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R Nitschke

Publications and source records attributed to R Nitschke.

At least 55 records · Page 3Linked to original sources
PubMed1997

Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system.

Transcellular Ca2+ transport in the distal nephron involves passive Ca2+ influx at the apical membrane, diffusion through the cytosol and active extrusion across the opposing basolateral membrane. The molecular identity of the apical Ca2+ entry step is still elusive, but its regulatory aspects have been analyzed in the present study. To this end, rabbit connecting and cortical collecting tubular cells were cultured on permeable and transparent supports and the apical Ca2+ influx was deduced from Mn2+ quenching of Ca2+ independent Fura-2 fluorescence, while the intracellular Ca2+ concentration ([Ca2+]i) was measured simultaneously. In parallel experiments, transcellular Ca2+ transport was determined isotopically as 45Ca2+ flux from the apical to basolateral compartment. Decreasing the apical pH from 7.4 to 5.9 inhibited transcellular Ca2+ transport by 53 +/- 1%, whereas apical Ca2+ influx was reduced by 39 +/- 7% and [Ca2+]i decreased by 18 +/- 3%. Reversal of the Na+/Ca2+ exchanger by iso-osmotic replacement of Na+ by N-methyl-D-glucamine in the basolateral compartment resulted in 50 +/- 5% inhibition of Ca2+ transport, 46 +/- 3% reduction of apical Ca2+ influx and 60 +/- 3% increase in [Ca2+]i. In the absence of basolateral Ca2+, however, this manoeuvre decreased [Ca2+]i by 21 +/- 8%, while Ca2+ transport and apical Ca2+ influx were reduced by the same magnitude as in the presence of Ca2+, that is by 53 +/- 6% and 45 +/- 4%, respectively. Stimulation of adenylyl cyclase with forskolin (10(-5) M) increased transcellular Ca2+ transport by 108 +/- 40%, stimulated apical Ca2+ influx by 120 +/- 17% and increased [Ca2+]i by 110 +/- 2%. In conclusion, the apical Ca2+ influx is regulated by apical pH, intracellular cAMP and basolateral Na+/Ca2+ exchanger activity, and is coupled in an 1:1 fashion to the rate of transepithelial Ca2+ transport.

Animals↗
PubMed1997

Capacitative Ca2+ entry (CCE) induced by luminal and basolateral ATP in polarised MDCK-C7 cells is restricted to the basolateral membrane.

In previous studies we have characterised various properties of capacitative Ca2+ entry (CCE) in different epithelia. After Ca2+ store depletion with PLC/InsP3-coupled agonists or by inhibition of store Ca2+ uptake, with for example thapsigargin, Ca2+ influx is activated. This leads to a sustained cellular response (e.g. NaCl secretion). In the present study, we have investigated CCE in polarised MDCK-C7 cells grown on permeable supports in a chamber allowing for separate luminal and basolateral perfusion. The transepithelial resistance (Rte) and voltage (Vte) were measured simultaneously to verify the tightness of the epithelial monolayers. MDCK-C7 cells grew to very tight monolayers (Rto > 3000 omega.cm2). Apical ATP (100 mumol/l) led to a biphasic [Ca2+]i increase. Removal of apical Ca2+ in the continuous presence of ATP did not reduce the stimulated plateau. However, removal of Ca2+ from the basolateral side rapidly and completely interrupted the [Ca2+]i plateau to below basal values ([Ca2+]i decrease during plateau phase after removal of basolateral Ca2+ = 213 +/- 15 nmol/l, n = 9). Furthermore, MDCK-C7 responded to basolateral ATP (100 mumol/l) with a biphasic [Ca2+]i transient. Again the plateau phase of the ATP-induced [Ca2+]i effect was fully dependent on the presence of basolateral but not apical Ca2+ ([Ca2+]i decrease during plateau phase after removal of basolateral Ca2+ = 196 +/- 5 nmol/l, n = 10). Receptor-independent depletion of cytosolic Ca2+ stores with thapsigargin from both sides led to a rise in [Ca2+]i, which was also exclusively dependent on the presence of basolateral Ca2+ (n = 8). These data indicate that MDCK-C7 cells express luminal and basolateral P2-receptors coupled to PLC/InsP3/Ca2+. ATP applied from both sides induced a sustained [Ca2+]i plateau which was due to transmembrane Ca2+ influx. The ATP- and thapsigargin-induced Ca2+ influx pathway was exclusively located in the basolateral membrane.

Adenosine Triphosphate↗
PubMed1997

Angiotensin II increases the cytosolic calcium activity in rat podocytes in culture.

In the glomerulus, angiotensin II (Ang II) reduces the ultrafiltration coefficient and enhances the filtration of macromolecules. During glomerular injury, inhibition of the renin-angiotensin system by angiotensin-converting-enzyme inhibitors reduces proteinuria and retards the progression to end-stage renal insufficiency. The mechanisms by which Ang II modulates glomerular function are still a matter of investigation. To study whether Ang II may regulate the cytosolic calcium activity ([Ca2+]i) in podocytes, these cells were propagated in short-term culture and the effect of Ang II was examined with the Fura-2 microfluorescence technique in single podocytes. The cellular identity of cultured podocytes was proven by the expression of WT-1 and pp44, specific antibodies against podocytes in vivo. Ang II led to a concentration-dependent, reversible and slow increase of [Ca2+]i with an EC50 of 3 nmol/liter Ang II (N = 229). Ten nmol/liter Ang II increased [Ca2+]i from 41 +/- 9 to 260 +/- 34 nmol/liter (N = 210). In a solution with an extracellular reduced Ca2+ concentration of 10 micromol/liter, Ang II-mediated [Ca2+]i increase was significantly reduced by 60 +/- 20% (N = 12), indicating that the [Ca2+]i increase was due to a Ca2+ influx from the extracellular space and a release of Ca2+ from intracellular stores. Flufenamate, an inhibitor of non-selective ion channels, significantly inhibited Ang II-mediated increase of [Ca2+]i (IC50 = 20 micromol/liter, N = 29), whereas the L-type Ca2+ channel blocker nicardipine even in high concentrations of > 1 micromol/liter had only a small inhibitory effect. The AT1 receptor antagonist losartan inhibited Ang II-mediated [Ca2+]i increase with an IC50 of about 0.3 nmol/liter (N = 35). The data suggest that Ang II increases [Ca2+]i in podocytes by an influx of Ca2+ through non-selective channels and by a release of Ca2+ from intracellular stores. The effect of Ang II is mediated via an AT1 receptor.

Angiotensin II↗
PubMed1996

8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29.

8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) is a widely used pharmacological tool to investigate the involvement of intracellular Ca2+ stores in cellular responses. In this study we investigate the effect of TMB-8 as a putative inhibitor of "Ca2+ signalling" in single fura-2 loaded HT29 colonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 mumol/l intracellular Ca2+ ([Ca2+]i) transient with an IC50 of 20 mumol/l. However, [Ca2+]i transients induced by other phospholipase C coupled agonists ATP (10 mumol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mumol/l). The agonist-induced [Ca2+]i transients remained equally unaffected by 100 mumol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mumol/l for ATP (n = 4) or 1 nmol/l for NT (n = 4). The competitive nature of the TMB-8-induced inhibition of the CCH-induced [Ca2+]i transient was demonstrated by examining the agonist at various concentrations in absence and presence of the antagonist. High TMB-8 concentrations (100 mumol/l) alone induced a small [Ca2+]i increase (delta[Ca2+]i: 40 +/- 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization (delta pH: 0.1 +/- 0.02, n = 7) occurring simultaneously with the increase in [Ca2+]i. From these results we draw the following conclusions: (1) In sharp contrast to a large number of other studies, but in agreement with studies in other types of cells, these results substantially challenge the value of the "tool" TMB-8 as an "intracellular Ca2+ antagonist"; (2) TMB-8 acts a muscarinic receptor antagonist at the M3 receptor; (3) TMB-8 does not influence the release of Ca2+ from intracellular stores when IP3 signal transduction is activated by ATP or NT; (4) TMB-8 as a weak organic base alkalinizes the cytosol at high concentrations; and (5) TMB-8 induces small [Ca2+]i transients at higher concentrations.

Adenosine Triphosphate↗
PubMed1996

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells.

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pHi) and intracellular Ca2+ activity ([Ca2+]i) of HT29 cells was investigated microspectrofluorimetrically using pH- and Ca2+- sensitive fluorescent indicators, [i.e. 2', 7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (Vm) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pKa value. Trimethylamine (20 mmol/l) increased pHi by 0.78 +/- 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pHi and a slow and incomplete recovery: pHi stayed 0.26 +/- 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pHi. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca2+]i transient which reached a peak value within 10-25 s and then slowly declined to a [Ca2+]i plateau. The initial Delta[Ca2+]i induced by trimethylamine (20 mmol/l) was 160 +/- 15 nmol/l (n = 17). The [Ca2+]i peak was independent of the Ca2+ activity in the bath solution, but the [Ca2+]i plateau was significantly lower under Ca2+-free conditions and could be immediately interrupted by application of CO2 (10%; n = 6), a manoeuvre to acidify pHi in HT29 cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca2+ stores completely abolished this [Ca2+]i transient. Tetramethylamine led to higher [Ca2+]i changes than the other amines tested and only this transient could be completely blocked by atropine (10(-6) mol/l). Trimethylamine (20 mmol/l) hyperpolarized Vm by 22.5 +/- 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 +/- 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pHi changes, release Ca2+ from intracellular, inositol-1,4, 5-trisphosphate-sensitive Ca2+ stores and increase Ca2+ influx into HT29 cells. The latter may be related to both the store depletion and the hyperpolarization.

Alkalies↗
PubMed1996

Attenuation of stimulated Ca2+ influx in colonic epithelial (HT29) cells by cAMP.

In HT29 colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca2+ activity ([Ca2+]i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P3-) mediated Ca2+ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca2+ influx. The present study was undertaken to examine the effect of increased adenosine 3',5'-cyclic monophosphate (cAMP) (forskolin 10 micromol/l = FOR) on the Ca2+ transient in the presence of CCH (100 micromol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca2+ ([Ca2+]i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K+ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca2+]i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca2+]i uniformly from a fluorescence ratio (345/380) of 2.9 +/- 0.12 to 1.8 +/- 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (Vm) of the patch-clamped cells significantly and reversibly from -54 +/- 7.4 to -27 +/- 5.9 mV (n = 6). In additional experiments Vm was depolarized by 15-54 mV by various increments in the bath K+ concentration. This led to corresponding reductions in [Ca2+]i. Irrespective of the cause of depolarization (high K+ or FOR) there was a significant correlation between the change in Vm and change in [Ca2+]i. These data indicate that the cAMP-mediated attenuation of Ca2+ influx is caused by the depolarization produced by this second messenger.

Adenosine Triphosphate↗
PubMed1996

Regulation of the intracellular calcium concentration in epithelial cells.

Epithelia, like other vertebrate cells, are equipped with an extensive network of signaling pathways, involved in the regulation of a large variety of distinct cellular functions. Amongst many the intracellular Ca2+ [Ca2+]i signal is the most common one. On the one hand, [Ca2+]i is involved in regulation functions of general cell physiology, such as the control of cell volume, cell growth or differentiation. On the other hand, [Ca2+]i controls epithelia-specific functions like the regulation of transepithelial solute transport. This short overview addresses the various aspects of Ca2+ signaling in epithelia. It is emphasized that [Ca2+]i is a spatially restricted signal, permitting the distinct regulation of one cellular response. Evidence will be discussed indicating that the functional epithelial polarity is paralleled by polarized membrane receptors and polarized [Ca2+]i signals, regulating the apical and basolateral membranes differentially.

Animals↗
PubMed1995

Cyclophosphamide/doxorubicin vs. cisplatin/teniposide in the treatment of children older than 12 months of age with disseminated neuroblastoma: a Pediatric Oncology Group Randomized Phase II study.

This prospective study was designed to estimate the response rates and to compare two drug pairs, cyclophosphamide/doxorubicin (Cy/A) and cisplatin/teniposide (P1/VM) in previously untreated patients with disseminated neuroblastoma > 12 months of age at diagnosis. Estimated complete clinical response rates after five courses of therapy were 13% (70 patients) and 22% (64 patients) for Cy/A and P1/VM, respectively (P = 0.17). After surgical removal of residual tumors in patients with partial response, the complete response rates were 27% and 34% (P = 0.50), respectively. The overall CR/PR rates after induction and surgery were 59% and 73% (P = 0.077). There was no significant difference in event free survival (P = 0.48) or survival (P = 0.40). Five year survival on the two arms were 14% (SE = 5%) and 12% (SE = 4%), respectively. Toxicity was significant but manageable. The Cy/A arm had significantly higher hematopoietic toxicity but significantly lower GI toxicity. Significant allergic reactions were seen with the P1/VM arm, none in the Cy/A arm. Given the activity of these two regimens, further therapy with a combination of these regimens is suggested.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1995

Results of pediatric oncology group protocol 8104 for infants with stages D and DS neuroblastoma.

PURPOSE: We determined the complete response and survival rates for infants with disseminated (stage D) neuroblastoma that followed therapy identical to that for regional disease. In those infants whose disease excluded cortical bone metastases (stage DS), we determined complete response rates achieved either spontaneously or with stage D therapy. PATIENTS AND METHODS: Eighty-eight patients with metastatic disease received induction chemotherapy followed by a second operation, the results of which determined additional therapy. Twenty-five patients were observed after diagnosis, without chemotherapy, until a second operation. RESULTS: The complete response (CR) rates for patients with stage D disease after induction chemotherapy and postinduction surgery were 26% and 52%, respectively, and for immediately treated patients with stage DS disease 69% and 77%, respectively. Fifty-four percent of initially observed patients with stage DS disease achieved CR after a second operation; 44% were never treated beyond these two operations. Five-year actuarial survival rates for patients with stage D and for all those with stage DS disease were 60% (SE = 6%) and 90% (SE = 5%), respectively. CONCLUSIONS: Improved survival rates for patients with stage D disease were achieved on this protocol but remained considerably lower than those for infants with less extensive disease. Rates of survival for patients with stage DS disease were achieved with therapy less aggressive than in published series.

Child↗
PubMed1994

A pilot study of isotretinoin in the treatment of juvenile chronic myelogenous leukemia.

BACKGROUND: Juvenile chronic myelogenous leukemia (CML) is a rare myeloproliferative disease of infants and young children for which there is no effective therapy other than allogeneic bone marrow transplantation. In vitro, isotretinoin (13-cis-retinoic acid) attenuates both the spontaneous proliferation of leukemic peripheral-blood progenitor cells (granulocyte-macrophage colony-forming units) and their selective hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). We conducted a pilot study to evaluate the clinical efficacy of isotretinoin in juvenile CML. METHODS: To be eligible the patients had to have newly diagnosed untreated disease, leukocytosis with monocytosis, marrow with less than 25 percent blasts, hepatosplenomegaly, no chromosomal abnormalities, and negative viral cultures and antibody titers. Isotretinoin was administered orally in single daily doses of 100 mg per square meter of body-surface area. When possible, patients subsequently underwent bone marrow transplantation. RESULTS: Ten children (median age, 10 months) were enrolled in the study. In all 10 there was spontaneous colony formation of leukemic progenitor cells in vitro. In the eight patients tested there was hypersensitivity to GM-CSF. The only toxic effect of isotretinoin therapy was cheilitis in two patients. Four children had disease progression. Two children had complete responses to isotretinoin (normalization of the white-cell count and disappearance of organomegaly), three had partial responses (more than a 50 percent reduction in the white-cell count and degree of organomegaly), and one had a minimal response (more than a 50 percent reduction in the white-cell count, but a 26 to 50 percent reduction in the degree of organomegaly). The median duration of response was 37 months (range, 6 to 83). Three of the four children who had a complete or partial response and who did not undergo bone marrow transplantation were alive 36 to 83 months after the diagnosis of juvenile CML. The spontaneous colony formation in vitro was reduced in samples from the five patients in whom this factor was reassessed during treatment. There was also a reduction in the hypersensitivity of leukemic progenitor cells to GM-CSF in the two patients retested. CONCLUSIONS: Isotretinoin can induce durable clinical and laboratory responses in patients with juvenile CML.

Child, Preschool↗
PubMed1994

pH regulation in HT29 colon carcinoma cells.

The pH regulation in HT29 colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pHi) was 7.21 +/- 0.07 (n = 22) in HCO3(-)-containing and 7.21 +/- 0.09 (n = 12) in HCO3(-)-free solution. HOE-694 (10 mumol/l), a potent inhibitor of the Na+/H+ exchanger, did not affect control pHi. As a means to acidify cells we used the NH4+/NH3 (20 mmol/l) prepulse technique. The mean peak acidification was 0.37 +/- 0.07 pH units (n = 6). In HCO3(-)-free solutions recovery from acid load was completely blocked by HOE-694 (1 mumol/l), whereas in HCO3(-)-containing solutions a combination of HOE-694 and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na(+)-dependent in HCO3(-)-containing and HCO3(-)-free solutions. Removal of external Cl- caused a rapid, DIDS-blockable alkalinization of 0.33 +/- 0.03 pH units (n = 15) and of 0.20 +/- 0.006 pH units (n = 5), when external Na+ was removed together with Cl-. This alkalinization was faster in HCO3(-)-containing than in HCO3(-)-free solutions. The present observations demonstrate three distinct mechanisms of pHi regulation in HT29 cells: (a) a Na+/H+ exchanger, (b) a HCO3-/Cl- exchanger and (c) a Na(+)-dependent HCO3- transporter, probably the Na(+)-HCO3-/Cl- antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1993

Ifosfamide/etoposide combination in the treatment of recurrent malignant solid tumors of childhood. A Pediatric Oncology Group Phase II study.

BACKGROUND: The prognosis for children with recurrent or resistant malignant solid tumors remains dismal. More effective rescue therapy is needed for these children. METHODS: Between August 1987 and November 1990, 311 children with recurrent or resistant malignant solid tumors were treated by investigators in the Pediatric Oncology Group with intravenous infusions of 2.0 g/m2 of ifosfamide and 100 mg/m2 of etoposide (VP-16) plus mesna as uroprotection three times daily, with courses being repeated every 14-21 days for as long as the patients responded to therapy. RESULTS: Seventy-four percent of the 294 assessable patients entered in the study had metastatic disease and previously had been treated heavily. The complete response/partial response rate was 30%, and the overall response rate was 39.5%. Toxic effects included nephrotoxicity, mild liver dysfunction, neurotoxicity, and myelosuppression. Sixty-eight percent had an absolute neutrophil count (ANC) of less than 500/microliters. In 1606 courses of therapy administered, only 3.6% of patients developed a bacterial infection. Only two patients died of gram-negative sepsis. Four percent of the patients had gross hematuria (> 50 erythrocytes/high-power field), and 18.5% had microscopic hematuria (< 20 erythrocytes/high-power field). Fanconi syndrome developed in eight children. CONCLUSIONS: Ifosfamide/VP-16 is an active combination in children with recurrent malignant solid tumors. Although it was myelosuppressive, the incidence of infection was quite low (3.6%). Mesna was very effective in preventing the development of hematuria.

Adolescent↗
PubMed1993

Intracellular Ca2+ transients in HT29 cells induced by hypotonic cell swelling.

Cell swelling induced by hypotonic solution led to an osmolality-dependent increase in intracellular Ca2+ activity ([Ca2+]i) in HT29 cells. At moderate reductions in osmolality from 290 to 240 or 225 mosmol/l in most cases only a small monophasic increase of [Ca2+]i to a stable plateau of 10-20 nmol/l above resting [Ca2+]i was observed. Lower osmolalities resulted in a triphasic increase of [Ca2+]i to a peak value. In a first phase after the volume change, lasting 20-40 s, [Ca2+]i increased slowly by about 30 nmol/l. Thereafter [Ca2+]i increased more rapidly within 20-30 s to a peak value. This peak was 189 +/- 45 nmol/l (190 mosmol/l, n = 9) and 243 +/- 41 nmol/l (160 mosmol/l, n = 20) above resting [Ca2+]i. The peak was then followed by a decline of [Ca2+]i over the next 2-3 min to a stable plateau value of 28 +/- 6 (n = 6) and 32 +/- 11 nmol/l (n = 11) above resting [Ca2+]i at 190 and 160 mosmol/l, respectively. The plateau lasted as long as the hypotonic solution was present. Under Ca(2+)-free bath conditions the peak value for the cell-swelling-induced [Ca2+]i transient was reached significantly later (60-100 s, compared to 40-60 s under control conditions). The peak values under Ca(2+)-free conditions were not significantly lower. This indicates that the [Ca2+]i peak was mostly of intracellular origin. No [Ca2+]i plateau phase was observed under Ca(2+)-free bath conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1993

Evidence for agonist-induced export of intracellular Ca2+ in epithelial cells.

There is increasing evidence that some agonists not only induce intracellular Ca2+ increases, due to store release and transmembranous influx, but also that they stimulate Ca2+ efflux. We have investigated the agonist-stimulated response on the intracellular Ca2+ activity ([Ca2+]i) in the presence of thapsigargin (10(-8) mol/l, TG) in HT29 and CFPAC-1 cells. For CFPAC-1 the agonists ATP (10(-7)-10(-3) mol/l, n = 9), carbachol (10(-6)-10(-3) mol/l, n = 5) and neurotensin (10(-10)-10(-7) mol/l, n = 6) all induced a concentration-dependent decrease in [Ca2+]i in the presence of TG. Similar results were obtained with HT29 cells. This decrease of [Ca2+]i could be caused by a reduced Ca2+ influx, either due to a reduced driving force for Ca2+ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca2+ permeability. Using the fura-2 Mn2+ quenching technique we demonstrated that ATP did not slow the TG-induced Mn2+ quench. This indicates that the agonist-induced [Ca2+]i decrease in the presence of TG was not due to a reduced influx of Ca2+ into the cell, but rather due to stimulation of Ca2+ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of -49 +/- 3.6 mV (n = 9). Within the first 3 min ATP was still able to induce a depolarization which could be attributed to an increase in Cl- conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Agonist-induced intracellular Ca2+ transients in HT29 cells.

In the present study we have investigated the mechanism of intracellular Ca2+ activity ([Ca2+]i) changes in HT29 cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca2+]i was measured with the fluorescent Ca2+ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1-40Hz). ATP and CCH induced not only a dose-dependent [Ca2+]i peak response, but also changes of the plateau phase. The [Ca2+]i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca2+]i plateau increased at higher CCH concentrations. NT showed (from 10(-10) to 10(-7) mol/l) in most cases only a [Ca2+]i spike lasting 2-3 min. The [Ca2+]i plateau induced by ATP (10(-6) mol/l) and CCH (10(-5) mol/l) was abolished by reducing the Ca2+ activity in the bath from 10(-3) to 10(-4) mol/l (n = 7). In Ca(2+)-free bathing solution the [Ca2+]i peak value for all three agonists was not altered. Using fura-2 quenching by Mn2+ as an indicator of Ca2+ influx the [Ca2+]i peak was always reached before Mn2+ influx started. Every agonist showed this delayed stimulation of the Ca2+ influx with a lag time of 23 +/- 1.5 s (n = 15) indicating a similar mechanism in each case. Verapamil (10(-6)-10(-4) mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca2+]i increase. Short pre-incubation with verapamil augmented the effect on the [Ca2+]i peak, whereas no further influence on the plateau was observed. Ni2+ (10(-3) mol/l) reduced the plateau value by 70%.

Adenosine Triphosphate↗
PubMed1993

Thoracic neuroblastoma: a Pediatric Oncology Group study.

Ninety-six patients with thoracic neuroblastoma were studied in a prospective fashion. Median age at presentation was 0.9 years. Forty-eight percent of the patients presented with stage A disease, 20% stage B, 13% stage C, 17% stage D, and 2% stage DS. Seventy-five patients have been followed for greater than 4 years. A posterior mediastinal mass was diagnosed on incidental chest roentgenograms performed for nontumor-related symptoms in 49% of the cases. Sixteen percent of the cases presented with neurological symptoms and 14% of the patients presented with acute respiratory distress. Urinary catecholamines were elevated in 76% of the cases. Complete surgical resection was carried out in 47% of the cases, while incomplete resection or biopsy was performed in 45%. No operation was performed in 3 patients. Minor surgical complications occurred in 20% of the patients, and 3% of the patients had significant perioperative complications. One patient died as a complication of therapy. Overall actuarial survival was 88% at 4 years. This study confirms the favorable outcome in children with mediastinal neuroblastoma. The basic biology of thoracic neuroblastomas seems to differ from that of other sites in that the majority of patients present at a younger age with localized disease or regional lymph node metastases, and have an improved survival even after correcting for age and stage. While complete excision is recommended, if possible, radical surgical procedures are not indicated since an excellent prognosis is associated with combined modality therapy.

Age Factors↗