytes.

Astrocytoma↗

PubMed1988

Lithium transport in human fibroblasts: relationship to RBC lithium transport and psychiatric diagnoses.

Cultured fibroblasts were prepared from six normal controls, five DSM-III manic patients, and six DSM-III schizophrenic patients. Lithium (Li+) uptake, 24-hour Li+ ratios, and steady-state membrane potential were measured in these cell lines. The uptake of 10 mM Li+ reached maximum at 2 hours, with an intracellular concentration of approximately 15 mM. No significant difference in uptake was found among subject groups. Twenty-four hour Li+ (ratio of intracellular/extracellular Li+) ratios were determined by incubating the cell lines for 24 hours in the presence of 2 mM Li+. No significant difference was observed among groups; nor was there any significant correlation between the fibroblast 24-hour ratios and 24-hour in vitro ratios determined in donor red cells. The relationship between membrane potential and the 24 hour Li+ ratio in fibroblasts was determined. The average potential in these cell lines was -56 mV and was not affected by Li+ treatment. No correlation between the Li+ ratio and membrane potential was found.

Adult↗

PubMed1988

Pathways of Cl- transport in human fibroblasts.

Three pathways of Cl- efflux were identified in normal human fibroblasts. Twenty percent of the total Cl- efflux is via an electrically conductive pathway with an efflux constant of 0.016 min-1. This pathway is insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and bumetanide but is partially inhibited by anthracene-9-carboxylic acid. Twenty-five percent of the Cl- efflux occurs via Cl- with cation cotransport having an efflux constant of 0.020 min-1. This pathway is inhibited by bumetanide and is dependent on the simultaneous presence of Na+, K+, and Cl-. Under basal conditions, the energetics of this pathway indicate that it is operating close to equilibrium. Fifty percent of the Cl- efflux occurs via an anion exchange pathway having an efflux constant of 0.040 min-1 that is inhibited by DIDS or by removal of Cl- from the extracellular medium. Together these pathways account for 95% of the total Cl- efflux.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1988

Regulation of conductive Cl- transport in human fibroblasts.

Under normal growth conditions, approximately 20% of the efflux of Cl- from human fibroblasts occurs via an electrically conductive pathway or Cl- channel. This basal Cl- conductance is insensitive to the Cl- -anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and to the Cl- -cation cotransport inhibitor bumetanide. Exposure of the cells to dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) for 15 min increases the electrically conductive component of Cl- efflux by approximately 20%. Unlike the basal Cl- conductance, the cAMP-activated channel is DIDS sensitive, indicating that cAMP activates a different Cl- pathway from the one responsible for the basal Cl- conductance. Elevation of intracellular Ca2+ by addition of the ionophore A23187 also stimulates Cl- efflux via a DIDS inhibitable, electrically conductive Cl- pathway. That the cAMP- and Ca2+-stimulated pathways are different is suggested by the observation that simultaneous exposure of cells to optimal levels of dibutyryl cAMP and A23187 results in an increased Cl- efflux equal to the sum of the two factors acting independently. Prostaglandin E1, a known activator of adenylate cyclase, also elevates the levels of intracellular free Ca2+ in these cells and concomitantly activates both the cAMP- and the Ca2+-stimulated Cl- channels. Although regulated, Cl- channels are known to function in the modulation of nerve and muscle excitability, their role in fibroblast function is not clear.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1987

Identification of a defective cAMP-stimulated Cl- channel in cystic fibrosis fibroblasts.

Cl- efflux from normal human fibroblasts is stimulated by elevation of cAMP and by elevation of intracellular free Ca2+. In both cases the stimulated Cl- transport occurs via electrically conductive pathways. In six lines of normal human fibroblasts, dibutyryl cAMP increased total Cl- efflux by an average of 13%. In six lines of fibroblasts from patients with cystic fibrosis, dibutyryl cAMP was without effect. The electrically conductive component of Cl- transport was increased an average of 30% by dibutyryl cAMP in normal cells and was unaffected by dibutyryl cAMP in cystic fibrosis cells. Stimulation of the Ca2+-sensitive Cl- channel by addition of A23187 increased Cl- efflux by an average of 30% in normal and 30% in cystic fibrosis fibroblasts. The data indicate that there is a defect in a cAMP-activated Cl- channel in cystic fibrosis fibroblasts.

Bucladesine↗

PubMed1979