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PubMed1991

Calcium signalling and the triggering of secretion in adrenal chromaffin cells.

The pivotal intracellular message for triggering catecholamine release from bovine adrenal chromaffin cells is an elevation in the concentration of cytosolic free Ca2+ ([Ca2+]i). Studies using video-imaging techniques have shown that a rise in [Ca2+]i at the cell periphery, that is due to Ca2+ entry, is the major activating signal for exocytosis. The cytoskeleton has been identified as a major regulatory site of exocytosis, with Ca(2+)-induced disruption of the cortical actin network being required in order that previously restrained granules may have access to their exocytotic sites. The Ca(2+)- and phospholipid-dependent annexin protein, calpactin, has been strongly implicated in a late stage of interaction between granules and the plasma membrane by both ultrastructural and biochemical studies.

Adrenal Glands↗
PubMed1995

Calcium signalling during mammalian fertilization.

The fertilized mammalian egg is a nice model system for analysing spatiotemporal Ca2+ signalling in the intact cell. Hamster eggs show repetitive Ca2+ transients, associated in the initial response with Ca2+ waves which begin from the site of sperm attachment and are propagated across the deep cytoplasm to the opposite pole. In unfertilized eggs, a regenerative Ca2+ wave is induced by injection of either inositol 1,4,5-trisphosphate (InsP3) or Ca2+, and Ca2+ oscillations are produced by continuous injection of InsP3. These Ca2+ waves and oscillations in both fertilized and unfertilized eggs are inhibited in a dose-dependent manner by a monoclonal antibody to the type 1 InsP3 receptor. Ryanodine receptors (both skeletal and cardiac types) are not detected by physiological or immunoblot analyses. Positive and negative feedback between cytosolic Ca2+ and Ca2+ release from InsP3-sensitive pools accounts for the spatiotemporal Ca2+ signalling. In addition to intracellular Ca2+ release, Ca2+ entry from outside the egg is necessary to refill the Ca2+ pools and maintain Ca2+ oscillations. Evidence suggests that inositol 1,3,4,5-tetrakisphosphate activates the Ca2+ influx. The signal transduction process leading to the production of InsP3 and the mechanism of egg activation following the Ca2+ response still remain to be elucidated.

Animals↗
PubMed1991

Effects of surgical sympathetic denervation on myo-inositol trisphosphate production and contraction in the dilator and sphincter smooth muscles of the rabbit iris: evidence for interaction between the cyclic AMP and calcium signaling systems.

The effects of norepinephrine (NE), carbachol (CCh), NaF, 3-isobutyl-1-methylxanthine (IBMX), and high K+ concentration (80 mM) depolarization on inositol trisphosphate (IP3) accumulation, cyclic AMP (cAMP) formation, and contraction were investigated in the dilator and sphincter smooth muscles of the sympathetically denervated as well as the normal rabbit eye. (a) In the denervated dilator muscle, NE-stimulated IP3 production and contraction are enhanced. (b) In the sphincter muscle of rabbits that have undergone sympathetic denervation. CCh-stimulated IP3 production and contraction are attenuated. (c) The increase in tension by a maximal effective dose of NaF (209 mM) in the dilator was 12.5 and 18 mg of tension/mg wet weight in normal and denervated tissue, respectively, and in the sphincter was 33.8 and 15.2 mg of tension/mg wet weight in normal and denervated tissue, respectively. NaF had no effect on cAMP formation. (d) Addition of NE had no effect on cAMP formation in both the normal and denervated dilator, whereas basal and IBMX-induced cAMP formation increased. in the denervated sphincter over that of the normal tissue by 15 and 60%, respectively. (e) Isoproterenol (5 microM) increased cAMP formation in the normal and denervated sphincter by 47 and 91%, respectively. (f) Whereas CCh inhibits cAMP formation in the normal sphincter, it lost its inhibitory effect in the sphincter with denervation. (g) IBMX (0.1 mM) attenuated the CCh-stimulated IP3 production and contraction of the sphincter by approximately 30% of their respective controls. (h) High K+ concentration depolarization attenuated contraction in both dilator and sphincter muscles with denervation. These observations suggest that an increase in the level of cAMP in the iris sphincter due to sympathetic denervation could lead to inhibition of phospholipase C (or other target sites, such as phosphorylation of the muscarinic receptor, Gp protein itself, myosin light chain kinase, or the IP3 receptor), IP3 production, and contraction. In conclusion, we suggest that the supersensitivity and subsensitivity observed after surgical sympathetic denervation of the iris dilator and sphincter muscles, respectively, are caused by alterations in the efficiency of coupling, probably through the Gp proteins, between their respective receptors and the breakdown of polyphosphoinositides by phospholipase C. In addition, we propose that the sympathetic nervous system can regulate, through alterations in cAMP levels, the muscarinic stimulation of IP3 accumulation and contraction in the iris sphincter. These findings add further support to the hypothesis that there are reciprocal interactions between the cAMP and IP3-Ca2+ signaling systems and the contractile response in the iris smooth muscle.

1-Methyl-3-isobutylxanthine↗
PubMed2002

Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells: the role of the actin cytoskeleton.

Loading of the spine alters the osmotic environment in the intervertebral disk (IVD) as interstitial water is expressed from the tissue. Cells from the three zones of the IVD, the anulus fibrosus (AF), transition zone (TZ), and nucleus pulposus (NP), respond to osmotic stress with altered biosynthesis through a pathway that may involve calcium (Ca(2+)) as a second messenger. We examined the hypothesis that IVD cells respond to hyperosmotic stress by increasing the concentration of intracellular calcium ([Ca(2+)](i)) through a mechanism involving F-actin. In response to hyperosmotic stress, control cells from all zones decreased in volume and cells from the AF and TZ exhibited [Ca(2+)](i) transients, while cells from the NP did not. Extracellular Ca(2+) was necessary to initiate [Ca(2+)](i) transients. Stabilization of F-actin with phalloidin prevented the Ca(2+) response in AF and TZ cells and decreased the rate of volume change in cells from all zones, coupled with an increase in the elastic moduli and apparent viscosity. Conversely, actin breakdown with cytochalasin D facilitated Ca(2+) signaling while decreasing the elastic moduli and apparent viscosity for NP cells. These results suggest that hyperosmotic stress induces volume change in IVD cells and may initiate [Ca(2+)](i) transients through an actin-dependent mechanism.

Actins↗
PubMed1993

P2-purinergic receptors in human breast tumor cells: coupling of intracellular calcium signaling to anion secretion.

ATP increases intracellular Ca++ ([Ca++]i) by activating different P2-purinoreceptors. Because ATP increases Cl- secretion in cystic fibrosis (CF)-affected epithelia, the current study was designed to establish the link between these two events. Studies were done in epithelial, human MCF-7 breast tumor cells in which the presence of mRNA transcripts encoding CF transmembrane conductance regulator was initially established. Changes in [Ca++]i were measured in single cells by fluorescence microscopy; anion transport was measured by 125I efflux. ATP stimulated concentration-dependent increases in [Ca++]i and 125I efflux from MCF-7 cells. The relative order of agonist potency of various selective P2-purinoreceptor agonists in increasing [Ca++]i and 125I efflux was: UTP > or = ATP > ADP = AMP; 2-chloro-ATP, 2-methylthio-ATP and AMP-phencyclidine were considerably less potent than ATP. The Ca++ ionophore ionomycin increased both intracellular [Ca++]i and 125I secretion. Exposing cells to the intracellular chelator ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetra-acetic acid (EGTA)-acetoxymethylester decreased (AM) decreased ATP- and ionomycin-stimulated 125I efflux. Extracellular EGTA did not alter the Ca++ response to ATP, but inhibited the response to ionomycin. The chelator inhibited both ATP- and ionomycin-induced 125I secretion. Exposure of cells to nifedipine did not affect the responsiveness of MCF-7 cells to ATP. The anion transport antagonist 4,4'-diisothiocyananatostilbene-2,2'-disulfonic acid partially inhibited ATP- and cationophore-stimulated increases in [Ca++]i and 125I secretion. The data suggest that activation of P2 receptors in MCF-7 cells leads to an increase in anion transport as a result of the ability of ATP to increase [Ca++]i; moreover, anion channel antagonists may produce their inhibitory effect on 125I secretion, in part, by blocking agonist-induced intracellular Ca++ signaling.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

Gap junctions mediate intercellular calcium signalling in cultured articular chondrocytes.

Gap junction-mediated intercellular communication has been implicated in a variety of cellular functions. Among these, signal transduction can be coordinated among several cells due to gap junctional permeability to intracellular second messengers. Chondrocytes from articular cartilage in primary culture respond to extracellular ATP by rhythmically increasing their cytosolic Ca2+ concentration. Digital imaging fluorescence microscopy of Fura-2 loaded cells was used to monitor Ca2+ in confluent and semi-confluent cell layers. Under these conditions, Ca2+ spikes propagate from cell to cell giving rise to intercellular Ca2+ waves. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in scrape-loading experiments. Intercellular dye transfer was blocked by the gap junction inhibitor 18 alpha-glycyrrhetinic acid. In imaging experiments, the inhibitor caused the loss of synchrony of ATP-induced Ca2+ oscillations, and blocked the intercellular Ca2+ propagation induced by mechanical stimulation of a single cell in a monolayer. It is concluded that gap junctions mediate intercellular signal transduction in cartilage cells and may provide a mechanism for co-ordinating their metabolic activity.

Adenosine Triphosphate↗
PubMed1990

Carmustine augments the effects of tert-butyl hydroperoxide on calcium signaling in cultured pulmonary artery endothelial cells.

The effects of oxidant stress and inhibition of glutathione reductase on the bradykinin-stimulated changes in cytosolic free Ca2+ concentration ([Ca2+]i) of calf pulmonary artery endothelial cells were determined using the intracellular fluorescent probe, fura-2. Changes in [Ca2+]i upon stimulation with bradykinin were measured after incubation of cells with the chemical oxidant tert-butyl hydroperoxide (0.4 mM) for various times. After 60 min, bradykinin-stimulated Ca2+ influx was significantly decreased. With more prolonged incubations with the peroxide, bradykinin had little effect on cytosolic calcium concentration. Preincubation of cells with the glutathione reductase inhibitor, carmustine, led to elevated basal [Ca2+]i, yet the cells remained responsive to bradykinin. However, incubation of carmustine-treated cells with tert-butyl hydroperoxide for 30 min dramatically reduced both bradykinin-stimulated release of Ca2+ from internal stores and influx of Ca2+ from the extracellular space. These results suggest that inhibition of glutathione reductase alters cytosolic Ca2+ homeostasis and enhances the effects of oxidative stress on signal transduction in vascular endothelial cells.

Animals↗
PubMed1993

Role of lipid peroxidation in tert-butylhydroperoxide-induced inhibition of endothelial cell calcium signaling.

The potential of lipid peroxidation in inhibition of Ca++ signaling by the membrane-permeant oxidant, tertiary butylhydroperoxide (tert-buOOH), was investigated in calf pulmonary vascular endothelial cells. The oxidant dose-dependently increased lipid peroxidation between concentrations of 10(-5) and 10(-3) M, with an ED50 of approximately 0.05 mM. In addition, the effect of tert-buOOH was time-dependent through the experimental period (3 h). Preincubation of cells with the 21-amino-steroid compound, 21-[4-(5,6-bis(diethylamino)-2-pyridinyl)-1-piperazinyl]-16 alpha-methyl-pregna-1,4,9(11)-triene-3,20-dione hydrochloride (U74500A), reduced tert-buOOH-induced lipid peroxidation to undetectable levels. The effect of U74500A was dose dependent with an IC50 of approximately 10(-6) M. Brief incubation of cells with the aminosteroid resulted in greater than 90% inhibition of lipid peroxidation during subsequent 2-h incubations with tert-buOOH and addition of U74500A during treatment of cells with tert-buOOH halted further lipid peroxidation. In contrast, the iron-containing moiety, hemin, potentiated the effect of tert-buOOH on lipid peroxidation. The ED50 of hemin was approximately 10(-6) M when cells were preincubated with this agent before treatment with tert-buOOH. The potentiating effect of hemin was time-dependent and reached a near maximum upon incubation of cells for 1 h before tert-buOOH. Preincubation of cells with U74500A before treatment with hemin and tert-buOOH decreased lipid peroxidation by 75%. Ca++ signaling was monitored in-cells loaded with the Ca(++)-sensitive fluorescent indicator, fura-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

The calcium signal and phosphatidylinositol breakdown in 2H3 cells.

Phosphatidylinositol (PI) and its phosphorylated derivatives are rapidly broken down in 2H3 cells stimulated with antigen, with a time course which coincides with the generation of the Ca signal. Stimulated PI breakdown is absolutely dependent on Ca2+ in the medium with a concentration dependence similar to that of the Ca signal and histamine release described in the preceding paper. However, PI breakdown does not depend on the rise in free cytoplasmic Ca2+ concentration in stimulated cells over the range 100 nM to 1 microM. Thus, stimulation by the ionophore A23187 causes only a small increase in PI breakdown and the Ca signal stimulated by antigen can be selectively blocked with appropriate concentrations of Zn2+ (100 microM) or La3+ (10-100 microM) which have small or negligible effects on stimulated PI breakdown. Both PI breakdown and the Ca signal appear to depend on a common external Ca2+ site (or sites) with Km approximately equal to 0.4 mM, and the data are consistent with either independent activation of PI phosphodiesterase and the Ca signal after antigenic stimulation, or with PI breakdown as a component of the mechanism by which the Ca signal is generated.

Animals↗
PubMed1997

Mechanisms of calcium signaling by cyclic ADP-ribose and NAADP.

Cells possess various mechanisms for transducing external signals to intracellular responses. The discovery of inositol 1,4,5-trisphosphate (IP3) as a messenger for mobilizing internal Ca2+ stores has centralized Ca2+ mobilization among signaling mechanisms. Results reviewed in this article establish that, in addition to IP3, the internal Ca2+ stores can be mobilized by at least two other molecules, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), via totally independent mechanisms. Cyclic ADP-ribose is a newly discovered cyclic nucleotide derived from NAD, but, unlike adenosine 3',5'-cyclic monophosphate, its main signaling function is modulation of Ca(2+)-induced Ca2+ release, a major mechanism of Ca2+ mobilization in addition to the IP3 pathway. Evidence shows that cADPR may in fact be responsible for mediating the Ca(2+)-mobilizing activity of the gaseous messenger nitric oxide. Cells responsive to cADPR are widespread and include species from plant to mammal, indicating the generality of cADPR as a signaling molecule. In addition to cADPR, NAADP, a metabolite of NADP, can also mobilize Ca2+ stores. The release mechanism and the stores on which NAADP acts are distinct from cADPR and IP3. Nicotinic acid adenine dinucleotide phosphate may play a role in generating Ca2+ oscillations, since liberation of NAADP in live cells by photolyzing its caged analog produces long lasting Ca2+ oscillations. These two new Ca2+ agonists are intimately related, since the same metabolic enzymes can, under appropriate conditions, synthesize either one, suggesting a unified mechanism may regulate both pathways. Elucidation of these two new Ca2+ mobilization pathways is likely to have an important impact on our understanding of cellular signaling mechanisms.

ADP-ribosyl Cyclase↗
PubMed1984

The mechanism of the calcium signal and correlation with histamine release in 2H3 cells.

Rat basophil leukemic (2H3) cells ( Siraganian , R.P., McGivney , A., Barsumian , E. L., Crews, F. T., Hirata , F., and Axelrod , J. (1982) Fed. Proc. 41, 30-34) loaded with fluorescent Ca2+ indicator quin 2 ( Tsien , R. Y. (1980) Biochemistry 19, 2396-2404) showed a rapid increase in free cytosol calcium concentration [( Ca]i) when histamine release was induced. Intracellular quin 2 concentrations up to 7 mM did not affect release of histamine in response to antigen (aggregated ovalbumin) or concanavalin A with cells primed with antigen-specific monoclonal IgE, or in response to Ca2+ ionophores. The [Ca]i increased from approximately 105 nM to a maximum of approximately 1200 nM within 2 to 3 min after antigenic stimulation and then declined slowly over 30 min toward the level in unstimulated cells. Histamine release was most rapid as [Ca]i reached the maximum value and then decreased continuously with [Ca]i over the subsequent 30 min. Neither the Ca signal nor histamine release was observed when the Ca2+ concentration in the medium [( Ca]o) was less than 50 microM, but both responses were restored on readdition of Ca2+ to 1 mM. The maximal Ca signal was obtained when [Ca]o was approximately greater than 1 mM and was half-maximal at [Ca]o congruent to 0.4 mM. In marked contrast [Ca]i in unstimulated cells varied very little with [Ca]o from 0.1 to 1 mM. Maintenance of the Ca signal required the continuous presence of stimulating ligand, external Ca2+, and the maintenance of cellular ATP; metabolic inhibitors blocked or reversed the Ca signal. La+ ions also caused a rapid and reversible block of the Ca signal and histamine release. The data are interpreted in a model in which the Ca signal is generated by a La3+-sensitive signal influx pathway that is functionally independent of the normal Ca2+ influx pathway in unstimulated cells, and that allows a 10-fold or greater increase in rate of Ca2+ entry. The Ca signal is maintained dynamically by the balance between the increased Ca2+ influx and active Ca2+ efflux across the plasma membrane.

Aminoquinolines↗
PubMed1994

Enhanced calcium signalling events in neuroblastoma x glioma hybrid NG108-15 cells after treatment with dibutyryl cyclic AMP.

The effects of dibutyryl cyclic AMP (dbcAMP) treatment on Ca2+ channel activities, Ca2+ accumulation by intracellular Ca2+ pools, and sizes of IP3- and GTP-releasable pools in neuroblastoma x glioma hybrid NG108-15 cells were studied. High extracellular K+ induced a greater rise in intracellular calcium concentration ([Ca2+]i) in dbcAMP-treated cells than in control cells. In dbcAMP-treated cells, the initial phase of the high K(+)-induced [Ca2+]i rise displayed a much higher sensitivity to omega-conotoxin than it did in control cells, whereas the plateau phase of the [Ca2+]i rise was sensitive only to nifedipine. These results indicate that predominantly L-type Ca2+ channels exist in control cells, and that N-type channels develop only after dbcAMP treatment. In dbcAMP-treated cells, mitochondria showed an increased Ca2+ uptake capacity (5.3 nmol Ca2+/mg protein) compared with that in control cells (4.2 nmol Ca2+/mg protein). However, dbcAMP treatment did not cause significant change in the affinity for Ca2+. Dibutyryl cAMP treatment enhanced the Ca2+ accumulation activity by nonmitochondrial pools (from 0.84 to 0.97 nmol Ca2+/mg protein) and increased the affinity for Ca2+ (EC50 for Ca2+ decreased from 0.146 microM to 0.063 microM). Our data also indicate that the pool that is sensitive to both IP3 and GTP was enlarged. The affinities for IP3 and GTP in causing Ca2+ release remained the same before or after dbcAMP treatment.

Bucladesine↗
PubMed1996

Further identification of neurokinin receptor types and mechanisms of calcium signaling evoked by neurokinins in the murine neuroblastoma C1300 cell line.

It has been suggested that murine neuroblastoma C1300 cells express endogenous neurokinin NK2 receptors with features that differ from those of NK2 receptors characterized in other systems. In this study, we have further characterized the neurokinin receptor types present in this cell line. RNA blots showed that mRNAs of NK2 and NK3 receptors, but not of NK1 receptors, were expressed in C1300 cells. The increase in the cytosolic calcium concentration ([Ca2+]i) induced by 0.33 microM neurokinin A was completely inhibited by SR 48968, an NK2 receptor antagonist, whereas the partial response to 0.33 microM neurokinin B was unaffected, and the response was completely inhibited by SR 142801, and NK3 receptor antagonist. In addition, the [Ca2+]i increase by 0.33 microM senktide, an NK3 receptor agonist, was inhibited by SR 142801 but not by SR 48968. These findings indicated that C1300 cells endogenously express functional NK2 and NK3 receptors. It was also demonstrated that NK2 and NK3 receptors can be activated independently by 3.3 microM neurokinin A in the presence of 1.0 microM SR 142801 or 1.0 microM senktide, respectively. Therefore, the mechanisms of Ca2+ signaling mediated by endogenous NK2 and NK3 receptors were investigated. The independent activation of NK2 or NK3 receptors induced not only the [Ca2+]i increase, but also stimulated the formation of inositol trisphosphates; both these responses were inhibited by U73122, a phospholipase C (PLC) inhibitor. In addition, NK2 and NK3 receptor-mediated [Ca2+]i increase was partially attenuated in the absence of extracellular Ca2+ or in the presence of nickel, an inorganic Ca2+ influx blocker, but was unaffected by nifedipine and omega-conotoxin, L- and N-type voltage-dependent Ca2+ channel blockers, respectively. Furthermore, the depolarization by 60 mM K+ did not affect the [Ca2+]i. These findings suggested that the NK2 and NK3 receptor-mediated [Ca2+]i increase was due to the activation of PLC and was dependent on the mobilization of internal Ca2+ and the entry of extracellular Ca2+ through voltage-independent channels. This study showed that the C1300 cell line is a useful system with which to investigate pharmacological functions and signaling pathways of endogenous NK2 and NK3 receptors.

Animals↗
PubMed1997

Intracellular pH and calcium signalling in rat pancreatic acinar cells.

Intracellular free Ca2+ signals, which occur in many secretory cell types after the binding of some secretagogues to their membrane receptors, are due to Ca2+ mobilization from internal stores and Ca2+ influx from the extracellular space. There is also growing evidence for a modulatory role of intracellular pH in Ca2+ metabolism. In fact it has been proposed that Ca2+ stores in pancreatic acinar cells may be loaded by Ca2+/H+ exchange. The aim of this paper was to establish the effect of intracellular pH on Ca2+ signalling in pancreatic acinar cells. Application of the proton carrier nigericin impairs Ca2+ mobilization in response to cholecystokinin (CCK-8), and application of membrane-permeant bases or acids inhibits CCK-8-evoked intracellular Ca2+ oscillations. Both nigericin and a cell-permeant weak base release Ca2+ from internal stores. However, cytosolic acidification by removal of extracellular Na+ had no effect on the resting or stimulated cytosolic Ca2+ concentration. After depletion of Ca2+ stores by a maximal concentration of CCK-8, nigericin and ionomycin released a residual Ca2+ pool. Taken together, our results show that in pancreatic acinar cells Ca2+ signals require the existence of subcellular gradients of pH and indicate the presence of acidic pools of Ca2+.

Animals↗
PubMed1994

Calcium signalling in individual T-cells measured by confocal microscopy.

Laser-scanning confocal microscopy was used in conjunction with a highly fluorescent Ca2+ indicator fluo-3 to visualize real-time alterations in the intracellular Ca2+ concentration ([Ca2+]i) in individual living Jurkat T-cells during the first minutes of activation by phytohaemagglutinin (PHA) at the physiological temperature (37 degrees C). With a delay of 30-120 s, PHA induced a strong [Ca2+]i peak in the micromolar range (1-3 microM). The rise in [Ca2+]i lasted for 1-2 minutes, and was followed by a sustained plateau of elevated [Ca2+]i in the 0.2-0.5 microM range. Some cells (10-20%) responded with additional asynchronous 0.5-1.5 microM peaks during the plateau phase. These oscillations continued for 10-20 minutes. The spans of the peaks ranged from 30 to 100 s, intervals between peaks varied from 60 to 300 s. It was shown that the initial [Ca2+]i peak was associated with Ca2+ mobilisation from internal sources, whereas the plateau was maintained by an influx of Ca2+ from external medium. In K(+)-rich medium or in the presence of quinine, a K+ channel blocker, no secondary response to PHA-activation characterised by an elevated plateau was observed. The data suggest that the Ca2+ influx was dependent on the membrane potential and/or the extracellular K(+)-concentration. Optical sectioning showed that the intracellular Ca2+ distributed almost homogeneously throughout the cell volume both in control and in PHA-stimulated cells including those exhibiting Ca2+ oscillations. This suggests that Ca2+ signals are localized not only in cytoplasm at the cell plasma membrane but can be also transferred directly into the nucleus.

Aniline Compounds↗
PubMed1998

Calcium signaling in cultured human and rat duodenal enterocytes.

Vagal stimuli increase duodenal mucosal HCO-3 secretion and may provide anticipatory protection against acid injury, but duodenal enterocyte (duodenocyte) responses and cholinoceptor selectivity have not been defined. We therefore developed a stable primary culture model of duodenocytes from rats and humans. Brief digestion of scraped rat duodenal mucosa or human biopsies with collagenase/dispase yielded cells that attached to the extracellular matrix Matrigel within a few hours of plating. Columnar cells with villus enterocyte morphology that exhibited spontaneous active movement were evident between 1 and 3 days of culture. Rat duodenocytes loaded with fura 2 responded to carbachol with a transient increase in intracellular calcium concentration ([Ca2+]i), with an apparent EC50 of approximately 3 microM. In a first type of signaling pattern, [Ca2+]i returned to basal or near basal values within 3-5 min. In a second type, observed in cells with enlarged vacuoles characteristic of crypt cell morphology, the initial transient increase was followed by rhythmic oscillations. Human duodenocytes responded with a more sustained increase in [Ca2+]i, and oscillations were not observed. Rat as well as human duodenocytes also responded to CCK-octapeptide but not to vasoactive intestinal polypeptide. Equimolar concentrations (100 nM) of the subtype-independent muscarinic antagonist atropine and the M3 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide prevented the response to 10 microM carbachol, whereas the M1 antagonist pirenzepine and the M2 antagonists methoctramine and AF-DX 116BS had no effect at similar concentrations. Responses in rat and human duodenocytes were similar. A new agonist-sensitive primary culture model for rat and human duodenocytes has thus been established and the presence of enterocyte CCK and muscarinic M3 receptors demonstrated.

Animals↗
PubMed1992

Cholera toxin-sensitive 3',5'-cyclic adenosine monophosphate and calcium signals of the human dopamine-D1 receptor: selective potentiation by protein kinase A.

The signal transduction pathways of the dopamine-D1 receptor were investigated in two cell types stably transfected with the human D1 receptor cDNA, rat pituitary GH4C1 cells (GH4-hD1), and mouse Ltk-fibroblast cells (L-hD1). In both GH4-hD1 and L-hD1 cell lines, stimulation of the dopamine-D1 receptor induced a marked increase in cAMP accumulation. In addition, dopamine potentiated activation of L-type voltage-dependent calcium channels in a cAMP-dependent manner in GH4-hD1 cells. However, in L-hD1 cells, dopamine increased cytosolic free calcium concentrations ([Ca++]i) by mobilization of intracellular calcium rather than by calcium influx. This effect was correlated with a dopamine-induced enhancement of phospholipase C activity in L-hD1 cells. Pretreatment (24 h) with cholera toxin (CTX) was used to maximally activate the GTP-binding protein (G protein) Gs, causing a maximal elevation of cAMP levels and uncoupling the D1 receptor from Gs. The described actions of dopamine in both cell lines were abolished by pretreatment with CTX, indicating that CTX substrates (e.g. Gs) may mediate these actions. The blockade by CTX was not due to CTX-induced elevation of cAMP, since pretreatment with forskolin or 8-bromo-cAMP to activate cAMP-dependent protein kinase did not inhibit dopamine actions nor alter basal [Ca++]i. Pretreatment (1-3 h) of L-hD1 cells with forskolin (10 microM) or 8-bromo-cAMP (5 mM) altered neither the basal activity of phospholipase C nor basal [Ca++]i in L-hD1 cells but greatly enhanced the dopamine-induced increase of phosphatidyl inositol turnover and [Ca++]i. From these results we conclude that: 1) the dopamine-D1 receptor induces multiple and cell-specific signals, including elevation of cAMP levels in both GH and L cells, cAMP-dependent activation and potentiation of opening of L-type voltage-dependent calcium channel in GH cells, and a novel phosphatidyl inositol-linked mobilization of cellular calcium in L cells; 2) coupling of the D1 receptor to these responses involves CTX-sensitive proteins, possibly Gs; and 3) acute preactivation of cAMP-dependent protein kinase can markedly enhance, rather than attenuate, certain pathways of dopamine-D1 transmembrane signaling.

1-Methyl-3-isobutylxanthine↗