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PubMed1994

Calcium-mediated inhibition of phorbol ester and Tax trans-activation of the human T cell leukaemia virus type 1.

Human Jurkat T cells containing a stably integrated human T cell leukaemia virus type 1 (HTLV-1) long terminal repeat (LTR) reporter gene construct were used to study the role of calcium-dependent cellular activation pathways in LTR trans-activation. Treatment of these cells with the calcium ionophore ionomycin resulted in a reduced basal response of the LTR and reduced responses to 12-O-tetradecanoylphorbol-13-acetate-and Tax-mediated trans-activation. This effect was also observed for virus production in the HTLV-1-producing T cell line MT-2. Experiments designed to determine the events underlying this inhibition, using inhibitors of calcium-related events, revealed that the ionomycin-induced repression of the LTR was alleviated in all cases by cyclosporin A. This compound was also effective in preventing the ionomycin-induced reduction in virus production in MT-2 cells. These results suggest a role for calcium-related events in the down-regulation of HTLV-1 expression.

Calcium↗
PubMed1985

A combination of calcium ionophore and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulates the growth of purified resting B cells.

In this study we investigated whether the calcium ionophores A23187 and ionomycin can act synergistically with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to stimulate the growth of resting B lymphocytes purified from human tonsil cells. Ionomycin, A23187, and TPA added separately to cultures at doses of 0.4-1.6 micrograms/ml, 0.2-0.8 micrograms/ml, and 0.05-0.25 ng/ml respectively, did not induce DNA synthesis in resting B lymphocytes. In contrast, calcium ionophores at concentrations of 0.4-1.6 micrograms/ml ionomycin and 0.2-0.8 micrograms/ml A23187, in the presence of 0.05-4 ng/ml TPA, induced marked DNA synthesis and B-cell proliferation, as shown by analyses of incorporation of [3H]thymidine, growth kinetics, and the percentage of cells in the S and G2 + M phases of the cell cycle. These results show that the synergistic effects of calcium ionophores and TPA can bypass the requirement for antigen and exogenous growth factors in B-cell activation. These observations are similar to those obtained from studies of T lymphocytes by other workers.

B-Lymphocytes↗
PubMed1991

Expression of CD40 and CD43 during activation of human B lymphocytes.

CD40 and CD43 are two cell-surface glycoproteins that appear to be functionally involved in the growth stimulation of human B cells. Whereas CD40 is structurally similar to the NGF receptor and is present on all resting B cells, CD43 displays no homology to other known proteins and is expressed only on a subpopulation of these cells. To further understand the extra- and intracellular signals regulating these molecules and in which stage of activation they may play a role, we used various activation strategies and studied their expression on tonsillar B cells. As expected, activation of protein kinase C by TPA increased both CD40 and CD43. In contrast, a rise in intracellular Ca2+, e.g. by ionomycin, did not influence the expression of these antigens. However, in the presence of TPA, ionomycin further up-regulated CD43 but not CD40. Anti-IgM behaved similarly to ionomycin suggesting that the effect of this reagent was due primarily to its ability to increase intracellular Ca2+. Of three interleukins (IL-2, IL-4 and IL-6) only IL-4 had a significant effect when used alone in that it up-regulated CD40 but not CD43. However, in the presence of anti-IgM, both IL-2 and IL-4 synergistically up-regulated the two antigens. Complementation of antigen receptor stimulation with TPA or IL-4 increased CD40 during the first 24 h, whereas up-regulation of CD43 did not occur until 24 to 48 h after stimulation. With regard both to up-regulation in response to different stimuli and to kinetics, CD40 expression paralleled that of the early activation antigen CD23, whereas CD43 was induced in parallel with the transferrin receptor (CD71). Taken together, our results suggests that the expression of CD40 and CD43 is regulated by different intracellular signals and that CD40 may be important during early activation, whereas CD43 may have its major function during later stages of B-cell differentiation. These assumptions are in line with the observations that CD40 antibodies can directly activate resting B cells and that CD43 are retained on plasma cells.

Antibodies↗
PubMed1992

B lymphoblasts show oxidase activity in response to cross-linking of surface IgM and HLA-DR.

Human B lymphocytes express components of the superoxide generating system of phagocytes, NADPH oxidase. We studied regulation of this 'B-cell oxidase' during in vitro blast transformation, using Lucigenin-amplified chemiluminescence (CL) to detect superoxide release. While freshly isolated tonsil B lymphocytes showed no CL responses, culture with phorbol myristate acetate (PMA) and ionomycin induced susceptibility to CL triggering by anti-IgM and anti-HLA-DR. Maximal effects were observed after 3 days of culture with 0.4 ng/ml PMA + 1 microgram/ml ionomycin. Cells from such B lymphoblast cultures showed no CL responses to opsonized zymosan. In contrast, peripheral blood mononuclear cells, where monocytes are the predominant oxidant source, showed CL responses to opsonized zymosan but not to anti-IgM and anti-HLA-DR, either before or after culture with PMA + ionomycin. Culture of B cells with the surface immunoglobulin cross-linking agent staphylococcus aureus Cowan I also led to emergence of a CL, response to anti-IgM, which was enhanced by interferon-gamma. Interestingly, markedly fewer B blasts than freshly isolated B lymphocytes expressed cytochrome b-558 surface antigen. Thus, the B-cell oxidase is up-regulated during blast transformation and can be triggered via surface IgM and HLA-DR; however, this appears to be restricted to a subset of B lymphoblasts.

B-Lymphocytes↗
PubMed1999

Control of apical membrane chloride permeability in the renal A6 cell line by nucleotides.

1. The effect of extracellular nucleotides applied on the apical side of polarised A6 cells grown on permeant filters was investigated by measuring the changes in (i) the 36Cl efflux through the apical membranes, (ii) the intracellular chloride concentrations (aCli, measured with N-(6-methoxyquinolyl) acetoethyl ester, MQAE), (iii) ICl, the short-circuit current in the absence of Na+ transport and (iv) the characteristics of the apical chloride channels using a patch-clamp approach. 2. ATP or UTP (0.1-500 microM) transiently stimulated ICl. The sequence of purinergic agonist potencies was UTP = ATP > ADP >> the P2X-selective agonist beta,gamma-methylene ATP = the P2Y-selective agonist 2-methylthioATP. Suramin (100 microM) as the P2Y antagonist Reactive Blue 2 (10 microM) had no effect on the UTP (or ATP)-stimulated current. These findings are consistent with the presence of P2Y2-like receptors located on the apical membranes of A6 cells. Apical application of adenosine also transiently increased ICl. This effect was blocked by theophylline while the UTP-stimulated ICl was not. The existence of a second receptor, of the P1 type is proposed. 3. ATP (or UTP)-stimulated ICl was blocked by apical application of 200 microM N-phenylanthranilic acid (DPC) or 100 microM niflumic acid while 100 microM glibenclamide was ineffective. 4. Ionomycin and thapsigargin both transiently stimulated ICl; the nucleotide stimulation of ICl was not suppressed by pre-treatment with these agents. Chlorpromazin (50 microM), a Ca2+-calmodulin inhibitor strongly inhibited the stimulation of ICl induced either by apical UTP or by ionomycin application. BAPTA-AM pre-treatment of A6 cells blocked the UTP-stimulated ICl. Niflumic acid also blocked the ionomycin stimulated ICl. 5. A fourfold increase in 36Cl effluxes through the apical membranes was observed after ATP or UTP application. These increases of the apical chloride permeability could also be observed when following aCli changes. Apical application of DPC (1 mM) or 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB; 500 microM) produced an incomplete inhibition of 36Cl effluxes through the apical membranes in ATP-stimulated and in untreated monolayers. 6. In single channel patch-clamp experiments, an apical chloride channel with a unitary single channel conductance of 7.3 +/- 0.6 pS (n = 12) was usually observed. ATP application induced the activation of one or more of these channels within a few minutes. 7. These results indicate that multiple purinergic receptor subtypes are present in the apical membranes of A6 cells and that nucleotides can act as modulators of Cl- secretion in renal cells.

Animals↗
PubMed1990

Endothelin and a Ca2+ ionophore raise cyclic GMP levels in a neuronal cell line via formation of nitric oxide.

1. The vasoconstrictor peptide endothelin-1 caused a fast, transient rise in guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in a neuronal cell line (mouse neuroblastoma x rat glioma hybrid cells 108CC15). The mechanism of activation of guanylate cyclase by endothelin-1 was investigated. The endothelin-1-induced rise depended on the release of internal Ca2+. 2. The stimulation of cyclic GMP synthesis induced by endothelin-1 was suppressed after preincubating the cells in medium containing haemoglobin (IC50 3 microM). Similarly, pretreatment of the cells with the L-arginine analogues, L-canavanine (IC50 60 microM) or NG-monomethyl-L-arginine (IC50 2.5 microM), inhibited the cyclic GMP response to endothelin-1. Therefore, endothelin-1 activates guanylate cyclase most probably via formation of nitric oxide, which is released from L-arginine. 3. The Ca2+ ionophore ionomycin induced a transient rise in cyclic GMP levels, which was also suppressed by preincubation in the presence of either haemoglobin or the L-arginine analogues L-canavanine or NG-monomethyl-L-arginine. Therefore, we conclude that ionomycin can activate guanylate cyclase by a mechanism involving nitric oxide formation, similar to that induced by endothelin-1. 4. The alkaloid veratridine, which activates Na+ channels and also causes influx of Ca2+ induced a transient rise of cyclic GMP levels in the neuronal cell line. This stimulation was blocked by pretreating the cells with L-canavanine, NG-monomethyl-L-arginine or haemoglobin. 5. Loading the cells with the Ca2+ chelator BAPTA suppresed the cyclic GMP response to application of endothelin-1, ionomycin, or veratridine. Thus, in the neuronal cell line a rise in cytosolic Ca2 + activity seems to be sufficient to stimulate the nitric oxide forming enzyme which synthesizes the activator of soluble guanylate cyclase.

Animals↗
PubMed1994

Effect of raised extracellular calcium on cell spread area in quail medullary bone osteoclasts.

The present study reports on the effects of extracellular calcium ([Ca2+]o) elevation and ionomycin on cell spread area of medullary bone osteoclasts freshly isolated from egg-laying Japanese quail. The responses were compared with those demonstrated in osteoclasts cultured for periods of 5-8 days and also to those previously demonstrated in neonatal rat osteoclasts. Freshly isolated medullary bone osteoclasts, unlike rat osteoclasts, were refractory to 20 mM [Ca2+]o, in that they showed no change in cell spread area. They did, however, show a modest (15%) reduction in cell spread area to ionomycin (7-50 microM), applied for 15-30 min. When medullary bone osteoclasts were precultured for 5-8 days, they exhibited a well-developed response to 20 mM [Ca2+]o with a 46% reduction in cell spread area. They also showed a similar reduction in cell spread area in response to ionomycin (4 microM). It is concluded that, unlike freshly isolated neonatal rat osteoclasts, those obtained from quail medullary bone appear refractory to inhibitory factors such as [Ca2+]o. However, when the avian cells are cultured for a few days they appear to recover their ability to respond to [Ca2+]o.

Animals↗
PubMed1994

Calcium influx and release in isolated rat osteoclasts.

Intracellular and extracellular sources of cytosolic [Ca2+] elevation in isolated rat osteoclasts were explored by a comparison of fura-2 signals in response to application of the Ca2+ ionophore, ionomycin, in Ca(2+)-containing and in Ca(2+)-free bathing solutions. Cytosolic [Ca2+] transients persisted in osteoclasts bathed in Ca(2+)-free, EGTA-containing solutions. They consisted of a peak cytosolic [Ca2+bd elevation followed by a full decay to baseline and were refractory to manipulations of surface membrane potential through changes in extracellular [K+]. They disappeared upon intracellular Ca2+ store depletion through repeated ionophore applications. They were therefore attributable solely to intracellularly stored Ca2+. In contrast, the fura-2 peaks in osteoclasts exposed to Ca(2+)-containing solutions decayed to sustained levels. Cytosolic [Ca2+] responses then persisted with repeated ionomycin application. These latter phenomena are accordingly attributable to extracellular Ca2+ entry. Finally, restoration of extracellular [Ca2+] to 1.25 mM following the depletion of intracellular Ca2+ stores by treatment with ionomycin elicited a cytosolic [Ca2+] 'overshoot' consistent with capacitative Ca2+ entry via a cytosolic route. These results demonstrate a refillable intracellular source of cytosolic Ca2+ that could function in osteoclastic regulation.

Animals↗
PubMed1985

Responses to adenosine diphosphate in human platelets loaded with the fluorescent calcium indicator quin2.

ADP produces a rapid elevation in the concentration of cytoplasmic free calcium, [Ca2+]i, in quin2-loaded human platelets which begins within 1 s of stimulation and peaks after 10 s. In the presence of 1 mM-extracellular calcium, [Ca2+]i peaks at 670 +/- 50 nM in the absence and 610 +/- 30 nM in the presence of a cyclo-oxygenase inhibitor. The production of prostaglandin endoperoxides and thromboxane A2 are not required for stimulation of Ca2+ fluxes by ADP but appear to have a supportive role. In the absence of extracellular calcium ions and with 1 mM-extracellular EGTA, stimulation with ADP caused [Ca2+]i to peak at 160 +/- 20 nM in the absence and 150 +/- 10 nM in the presence of a cyclo-oxygenase inhibitor. ADP can cause the discharge of calcium ions from internal stores and does not require the prior formation of prostaglandin endoperoxides or thromboxane A2. The rise in [Ca2+]i in the presence of extracellular Ca2+ is sixfold larger than in the absence of extracellular Ca2+. This suggests that the major component of the ADP-stimulated rise in [Ca2+]i is caused by the influx of Ca2+ ions across the plasma membrane. Diltiazem, D600, nimodipine and nifedipine had little or no effect on resting or ADP-stimulated [Ca2+]i levels. Depolarization with potassium-rich media alone or in conjunction with valinomycin had no effect on basal [Ca2+]i and only a partial inhibitory effect on ADP-stimulated increases in [Ca2+]i. Depolarization had no effect on the ADP-stimulated rise in [Ca2+]i in Ca2+-free media. Hyperpolarization had no marked effect on the rise in [Ca2+]i produced by ADP in the presence of extracellular calcium. These results are consistent with there being no voltage-dependent channels in the platelet plasma membrane. Using ionomycin, a selective Ca2+ ionophore, and measuring both quin2 fluorescence and optical density of the suspension simultaneously, the threshold [Ca2+]i for shape change was determined to be 300 nM with half-maximal effect at 500 nM and maximal shape change at 800 nM. ADP produced maximal shape change confirmed by scanning electron microscopy with corresponding [Ca2+]i at below 200 nM. The level of [Ca2+]i required to produce aggregation using ionomycin was approximately 1 microM. ADP alone, or following a smaller rise in [Ca2+]i produced by ionomycin to disguise the effect of ADP, produced an aggregatory response at concentrations below 1 microM. These data indicate that excitatory mechanisms are involved producing shape change and aggregation to ADP other than a stimulated rise in [Ca2+]i.

Adenosine Diphosphate↗
PubMed1990

Effects of metabolic inhibition on the membrane properties of isolated mouse primary sensory neurones.

1. The patch-clamp technique has been used to investigate the mechanisms that couple membrane excitability to metabolism in neurones isolated from mouse dorsal root ganglia. 2. Blockade of electron transport by cyanide (CN-), reduction of the mitochondrial membrane potential with carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), removal of glucose or inhibition of glycolysis with idoacetic acid (IAA), all increased a K+ conductance (gK), which could be sufficient to shunt action potentials. 3. The K+ conductance was reduced by incubation of cells in Ca2(+)-free solutions or by increasing the Ca2+ buffering power of pipette-filling solutions. The Ca2+ ionophore, ionomycin, also increased a K+ conductance, and current fluctuation analysis showed that the channels carrying the current induced by both ionomycin and by CN- had a similar mean conductance of circa 9 pS. Thus, increased gK was a Ca2(+)-dependent K+ conductance, gK(Ca), reflecting a rise in resting [Ca2+]i. 4. The conductance was not affected by inclusion of ATP or an ATP-regenerating system in the pipette, suggesting that the underlying rise in [Ca2+] is not due directly to loss of ATP, and confirming that the increased gK is not carried through ATP-dependent K+ channels. 5. Voltage-gated K+ currents evoked by membrane depolarization were increased by CN- or glucose removal. The current-voltage relation of the increased gK mirrored the voltage dependence of Ca2+ entry, and thus reflects impaired cellular handling of the Ca2+ load imposed by depolarization. 6. The rise in [Ca2+]i and altered Ca2+ buffering capacity induced by metabolic blockade affected several other conductances: (i) a Ca2(+)-dependent chloride current was increased. (ii) Both the low-threshold transient and high-threshold sustained voltage-gated Ca2+ currents were attenuated and their thresholds were shifted in the hyperpolarizing direction. (iii) The inward current activated by hyperpolarization. IH, seen in large cells, was attenuated by either metabolic blockade or ionomycin. 7. The responses of these neurones to impaired metabolism thus depend largely on the effects of raised [Ca2+]i on the populations of channels expressed by the cells. These changes in membrane properties could account for some of the changes in neuronal behaviour seen during the clinical states of hypoxia or hypoglycaemia, underlying changes in central nervous system function.

Adenosine Triphosphate↗
PubMed1992

The source of calcium for muscarinic-mediated catecholamine release from cat adrenals.

1. In view of conflicting reports on the source of Ca2+ needed to trigger the secretory response to muscarinic stimulation of chromaffin cells, we have reinvestigated this problem in the cat adrenal gland perfused with oxygenated Krebs solution at 37 degrees C. Above a basal rate of secretion of 60 ng/30 s of total catecholamines, 5 s pulses of 100 microM-methacholine evoked 10-fold increases of secretion. This response was entirely mediated by muscarinic receptors, since it was blocked by submicromolar concentrations of atropine but not by d-tubocurarine. 2. Delayed application of methacholine pulses after Ca2+ removal from the Krebs solution led to a progressive decline of the secretory response with a t1/2 of 15 s. Secretion was blocked by 85% after a 60 s period of Ca2+ deprivation; extension of the external Ca2+ (Ca2+o) wash-out period up to 5 min did not further reduce the secretory response. 3. When EGTA (1 mM) was present in the 0 Ca2+ solution, the rate of decline of methacholine responses, as a function of the time of exposure to 1 mM-EGTA, was similar to that obtained with 0 Ca2+. Again, about 15-20% of the secretory response was resistant even to prolonged periods of washing out with the 0 Ca(2+)-EGTA solution. 4. The Ca2+ ionophore ionomycin (1 microM) first decreased and then accelerated the rate of decline of methacholine responses upon Ca2+o wash-out. Particularly relevant is the complete blockade of secretion when the Ca2+o wash-out is performed in the presence of this ionophore. This suggests the existence of a small intracellular functional Ca2+ store sensitive to ionomycin. 5. After abolition of the secretory response through 60 s periods of wash-out with a 0 Ca(2+)-EGTA-ionomycin solution, followed by delayed 5 s methacholine pulses after Ca2+o reintroduction, the glands instantly recovered their normal muscarinic-mediated secretory response. This suggests that upon muscarinic stimulation, Ca2+ required by the secretory machinery to trigger such response immediately comes from extracellular sources. How Ca2+o gains the cell interior so fast upon muscarinic stimulation is unknown; we have previously suggested that the muscarinic receptor in the cat chromaffin cell could be coupled to an ionophore channel which might be chemically activated by muscarinic agonists. 6. Secretory responses to 5 s pulses with 35 or 100 mM-K+ declined faster (t1/2 of 3 and 6 s, respectively) upon Ca2+o wash-out than those of methacholine.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed2004

Basolateral Na+-H+ exchanger-1 in rat taste receptor cells is involved in neural adaptation to acidic stimuli.

The role of basolateral Na(+)-H(+) exchanger isoform-1 (NHE-1) was investigated in neural adaptation of rat taste responses to acidic stimuli, by direct measurement of intracellular pH (pH(i)) in polarized taste receptor cells (TRCs) and by chorda tympani (CT) taste nerve recordings. In TRCs perfused with CO(2)/HCO(3)(-)-free solution (pH 7.4), removal of basolateral Na(+) decreased pH(i) reversibly and zoniporide, a specific NHE-1 blocker, inhibited the Na(+)-induced changes in pH(i). The spontaneous rate of TRC pH(i) recovery from NH(4)Cl pulses was inhibited by basolateral zoniporide with a K(i) of 0.33microm. Exposure to basolateral ionomycin, reversibly increased TRC Ca(2+), resting pH(i), and the spontaneous rate of pH(i) recovery from an NH(4)Cl pulse. These effects of Ca(2+) on pH(i) were blocked by zoniporide. In in vivo experiments, topical lingual application of zoniporide increased the magnitude of the CT responses to acetic acid and CO(2), but not to HCl. Topical lingual application of ionomycin did not affect the phasic part of the CT responses to acidic stimuli, but decreased the tonic part by 50% of control over a period of about 1 min. This increased adaptation in the CT response was inhibited by zoniporide. Topical lingual application of 8-CPT-cAMP increased the CT responses to HCl, but not to CO(2), and acetic acid. In the presence of cAMP, ionomycin increased sensory adaptation to HCl, CO(2), and acetic acid. Thus, cAMP and Ca(2+) independently modulate CT responses to acidic stimuli. While cAMP enhances TRC apical H(+) entry and CT responses to strong acid, an increase in Ca(2+) activates NHE-1, and increases neural adaptation to all acidic stimuli.

Acids↗
PubMed2006

Exocytotic release of ATP and activation of P2X receptors in dissociated guinea pig stellate neurons.

Activation of P2X receptors by a Ca(2+)- and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent release of ATP was measured using patch-clamp recordings from dissociated guinea pig stellate neurons. Asynchronous transient inward currents (ASTICs) were activated by depolarization or treatment with the Ca(2+) ionophore ionomycin (1.5 and 3 microM). During superfusion with a HEPES-buffered salt solution containing 2.5 mM Ca(2+), depolarizing voltage steps (-60 to 0 mV, 500 ms) evoked ASTICs on the decaying phase of a larger, transient inward current. Equimolar substitution of Ba(2+) for Ca(2+) augmented the postdepolarization frequency of ASTICs, while eliminating the larger transient current. Perfusion with an ionomycin-containing solution elicited a sustained activation of ASTICs, allowing quantitative analysis over a range of holding potentials. Under these conditions, increasing extracellular [Ca(2+)] to 5 mM increased ASTIC frequency, whereas no events were observed following replacement of Ca(2+) with Mg(2+), demonstrating a Ca(2+) requirement. ASTICs were Na(+) dependent, inwardly rectifying, and reversed near 0 mV. Treatment with the nonselective purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 microM) blocked all events under both conditions, whereas the ganglionic nicotinic antagonist hexamethonium (100 microM and 1 mM) had no effect. PPADS also blocked the macroscopic inward current evoked by exogenously applied ATP (300 microM). The presence of botulinum neurotoxin E (BoNT/E) in the whole-cell recording electrode significantly attenuated the ionomycin-induced ASTIC activity, whereas phorbol ester treatment potentiated this activity. These results suggest that ASTICs are mediated by vesicular release of ATP and activation of P2X receptors.

Adenosine Triphosphate↗
PubMed1989

Cyclic nucleotide-mediated effects of ANF and VIP on flounder intestinal ion transport.

The purpose of this study was to compare the effects of atriopeptin III (AP-III), vasoactive intestinal peptide (VIP), and ionomycin on Na and Cl influx and to correlate changes in transport with effects on intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 5'-cyclic monophosphate (cGMP) content of the tissue. In addition, the question of whether AP-III inhibits ion transport directly by acting on enterocyte receptors for AP-III or indirectly by stimulation of enteric nerves in the submucosa was also addressed. The results showed that AP-III, ionomycin, and bumetanide all inhibited the initial rate of Na and Cl influx, suggesting that they directly block Na-K-2Cl cotransport activity. VIP had no effect on unidirectional influx of Na and Cl. AP-III caused a fourfold increase in intracellular [cGMP] without any significant effect on [cAMP]. VIP stimulated [cAMP] by fourfold but had no effect on [cAMP]. Ionomycin had no effect on either [cAMP] or [cGMP]. Inhibition of transport by AP-III could not be blocked by tetrodotoxin (TTX), indicating that enteric nerves in the submucosa are not directly involved in mediating the effects of AP-III on epithelial ion transport. The observation that two classes of neuronal depolarizing agents (veratrine and scorpion venom) cause TTX-sensitive inhibition of basal ion transport establishes that NaCl absorption in flounder intestine is subject to regulation by enteric nerves located in the submucosa.

Animals↗
PubMed1990

Myosin light chain phosphorylation in human myometrial smooth muscle cells.

Ca2+/calmodulin-dependent phosphorylation of the 20-kDa regulatory light chain of myosin is of signal importance in the initiation of contraction in a number of smooth muscle tissues. In this investigation, we evaluated the relationship between intracellular free Ca2+/concentration [( Ca2+]i) and the extent of myosin light chain phosphorylation in cultured human myometrial smooth muscle cells. Treatment of myometrial cells with ionomycin caused a concentration- and time-dependent increase in [Ca2+]i and phosphorylation of myosin light chain. Temporally, the increases in light chain phosphorylation and [Ca2+]i in response to ionomycin were similar. In myometrial cells treated with ionomycin (10(-5) M) for 10 s, [Ca2+]i increased from 138 to 800 nM; in these same cells, myosin light chain phosphorylation increased from 5% to a maximum value of 54%. Half-maximal phosphorylation of myosin light chain was attained at 300 nM [Ca2+]i. Treatment of myometrial smooth muscle cells with prostaglandin (PG) F2 alpha (10(-8) M) and PGE2 (10(-8) M) caused a proportionate increase in [Ca2+]i and myosin light chain phosphorylation. In addition, [Ca2+]i and myosin light chain phosphorylation increased in response to oxytocin and angiotensin II. These findings indicate that a number of uterotonic agents effect an increase in [Ca2+]i, which in turn causes phosphorylation of myosin light chain. Furthermore, the concentration of Ca2+ in the cytoplasm is a primary determinant for myosin light chain phosphorylation in human myometrial smooth muscle cells.

Angiotensin II↗
PubMed1990

A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells.

We developed a convenient flux assay that permits simultaneous measurement of Cl and K conductance pathways in Cl-secreting epithelial cells. Monolayers of the colonic tumor cell line T84 were preloaded with 125I and 86Rb, and isotope effluxes were monitored by a sample-replace procedure. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agonists forskolin and prostaglandin E2 increased I efflux with little effect on Rb efflux, whereas the Ca-mediated agonists ionomycin, A23187, and carbachol increased both I and Rb effluxes. Simultaneous determinations of I and Cl or Rb and K effluxes indicated that I and Rb provide good measures of the effluxes of Cl and K, respectively. Forskolin- and ionomycin-stimulated I effluxes were inhibited by the Cl-channel blockers diphenylamine-2-dicarboxylate (DPC), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), and 2-[cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H- inden-5-yl)oxy]acetic acid (IAA-94) and by high external K. The Rb efflux evoked by ionomycin was inhibited by the K-channel blockers Ba and charybdotoxin. These findings suggest that I and Rb effluxes provide qualitative estimates of agonist-stimulated Cl and K conductance pathways. Thus this method can provide a simple and relatively inexpensive screening assay for Cl and K conductances in cultured cells to assess the effects of agonist, blockers, or genetic manipulations.

Barium↗
PubMed1992

Substance P induces whole cell current transients in RBL-2H3 cells.

To investigate the basis of interactions between nerves and mast cells, we tested the actions of the neuropeptide substance P (SP) on whole cell current characteristics of RBL-2H3 cells (homologous to mucosal mast cells). Control RBL cells showed a K(+)-dependent inwardly rectified current. SP (10(-6) M) caused transient, frequently repetitive increases in current amplitude, which at a membrane potential (Vm) of -80 mV rose by -1,020.0 +/- 223.4 pA after SP application compared with -6.8 +/- 1.7 pA for control. This response was characterized by a lag phase of 102 +/- 16 s. Seventeen percent of cells showed spontaneous transients in the current amplitude from the beginning of the recording. After SP administration, the amplitude of these transients increased by 6.3 +/- 2.0-fold. Responses to SP were mimicked by the application of ionomycin. For both SP and ionomycin, there was a dose dependency of the lag phase. Removal of extracellular calcium abolished the response for 10(-6) M SP but not for 6.6 x 10(-6) M ionomycin. During current transients, the whole cell current had both inward and outward rectified components with the zero current Vm shifted from -87.3 +/- 3.2 mV at control to -10.8 +/- 1.7 mV. We compare the SP-evoked current responses in mucosal-type mast cells with those described in connective tissue type.

Animals↗
PubMed1993

Nitric oxide synthesis is impaired in glutathione-depleted human umbilical vein endothelial cells.

Human endothelial cells cultured from umbilical vein (HUVEC) were tested for their ability to synthesize nitric oxide (NO), which has been identified as an endothelium-derived relaxing factor. The synthesis of this free radical (detected as citrulline, which is produced stoichiometrically with NO from arginine) in HUVEC is Ca2+ dependent, is increased sevenfold by the calcium ionophore ionomycin, and accounts for most basal and ionomycin-induced guanosine 3',5'-cyclic monophosphate (cGMP) production. Loading of cells with reduced glutathione (GSH), but not with N-(2-mercaptopropionyl)- glycine (MPG), led to increased citrulline production, both basally and after ionomycin stimulation. When the cells were depleted of GSH by incubation with 1-chloro-2,4-dinitrobenzene (CDNB), citrulline synthesis and cGMP production were inhibited in a concentration-dependent way. CDNB was not cytotoxic and did not inhibit cGMP increase elicited by sodium nitroprusside; cell loading with GSH (but not with MPG) relieved the block of citrulline synthesis. These results suggest that GSH is necessary in HUVEC for NO synthesis rather than for the NO effect on guanylate cyclase.

Calcium↗