Cross-talk between calcium and cAMP-dependent intracellular signaling pathways. Implications for synergistic secretion in T84 colonic epithelial cells and rat pancreatic acinar cells.

Treatment of various cells with combinations of agents that increase either cAMP or cytosolic calcium can lead to synergistic responses. This study examined interactions, or cross-talk, between these two intracellular messengers and its implication for signaling in two secretory cell types, T84 human colonic epithelial cells and rat pancreatic acinar cells. T84 cell chloride secretion was measured in Ussing chambers. Acinar cell activation was monitored as amylase secretion. Cytosolic calcium was assessed via fura-2 microfluorimetry. A cell-permeant analogue of cAMP synergistically enhanced secretory responses to calcium-mobilizing hormones in both cell types, but paradoxically reduced overall calcium mobilization. The reduction in calcium mobilization could be attributed to an inhibition of calcium influx in T84 cells, although a different mechanism likely operates in acinar cells. The effects of the cAMP analogue were reproduced by other agents that increase cAMP. Furthermore, econazole, an inhibitor of calcium influx, potentiated secretory responses to calcium-dependent stimulation in T84 cells without itself inducing secretion. We conclude that there is cross-talk between calcium and cAMP-dependent signaling pathways at the level of second messenger generation in two secretory cell types. This cross-talk appears to regulate the extent of secretory responses.

Protein kinase C activity does not mediate the inhibitory effect of carbachol on chloride secretion by T84 cells.

Carbachol induces calcium-dependent chloride secretion and activates protein kinase C in T84 cells. However, prolonged stimulation with carbachol or direct activation of protein kinase C inhibits subsequent calcium-dependent chloride secretion. Furthermore, the ability of carbachol to elevate inositol tetrakisphosphate levels may be linked to inhibition of chloride secretion. Here we demonstrate that protein kinase C activation increases levels of inositol tetrakisphosphates (1,3,4,6- and 3,4,5,6-isomers) in T84 colonic epithelia. Furthermore, this corresponds to an inhibition of chloride secretion. However, protein kinase C is unlikely to mediate the analogous effects of carbachol. Neither the ability of carbachol to inhibit calcium-dependent chloride secretion nor its effects on inositol 3,4,5,6-tetrakisphosphate levels were reversed by staurosporine. Carbachol also has quantitatively and qualitatively different effects on inositol tetrakisphosphate isomers than protein kinase C activators. Thus protein kinase C activity can increase levels of various inositol tetrakisphosphate isomers within T84 cells but does not mediate carbachol-induced increases in these putative messengers. These data further support the hypothesis that inositol 3,4,5,6-tetrakisphosphate is a negative second messenger, uncoupling epithelial chloride secretion from changes in intracellular calcium.

Membrane-permeant derivatives of cyclic AMP optimized for high potency, prolonged activity, or rapid reversibility.

A novel membrane-permeant derivative of cAMP, cAMP acetoxymethyl ester (cAMP/AM), was synthesized via silylated intermediates. Its ability to induce Cl- secretion by T84 cells, a human colon cancer cell line, was compared with that of two other membrane-permeant cAMP derivatives that were recently introduced, N6,O2'-dibutyryl-cAMP acetoxymethyl ester (bt2cAMP/AM) and Sp-5,6-dichlorobenzimidazole-1-beta-D-ribofuranoside 3',5'-cyclic phosphorothioate (Sp-5,6-DCl-cBIMPS). All of these compounds are powerful activators of Cl- secretion when applied extracellularly, with EC50 values of 60 microM, 0.7 microM, and 3 microM, respectively. However, cAMP/AM was expected to be readily degraded inside cells, in contrast to the cyclophosphodiesterase-resistant Sp-5,6-DCI-cBIMPS or the only slowly metabolizable N6-butyryl-cAMP derived from bt2cAMP/AM. Reversibility of cAMP/AM action was demonstrated by wash-out experiments; Cl- secretion induced by high doses of cAMP/AM (100 microM) could be quickly abolished by rinsing of the cells, whereas similar experiments with bt2cAMP/AM and Sp-5,6-DCI-cBIMPS showed much slower decreases. Even more sensitive to residual cAMP derivatives was the synergistic effect of carbachol, which was applied after the incubation with membrane-permeant derivatives and their subsequent wash-out. Although doses of cAMP derivatives that barely activated Cl- secretion were readily capable of inducing a synergistic response with carbachol, cells incubated with high doses of cAMP/AM (100 microM) and subsequently washed showed only a nonsynergistic carbachol response, in contrast to cells incubated with bt2cAMP/AM or Sp-5,6-DCI-cBIMPS. We therefore characterize cAMP/AM as a membrane-permeant derivative of cAMP that is easily metabolizable inside cells and hence is most useful for applications where a transient intracellular cAMP signal is desired. In contrast, completely nonmetabolizable Sp-5,6-DCI-cBIMPS seems to be more useful in longer incubations that require steady levels of cAMP-dependent protein kinase activation. bt2cAMP/AM combines the advantages of intracellular trapping by ester hydrolysis and reduced cyclophosphodiesterase sensitivity of its active intracellular product, which probably lead to its particularly high potency.

Long-term uncoupling of chloride secretion from intracellular calcium levels by Ins(3,4,5,6)P4.

Osmoregulation, inhibitory neurotransmission and pH balance depend on chloride ion (Cl-) flux. In intestinal epithelial cells, apical Cl- channels control salt and fluid secretion and are, in turn, regulated by agonists acting through cyclic nucleotides and internal calcium ion concentration ([Ca2+]i). Recently, we found that muscarinic pretreatment prevents [Ca2+]i increases from eliciting Cl- secretion in T84 colonic epithelial cells. By studying concomitant inositol phosphate metabolism, we have now identified D-myo-inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4), as the inositol phosphate most likely to mediate this uncoupling. A novel, membrane-permeant ester prepared by total synthesis delivers Ins(3,4,5,6)P4 intracellularly and confirms that this emerging messenger does inhibit Cl- flux resulting from thapsigargin- or histamine-induced [Ca2+]i elevations.

Activation by calcium alone of chloride secretion in T84 epithelial cells.

1. The goal of this study was to determine if an increase in cytoplasmic calcium concentration ([Ca2+]i), in the absence of additional second messengers derived from membrane phospholipid turnover, is a sufficient signal to induce chloride secretion across monolayers of the human colonic epithelial line, T84. 2. Thapsigargin was used to increase [Ca2+]i by inhibiting the endomembrane Ca(2+)-ATPase. [Ca2+]i was monitored in monolayers by fura-2 fluorescence spectroscopy, chloride secretion by measuring changes in short circuit current (Isc) in modified Ussing chambers, and inositol phosphates were measured by radio-h.p.l.c. of extracts of cells prelabelled with [3H]-inositol. 3. Thapsigargin increased [Ca2+]i and Isc in parallel, without increasing any inositol phosphates. The effect of thapsigargin on Isc was abolished by the intracellular calcium chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N"-tetraacetic acid (BAPTA). 4. Increasing [Ca2+]i with thapsigargin did not prevent a subsequent calcium response to carbachol or histamine if extracellular calcium was available. In the absence of extracellular calcium, only one such release of calcium to hormonal stimulation occurred when cells were pretreated with thapsigargin, and a second response to either carbachol histamine was essentially abolished. 5. Addition of carbachol or histamine to thapsigargin-treated cells mounted in Ussing chambers caused a transient further increase in Isc followed by termination of the response, even though [Ca2+]i continued to rise. 6. We conclude that an elevation in [Ca2+]i is a sufficient signal to induce chloride secretion in T84 cells. Rather than being required to stimulate secretory responses, additional second messengers induced by hormonal secretagogues (such as inositol phosphates) may in fact serve to limit the secretory response.

Phosphatidic acid modulates Cl- secretion in T84 cells: varying effects depending on mode of stimulation.

Cl- secretion in T84 cells evoked by a stimulus that activates protein kinase C, carbachol, was associated with elevated levels of 32P-labeled phosphatidic acid (PA). PA's role in the regulation of Cl- secretion was explored by examining the effect of exogenous PA (10(-4) M) on Cl- secretion and intracellular Ca2+ levels ([Ca2+]i) in monolayers. PA potentiated the effect of carbachol on [Ca2+]i and Cl- secretion, although it did not stimulate Cl- secretion by itself. PA had divergent effects on cyclic nucleotide-dependent Cl- secretion. It delayed Cl- secretion induced by vasoactive intestinal polypeptide [VIP, adenosine 3',5'-cyclic monophosphate (cAMP) dependent] but potentiated that induced by the heat-stable enterotoxin of Escherichia coli (STa; guanosine 3',5'-cyclic monophosphate dependent). PA did not alter AMP or GMP levels, suggesting that PA acts at a site distal to the generation of these second messengers. PA caused a slight increase in phosphorylation of protein kinase C substrates but not of cAMP-dependent protein kinase substrates. However, PA is probably not acting through a classical protein kinase C pathway, because we have previously shown that phorbol esters inhibit carbachol's actions, and the protein kinase C inhibitor staurosporine failed to block the effect of PA on VIP- or STa-stimulated Cl- secretion. Thus PA differentially regulates stimulated Cl- secretion in T84 cells, depending on the nature of the agonist.

Acetoxymethyl esters of phosphates, enhancement of the permeability and potency of cAMP.

Acetoxymethyl esters of alkyl or aryl phosphates can be prepared by reacting their trialkylammonium or silver salts with acetoxymethyl bromide. Because acetoxymethyl esters are rapidly cleaved intracellularly, they facilitate the delivery of organophosphates into the cytoplasm without puncturing or disruption of the plasma membrane. In addition, acylation of free hydroxyls, for example with butyryl groups, is useful both for synthetic convenience and increased hydrophobicity of the permeant derivatives. The highly polar intracellular messengers cAMP and cGMP were thus converted into uncharged membrane-permeant derivatives. Extracellularly applied N6,2'-O-dibutyryl cAMP acetoxymethyl ester (Bt2cAMP/AM) is shown to simulate intracellular cAMP in three model systems, namely dissociation of cAMP-dependent protein kinase in fibroblasts, activation of Cl- secretion of monolayers of the human colon epithelial cell line T84, and dispersion of pigment granules in angel fish melanophores. Bt2cAMP/AM is effective at concentrations two or three orders of magnitude less than those required for commonly used membrane-permeant cAMP derivatives such as Bt2cAMP, 8-Br-cAMP, and 8-pCPT-cAMP lacking the acetoxymethyl ester. This methodology should be of general utility for the intracellular delivery of phosphate-containing second messengers.

Elevation of inositol tetrakisphosphate parallels inhibition of Ca(2+)-dependent Cl- secretion in T84 cells.

Carbachol and histamine both stimulate calcium-dependent chloride secretion in the colonic epithelial cell line, T84. However, pretreatment of cell monolayers with carbachol blocks subsequent chloride secretion induced by thapsigargin but not the calcium elevation stimulated by this agent, whereas histamine pretreatment blocks neither thapsigargin-induced chloride secretion nor calcium elevation. To examine whether inositol phosphate metabolism might account for this difference, we measured levels of radiolabeled inositol phosphates: Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins-(1,3,4,6)P4, Ins(3,4,5,6)P4, InsP5, and InsP6 after cell stimulation. Although both carbachol and histamine increase Ins (1,4,5)P3 at 5 s, there is a greater and more persistent increase in the levels of Ins(1,3,4)P3 and InsP4 at later time points after carbachol than histamine, which corresponded to the suppression of the chloride secretory response.

Dual effects of a phorbol ester on calcium-dependent chloride secretion by T84 epithelial cells.

Ca(2+)-dependent secretagogues (e.g., carbachol, histamine, ionomycin, and 4-bromo-A23187) have relatively transient effects on chloride secretion, even if there is a sustained increase in cytosolic calcium ([Ca2+]i) (as for the ionophores). Because these agents increase both [Ca2+]i and protein kinase C (PKC) activity, chloride secretion might be stimulated by [Ca2+]i and terminated by PKC activity. We tested the effect of a PKC activator, phorbol 12-myristate 13-acetate (PMA), on Cl- secretion by T84 cell monolayers by measuring short-circuit current (Isc). PMA alone had no effect on Isc but potentiated increases in Isc when added 10 min or less before Ca(2+)-dependent secretagogues. Chelation of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid inhibited the increases both in [Ca2+]i and Isc induced by carbachol with or without brief PMA pretreatment. Longer preincubations with PMA inhibited Isc responses to Ca(2+)-dependent secretagogues, even when increased [Ca2+]i was sustained by ionophores. Inhibitors of PKC could reverse the inhibitory effect of PMA but did not reverse the potentiating effect. The effects of PMA on Cl- secretion were reproduced by 1,2-dioctanoyl-sn-glycerol and were mirrored by effects on K+ channel opening. Thus PMA has dual effects on chloride secretion. Initially, it exerts a stimulatory action and subsequently an inhibitory action. The stimulatory effect only occurs if Ca(2+)-dependent secretion is ongoing. The inhibitory effect of PMA is mediated by PKC and cannot be overcome by increasing [Ca2+]i.