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S H Thompson

Publications and source records attributed to S H Thompson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Intracellular calcium signals in response to bradykinin in individual neuroblastoma cells.

The Ca indicator fura 2 was used to study the modulation of cytoplasmic Ca by bradykinin (Bk) in single N1E-115 murine neuroblastoma cells. Increases in cytoplasmic Ca in response to Bk were mediated by the B2 receptor subtype. Responses to high concentrations of Bk (1-100 nM) were homogeneous and characterized by a rapidly rising transient that decayed to baseline in the continued presence of agonist, with a half-time of 15 s. Responses to low concentrations of Bk (100-500 pM) were more heterogeneous, with longer latencies and often with oscillations. Pretreatment with thapsigargin for 20 min prevented the Ca response, showing that the Ca change results from intracellular Ca release. Removal of external Ca had little effect on the response to Bk, indicating that the agonist does not activate Ca influx. The extent of Ca release and refilling after Bk was tested with ionomycin. A saturating dose of Bk (20 nM) mobilizes > 90% of stored Ca within 30 s, and this is replaced slowly. Replacement of external Na by N-methyl-D-glucamine to block Na/Ca exchange affected the Ca response, causing decreases in latency and in the period of Ca oscillations and increases in overall duration and peak amplitude of the response.

Animals↗
PubMed1995

Calcium requirement for cGMP production during muscarinic activation of N1E-115 neuroblastoma cells.

Muscarinic agonists elicit large increases in intracellular Ca2+ and guanosine 3',5'-cyclic monophosphate (cGMP) in N1E-115 neuroblastoma cells. Both signals are blocked in cells loaded with the Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid showing that the increase in intracellular Ca2+ concentration ([Ca2+]i) is necessary to stimulate cGMP accumulation. Inhibition of nitric oxide synthase (NOS) blocks the cGMP response without affecting the peak amplitude of the intracellular Ca2+ signal, and it is concluded that Ca(2+)-dependent activation of NOS is required for cGMP production. cGMP accumulation is reduced by 60% when cells are bathed in Ca(2+)-free saline, but the peak change in [Ca2+]i is not affected. This suggests that Ca2+ influx is strongly coupled to the activation of cGMP production, even though it makes a smaller contribution to the intracellular Ca2+ signal than does Ca2+ release. Thapsigargin, which releases Ca2+ from intracellular stores, activates Ca2+ influx and increases cGMP. The cGMP increase is transient and follows approximately the same time course as Ca2+ store depletion. Ca2+ influx remains activated after store depletion, however, which indicates that influx alone cannot sustain cGMP production. It is concluded that summation of Ca2+ influx and Ca2+ release is necessary to reach a threshold Ca2+ level needed to stimulate cGMP accumulation. Because of the large contribution from Ca2+ influx, we suggest that NOS or a cofactor necessary for its activation may be located close to Ca2+ channels in the membrane.

Animals↗
PubMed1994

The aminoglycoside G418 suppresses muscarinic receptor-activated calcium release in stably transfected murine N1E-115 neuroblastoma cells.

The aminoglycoside G418 inhibited the release of calcium (Ca2+) from internal stores coupled to muscarinic receptors in murine N1E-115 neuroblastoma cells carrying the aminoglycoside resistance gene neomycin phosphotransferase (NPT). No significant effect was observed on responses coupled to histamine or bradykinin receptors. Cells were transfected using the eukaryotic expression vector pH beta APr-1-neo and selected using G418. Two groups were differentiated either in the continued presence of G418 or in the absence of G418. Carbachol (1 mM), histamine (200 microM) and bradykinin (100 nM) were administered to cells for thirty seconds and changes in [Ca2+]i were measured with fluorescence video microscopy of single cells loaded with the Ca2+ indicator fura-2. The effects of G418 on carbachol evoked Ca2+ release included a 73% reduction in the number of cells responding, a two fold increase in the time to reach half-maximal response, a 35% reduction of the peak [Ca2+]i in response to agonist and an elevation of resting [Ca2+]i from 99 +/- 14 nM (mean +/- S.E.M.) to 155 +/- 27 nM. Acute application (20 min) of G418 to transfected cells differentiated without G418 also reduced the percentage of cells responding to carbachol. This effect was less pronounced in non-transfected parent cells. Thus, the mechanism might involve a metabolite of G418 produced in cells expressing NPT. These results indicate that G418 attenuates Ca2+ release coupled to muscarinic receptors.

Animals↗
PubMed1994

Measurement of changes in functional muscarinic acetylcholine receptor density in single neuroblastoma cells using calcium release kinetics.

Calcium release in response to the activation of muscarinic M1 and histamine H1 receptors was studied in single N1E-115 cells using Fura-2 imaging. The objective was to relate changes in the kinetics of Ca release with reductions in functional receptor density resulting from receptor desensitization. Calcium release increased and its time course accelerated with increasing carbachol concentration with an EC50 = 96 +/- 8 microM. This value is similar to the binding KD (100 microM) and the similarity shows that the activation of calcium release is limited by the number of muscarinic receptors. In contrast, the EC50 for Ca release in response to histamine is 4.0 +/- 0.7 microM while the binding KD is 8.3 microM and, therefore, H1 receptors appear to be in approximately 2-fold excess over the minimum number necessary to fully engage the Ca release mechanism. Functional surface receptor number was assayed in the population of cells by counting the total number of cells responding to agonist. A 5 min exposure to 1 mM carbachol caused 12% of cells to lose their ability to respond to carbachol, with no change in their response to histamine. Interpolating from the dose-response curve taken before desensitization, this is equivalent to an average 23% reduction in the number of muscarinic receptors. In individual cells the latency to Ca release is dose-dependent in the absence of excess receptors. The loss of functional receptors was therefore estimated from the increase in latency after desensitization, and varied from 5-48% of receptors (22 +/- 18%). Muscarinic desensitization did not depend on IP3-evoked Ca release, Ca entry, protein kinase C, NO, or cGMP. We conclude that in a population, the number of cells responding and in single cells, the latency to Ca release can serve as measures of functional receptor density.

Animals↗
PubMed1994

Calcium current activated by muscarinic receptors and thapsigargin in neuronal cells.

The activation of muscarinic receptors in N1E-115 neuroblastoma cells elicits a voltage-independent calcium current. The current turns on slowly, reaches its maximum value approximately 45 s after applying the agonist, is sustained as long as agonist is present, and recovers by one half in approximately 10 s after washing the agonist away. The current density is 0.11 +/- 0.08 pA/pF (mean +/- SD; n = 12). It is absent in zero-Ca++ saline and reduced by Mn++ and Ba++. The I(V) curve characterizing the current has an extrapolated reversal potential > +40 mV. The calcium current is observed in cells heavily loaded with BAPTA indicating that the calcium entry pathway is not directly gated by calcium. In fura-2 experiments, we find that muscarinic activation causes an elevation of intracellular Ca++ that is due to both intracellular calcium release and calcium influx. The component of the signal that requires external Ca++ has the same time course as the receptor operated calcium current. Calcium influx measured in this way elevates (Ca++)i by 89 +/- 41 nM (n = 7). Thapsigargin, an inhibitor of Ca++/ATPase associated with the endoplasmic reticulum (ER), activates a calcium current with similar properties. The current density is 0.22 +/- 0.20 pA/pF (n = 6). Thapsigargin activated current is reduced by Mn++ and Ba++ and increased by elevated external Ca++. Calcium influx activated by thapsigargin elevates (Ca++)i by 82 +/- 35 nM. The Ca++ currents due to agonist and due to thapsigargin do not sum, indicating that these procedures activate the same process. Carbachol and thapsigargin both cause calcium release from internal stores and the calcium current bears strong similarity to calcium-release-activated calcium currents in nonexcitable cells (Hoth, M., and R. Penner. 1993. Journal of Physiology. 465:359-386; Zweifach, A., and R. S. Lewis, 1993. Proceedings of the National Academy of Sciences, USA. 90:6295-6299).

Animals↗
PubMed1994

Facilitation of calcium-dependent potassium current.

The activation of Ca-dependent K+ current, Ic, was studied in macropatches on the cell bodies of molluscan neurons. When a depolarizing voltage-clamp pulse was applied repeatedly, Ic facilitated in a manner that resembled the facilitation of synaptic transmitter release. Facilitation was characterized by an increase in Ic amplitude, a progressive increase in instantaneous outward current, and a decrease in utilization time. Experiments were done to investigate the mechanism responsible for Ic facilitation. Facilitation was reduced by microinjection of an exogenous Ca2+ buffer into the cytoplasm, indicating that facilitation is a Ca(2+)-dependent process. It was also reduced at elevated temperatures. Conversely, facilitation was greatly potentiated by blocking the Na/Ca exchange mechanism. It is concluded that the facilitation of Ca-dependent K+ current results from the accumulation of Ca2+ at the inner face of the membrane during the repeated activation of Ca2+ channels by depolarization. The Ca2+ indicator fluo-3 was used in fluorescence imaging experiments to measure changes in [Ca]i near the cell membrane during repeated depolarizing pulses and the interpretation of these results was aided by numerical simulations of Ca2+ accumulation, diffusion, and buffering in the peripheral cytoplasm. These experiments showed that the time course of Ic facilitation matches the time course of Ca2+ accumulation at the membrane. It was found that the strength of Ic facilitation varies among patches on the same neuron, suggesting that the accumulation of Ca2+ is not uniform along the inner surface of the membrane and that gradients in [Ca]i develop and are maintained during trains of depolarizing pulses. Potential mechanisms that may lead to local differences in Ca2+ accumulation and Ic facilitation are discussed.

Aniline Compounds↗
PubMed1993

Membrane toxicity of the protein kinase C inhibitor calphostin A by a free-radical mechanism.

The effects of calphostin A on cytoplasmic calcium levels, receptor-mediated calcium release, and membrane input resistance were measured in neuroblastoma cells. Calphostin A is a lipophilic, light-sensitive perylenequinone that generates singlet oxygen when illuminated. It inhibits the activity of protein kinase C (IC50 = 250 nM), but only in the presence of light. Phorbol esters normally attenuate carbachol-evoked calcium release. This effect was blocked by simultaneous exposure to light and calphostin A (40 nM) for 30 min. At higher doses (0.5-1 microM) calphostin A also approximately doubled the resting calcium level and decreased cell input resistance by 51%. These toxic effects did not occur in the dark or after preincubation with the antioxidant alpha-tocopherol. These data support the hypothesis that the calphostins act by partitioning into the membrane and producing singlet oxygen and endoperoxides which then irreversibly modify protein kinase C and other membrane proteins and lipids.

Animals↗
PubMed1993

Membrane toxicity of the protein kinase C inhibitor calphostin A by a free-radical mechanism.

The effects of calphostin A on cytoplasmic calcium levels, receptor-mediated calcium release, and membrane input resistance were measured in neuroblastoma cells. Calphostin A is a lipophilic, light-sensitive perylenequinone that generates singlet oxygen when illuminated. It inhibits the activity of protein kinase C (IC50 = 250 nM), but only in the presence of light. Phorbol esters normally attenuate carbachol-evoked calcium release. This effect was blocked by simultaneous exposure to light and calphostin A (40 nM) for 30 min. At higher doses (0.5-1 microM) calphostin A also approximately doubled the resting calcium level and decreased cell input resistance by 51%. These toxic effects did not occur in the dark or after preincubation with the antioxidant alpha-tocopherol. These data support the hypothesis that the calphostins act by partitioning into the membrane and producing singlet oxygen and endoperoxides which then irreversibly modify protein kinase C and other membrane proteins and lipids.

Animals↗
PubMed1992

Intracellular calcium release in N1E-115 neuroblastoma cells is mediated by the M1 muscarinic receptor subtype and is antagonized by McN-A-343.

Experiments using muscarinic receptor antagonists were done to determine which muscarinic receptor subtypes(s) mediate carbachol-evoked calcium release in N1E-115 cells. McN-A-343 and a new analog, (+/-)BN228, were weak antagonists and neither compound caused release on its own. The rank order of potency was 4-DAMP greater than pirenzepine greater than AFDX116 greater than (+/-)BN228 and McN-A-343. This profile, pirenzepine's high potency (19-fold greater than AFDX116) and its IC50 of 31 nM suggest that calcium release in this neuronal cell line is mediated by the M1 muscarinic receptor subtype.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1992

Correlation of oral disease with the Walter Reed staging scheme for HIV-1-seropositive patients.

This study correlates the prevalent oral disease findings in 390 patients seropositive for human immunodeficiency virus type 1 (HIV-1) with their level of staging (Walter Reed) and depletion of peripheral helper T lymphocytes (CD4+). Chronic lymphadenopathy of the head and neck was a common finding (59.2%) that occurred early in staging progression and did not correlate with depression of helper T-cell levels. Of the three prevalent oral disease findings (oral hairy leukoplakia (OHL), candidiasis, necrotizing ulcerative gingivitis [NUG]) only OHL and NUG were significantly correlated with helper T-cell depletion. The occurrence of visually detectable OHL and NUG corresponds to depletion of peripheral helper T-lymphocyte values in a range of 157 to 299 cells/mm3. This range may represent a more accurate value for biologically significant lymphocyte depletion than the Walter Reed value of 400 cells/mm3. The presence of OHL showed a weak statistical correlation with staging progression, indicating deteriorating immunoregulation. No cases of Kaposi's sarcoma or other HIV-1-associated oral diseases were observed in the sample population, regardless of the patient's staging category or peripheral helper T-lymphocyte count.

Adult↗
PubMed1992

Correlation of subclinical candidal colonization of the dorsal tongue surface with the Walter Reed staging scheme for patients infected with HIV-1.

The correlation between subclinical candidal colonization of the dorsal tongue surface and the Walter Reed staging scheme for patients positive for human immunodeficiency virus (HIV-1) antibody is reported. Of 76 cytologic smears of the dorsal tongue surface, 12 (16.2%) demonstrated subclinical colonization. The mean peripheral helper T-lymphocyte count for patients with subclinical colonization was 304 +/- 226 cells/mm3 and was not significantly different from seropositive patients without colonization (411 +/- 209 cells/mm3). The Walter Reed scheme for assessing progressive immunodysregulation did not significantly correlate with the presence of subclinical colonization. This study suggests a more complex pathogenesis for oral candidiasis in HIV-1-infected patients rather than a direct link to peripheral helper T-lymphocyte depletion below a count of 400 cells/mm3.

Adult↗
PubMed1992

A comparison of T4:T8 lymphocyte ratio in the periodontal lesion of healthy and HIV-positive patients.

Previous reports describe a characteristic, rapidly progressive, periodontitis that is unique to patients who are seropositive for HIV antibody (Western blot +). The purpose of this study was to compare the T4 and T8 lymphocyte subpopulations in the peripheral blood and periodontal lesions of these HIV patients with those of healthy controls. T-cell subsets in peripheral blood were quantified by flow cytometry. The values from this analysis were used to calculate the peripheral T4:T8 lymphocyte ratio for each patient. Gingival tissue (papilla) was obtained from 8 HIV+ patients and from 6 healthy HIV- control patients during routine gingival surgery. The T-cell subpopulations in the gingival tissue were determined using serial cryostat sections that were labeled with monoclonal antibodies for T4 and T8 cells and developed using an avidin-biotin-peroxidase system. Six sections were taken from each of the 14 tissue specimens (one per patient). The sections were examined at 450 x and the mean number of T4 and T8 cells calculated for each section. These mean values were then used to determine the T4:T8 lymphocyte ratio for each tissue specimen. The peripheral blood analysis revealed a mean serum T4:T8 ratio of (2.07 +/- 0.455) for the controls and (0.58 +/- 0.26) for the HIV patients. The significantly lower T4:T8 ratio in HIV patients is consistent with their diagnosis. Although the results indicated that the mean T4:T8 lymphocyte ratio in the gingiva of controls was highly variable (2.70 +/- 1.344), the gingiva of HIV patients consistently exhibited a complete absence of T-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1990

Intestinal permeability to intact lactose in newborns and adults.

Small amounts of lactose have been shown to be absorbed intact across the intestine and excreted unchanged in the urine of newborns and adults. We designed a study to quantitate the intestinal uptake and urinary excretion of this disaccharide in these age groups. Similar amounts of lactose were given orally to 17 term newborns (age: 24.8 +/- 3.0 h) as a standard infant formula, and to 15 adult lactose absorbers (age: 28.1 +/- 2.6 years) and 11 adult lactose malabsorbers (age: 24.7 +/- 2.9 years) as a 20% water solution. Following lactose ingestion, breath was collected every 30 or 60 min for 3 h and analyzed for hydrogen concentration. Urine was also collected, and lactose and creatinine concentrations were determined. Peak hydrogen concentration was less than 20 ppm above baseline in newborns and adult lactose absorbers and 85 +/- 14 ppm in adult lactose malabsorbers. Urinary lactose excretion, expressed as a function of body weight (mg/ml/kg b.w.), was substantially greater in newborns (4.2 +/- 0.82) than in adult lactose absorbers (0.29 +/- 0.07; p less than 0.001) and adult lactose malabsorbers (0.55 +/- 0.04; p less than 0.01). Similarly, urinary lactose excretion expressed as a ratio of urinary lactose to urinary creatinine (mg/mg) was increased (p less than 0.001) in newborns (2.05 +/- 0.26) when compared to adult lactose absorbers (0.11 +/- 0.02) and adult lactose malabsorbers (0.20 +/- 0.02). Our data demonstrate that the intestinal uptake and urinary excretion of intact lactose is significantly increased in newborns compared to adult subjects.

Adult↗
PubMed1989

Assessment of lactose absorption by measurement of urinary galactose.

Individuals with sufficient intestinal lactase hydrolyze ingested lactose to galactose and glucose and these monosaccharides are absorbed. Lactose is not digested completely when intestinal lactase activity is low and the disaccharide is malabsorbed. Breath hydrogen excretion after lactose ingestion is used commonly to diagnose lactose malabsorption. However, no direct tests are currently used to assess lactose absorption. We tested a new method of assessing lactose absorption in 26 healthy individuals. Each subject ingested 50 g of lactose. Participants were evaluated for lactose malabsorption using a standard 3-h breath hydrogen test. In addition, the urinary excretions of galactose, lactose, and creatinine were quantitated for 3-5 h after lactose ingestion. On the basis of breath hydrogen analysis after lactose ingestion, 12 individuals were lactose malabsorbers (defined as a rise in the breath hydrogen concentration of greater than 20 parts per million above the baseline value). The 14 subjects who did not malabsorb lactose by breath hydrogen testing (defined as a rise in the breath hydrogen concentration of less than or equal to 20 parts per million above the baseline value), had significantly more galactose in their urine 1, 2, and 3 h after lactose ingestion than lactose malabsorbers. The ratio of excreted lactose to excreted galactose was significantly decreased in lactose absorbers compared with lactose malabsorbers (p less than 0.001). Determination of the ratio of urinary galactose to urinary creatinine separated lactose absorbers from lactose malabsorbers completely (p less than 0.001). We conclude from this study that the determination of urinary galactose, urinary lactose/galactose ratio, and urinary galactose/creatinine ratio may be used to assess lactose digestion and absorption in healthy adults.

Adolescent↗
PubMed1989

Trenbolone alters the responsiveness of skeletal muscle satellite cells to fibroblast growth factor and insulin-like growth factor I.

The potential role of satellite cells in mediating the effect of trenbolone [17 beta-hydroxyestra-4,9-11-trien-3-one (TBOH)] on skeletal muscle hypertrophy was examined. Young female Sprague-Dawley rats received TBOH injections daily for 2 weeks; growth, body composition, and the composition of selected muscles were assessed. Treated rats grew more rapidly and deposited less body lipid and more protein. The semimembranosus muscle from treated rats was larger and had approximately 60% more DNA per muscle than muscles from control rats. The addition of trenbolone directly to the medium of cultured satellite cells did not stimulate cell proliferation, nor did it augment the stimulatory response of these cells to fibroblast growth factor (FGF) or insulin-like growth factor I (IGF-I). In contrast, satellite cells cultured from TBOH-treated rats exhibited greater proliferative responses to FGF and IGF-I than satellite cells from control rats. In addition, serum from TBOH-treated rats stimulated greater cell proliferation in satellite cell cultures than serum from control rats. These experiments suggest that one possible mechanism responsible for the ability of TBOH to stimulate skeletal muscle hypertrophy may be through enhanced proliferation and differentiation of satellite cells as a result of the increased sensitivity of these cells to IGF-I and FGF.

Animals↗