Stimulation of leucine transport by a mitogen through intracellular Ca2+ increase in human peripheral lymphocytes

The effect of concanavalin A and ionophore A23187 on leucine uptake by human peripheral lymphocytes has been examined. Preincubation of the cells with 32 micrograms/ml concanavalin A or 0.1 microM A23187 increased leucine uptake by 67% and 100%, respectively. Both concanavalin A and A23187 could, within 2 min, induce a more than 2-fold increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i). This increase by concanavalin A was completely blocked by the addition of 0.1 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) to incubation medium; TMB-8 partially blocked the action of A23187. The stimulation of leucine uptake by concanavalin A and A23187 was strongly inhibited by the presence of TMB-8 in the medium, whereas the basal uptake was not affected by this intracellular Ca2+ antagonist. Amiloride did not inhibit the stimulation of leucine uptake by concanavalin A. The concanavalin A- and A23187-induced elevation of [Ca2+]i was accompanied by membrane hyperpolarization. Concanavalin A-stimulated leucine uptake was greatly inhibited by the presence of an excess of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid. These results indicate that the increase in [Ca2+]i may function as a signal of the stimulation by mitogen of leucine uptake mediated by system L, finally inducing membrane hyperpolarization in human lymphocyte.     

Na+/K+/Cl- cotransport is stimulated by a Ca(++)-calmodulin-mediated pathway in BALB/c 3T3 fibroblasts.

In the present study, we investigated the role of intracellular Ca++ in the stimulation of the Na+/K+/Cl- cotransport in synchronized BALB/c 3T3 cells. The Na+/K+/Cl- cotransport was stimulated by the growth factors EGF, TGF-alpha, IGF-1, and IGF-2, which do not activate protein kinase C, but do induce a transient increase in free cytoplasmic Ca++. In addition, direct activation of protein kinase C by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) did not affect the Na+/K+/Cl- cotransport activity of quiescent cells. The Na+/K+/Cl- cotransport was also stimulated by the above mitogens in cells pretreated with the phorbol ester TPA. This treatment led to a progressive decline in the activity of cellular protein kinase C. This result implies that cells deficient in protein kinase C may still support stimulation of the Na+/K+/Cl- cotransport. Taken as a whole, these findings suggest that the Na+/K+/Cl- cotransport is stimulated predominantly by a protein kinase C-independent mechanism in BALB/c 3T3 fibroblasts. Both the intracellular Ca++ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and two potent calmodulin antagonists, trifluoperazine (TFP) and chloropromazine (CP), blocked serum- and mitogen-stimulated Na+/K+/Cl- cotransport. These results suggest that the Na+/K+/Cl- cotransport is stimulated by an increase of intracellular Ca++ and subsequently by a Ca(++)-calmodulin-mediated pathway in the synchronized BALB/c 3T3 fibroblasts.     

The role of cAMP and calcium in the stimulation of proliferation of immature erythroblasts by erythropoietin

The hypothesis that cAMP or calcium are the second messengers of erythropoietin (Epo) was tested on fractionated, Epo-responsive immature erythroblasts from anemic rabbit bone marrow by examining whether the proliferative effects of the hormone could be mimicked by agents that increase the intracellular concentration of cAMP or Ca2+. None of the compounds tested (including 10(-6)-10(-4) M db-cAMP, forskolin, isoprenaline or 10(-7)-10(-6) M of the calcium ionophore A23187) alone or in combination could either initiate or potentiate the mitogenic action of the hormone. Furthermore, addition of 0.2 U/ml erythropoietin produced no permanent or transient increase in the uptake of 45Ca2+ by erythroblasts at 37 degrees C. However, cells cultured with imidazole or cordycepin (which reduce the level of intracellular cAMP), or with the calcium chelator EGTA, or the drugs verapamil or TMB-8 (which interfere with the utilization of extracellular or intracellular calcium) showed a decreased stimulation of DNA synthesis by Epo. Finally, the tumour promoter phorbol ester TPA could partially mimic the action of Epo when added to cultures containing more immature progenitor cells. We conclude then that an artificial increase in the cytoplasmic concentration of either cAMP or Ca2+ is not sufficient to elicit the proliferation of Epo-responsive cells.     

Permissive role of calcium in the inhibition of T cell mitogenesis by calmodulin antagonists

The importance of Ca++ in the initiation of lymphocyte activation and mitogenesis has been supported by several studies. Because calmodulin functions as the intracellular mediator of the effects of Ca++, it likely plays a major role in the regulation of lymphocyte function. We have examined the effects of known calmodulin antagonists, the phenothiazines, on lectin-induced T cell mitogenesis and have shown a central role for Ca++ uptake in the expression of a phenothiazine-sensitive stage after lectin activation. The drug effects were observed only if the cells were previously activated by PHA or the ionophore A23187, and only in the presence of Ca++. These effects were restricted to a defined time period (5 hr) after lectin activation. The data support the concept that calmodulin is the target for the phenothiazine effects and demonstrate the permissive role of Ca++ in the mediation of these events.     

Stimulatory and inhibitory effects of TMB-8 on pancreatic enzyme secretion

The putative intracellular calcium antagonist 3,4,5-trimethoxybenzoate 8-(diethylamino)-octyl ester (TMB-8) affects carbachol-induced enzyme secretion from rabbit pancreatic acini in a different way than it does that induced by either the C-terminal octapeptide of cholecystokinin (CCK-8), the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) or the calcium ionophore, A23187. In the presence of TMB-8 the dose-response curve for carbachol-induced amylase release shifts to the right, suggesting competitive antagonism at the muscarinic receptor. The hypothesis that TMB-8 acts as a muscarinic receptor antagonist is supported by the observation that TMB-8 dose-dependently inhibits the carbachol-, but not CCK-8-induced increases in cytosolic free calcium, measured in acinar cells by means of the fluorescent calcium indicator quin2. At a concentration of 100 microM, TMB-8 maximally potentiates the secretory response to suboptimal, but not (supra)optimal, concentrations of CCK-8. At the same concentration the drug also potentiates TPA- and A23187-induced enzyme secretion. Cytosolic free calcium levels and CCK-8-induced increases in cytosolic free calcium remain unaffected by 100 microM TMB-8. The above results strongly suggest that potentiation occurs at or beyond the site of interaction between the diacylglycerol- and the Ca2+-activated pathways. At concentrations beyond 100 microM the potentiating effect of TMB-8 declines and, finally, at a concentration of 500 microM the drug completely abolishes the secretory response to CCK-8 and TPA. Basal enzyme secretion, however, remains unaffected. At 500 microM severe side effects are observed as is shown by Trypan blue uptake, lactic dehydrogenase release and release of trapped quin2. It is concluded that at lower concentrations TMB-8 does not act as a specific intracellular calcium antagonist in pancreatic enzyme secretion and that inhibitory effects obtained with rather high concentrations of this drug should be treated with caution.     

c-fos and c-myc gene activation, ionic signals, and DNA synthesis in thymocytes

Among the earliest responses to mitogens that have been detected in normal quiescent cells are ionic changes: we have described rapid increases in the cytosolic free Ca2+ concentration ([Ca]i) and in the intracellular pH (pHi) in mitogen-stimulated thymocytes and fibroblasts (Hesketh, T. R., Moore, J. P., Morris, J. D. H., Taylor, M. V., Rogers, J., Smith, G. A., and Metcalfe, J. C. (1985) Nature 313, 482-484). Here we investigate the relationship between these ionic signals and the subsequent expression of the c-fos and c-myc proto-oncogenes in murine thymocytes. We show that the plant lectin concanavalin A (ConA), the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the Ca2+-ionophore A23187 each causes a rapid increase in both c-fos and c-myc mRNAs. The activation of both genes is completely dependent on the extracellular Ca2+ concentration ([Ca]o) for A23187 and independent of [Ca]o for TPA. Activation of c-myc, but not c-fos, by ConA is partially dependent on [Ca]o. The pHi increases generated by ConA or TPA are not necessary for expression of mRNA from either gene in response to these mitogens. Exogenous 8-bromo-cyclic AMP (but not 8-bromo-cyclic GMP) inhibits the c-myc responses to ConA and TPA. The data also show that neither early c-fos nor c-myc expression is sufficient to commit the cells to DNA synthesis.     

Ca++ regulation of paracellular permeability in the middle intestine of the eel, Anguilla anguilla

The role of Ca++ on the regulation of the paracellular pathway permeability of the middle intestine of Anguilla anguilla was studied by measuring the transepithelial resistance and the dilution potential, generated when one half of NaCl in the mucosal solution was substituted iso-osmotically with mannitol, in various experimental conditions altering extracellular and/or intracellular calcium levels. We found that removal of Ca++ in the presence of ethylene glycol-bis(beta-aminoethyl ether) (EGTA) from both the mucosal and the serosal side, but not from one side only, reduced both the transepithelial resistance and the magnitude of the dilution potential. The irreversibility of this effect suggests a destruction of the organization of the junction in the nominal absence of Ca++. However a modulatory role of extracellular Ca++ cannot be excluded. The decrease of the intracellular Ca++ activity, produced by using verapamil to block the Ca++ entry into the cell, or by adding 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (hydrochloride) (TMB-8), an inhibitor of Ca++ release from the intracellular stores, reduced both the transepithelial resistance and the magnitude of the dilution potential, indicating a role of cytosolic Ca++ in the modulation of the paracellular permeability. However the rise of calcium activity produced by the Ca++ ionophore calcimycin (A23187) evoked an identical effect, suggesting that any change in physiological intracellular Ca++ activity alters the paracellular permeability.     

Protein kinase C and calcium ion in mitogenic response of macrophage-depleted human peripheral lymphocytes

For mitogenic response of macrophage-depleted human peripheral lymphocytes, 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG) and Ca2+ ionophore are both needed in addition to a small quantity of plant lectin, phytohemagglutinin (PHA). PHA alone is not sufficient to produce the cellular response. The addition of TPA or OAG to these cells induces the activation of protein kinase C as assayed by the phosphorylation of its endogenous substrates. Apparently, TPA or OAG and A23187 together substitute for macrophages and act synergistically to potentiate the DNA synthesis of this lymphocyte preparation. The results suggest that protein kinase C activation and Ca2+ mobilization are essential and that additional receptor occupation by PHA is necessary for producing cell proliferation.     

Calcium dependence of the stimulatory action of hypertonicity on renal medullary prostaglandin synthesis

Previous studies have shown that hypertonic mannitol or NaCl increases the release of [3H]arachidonate and immunoreactive prostaglandin E in inner medullary slices incubated in Ca2+-free media containing EGTA. By contrast, the stimulation of these parameters by ionophore A23187 and by arginine-vasopressin are abolished in Ca2+-free media plus EGTA. In the present study, the effects of Ca2+ deprivation and the intracellular Ca2+ antagonist TMB-8 [8-N,N-diethylamino)octyl-3,4,5 -trimethoxybenzoate-HCl) were further examined to assess the Ca2+ dependence of the actions of different stimuli of prostaglandin E synthesis in rat renal inner medulla. Ca2+-free media without EGTA abolished increases in [3H]arachidonate and immunoreactive prostaglandin E release induced by ionophore A23187, but not those induced by arginine-vasopressin, suggesting that different pools of Ca2+ subserve expression of the actions of these two stimuli. At low concentrations, TMB-8 (10-25 microM) inhibited increases in [3H]arachidonate and immunoreactive prostaglandin E release induced by arginine-vasopressin, but did not influence effects of Ca2+ plus ionophore A23187 or hypertonicity on these parameters. At higher concentrations (100-500 microM), TMB-8 suppressed effects of ionophore A23187, hyperosmolar NaCl and mannitol on immunoreactive prostaglandin E and [3H]arachidonate release from slices. The effects of a sub-optimal inhibitory concentration of TMB-8 on ionophore A23187 actions were overcome by increasing Ca2+ in the media from 1.5 to 5 mM. Ca2+ deprivation, or concentrations of EGTA or TMB-8, that were effective in suppressing increases in immunoreactive prostaglandin E induced by ionophore A23187, arginine-vasopressin or hypertonicity, did not modify increases in immunoreactive prostaglandin E induced by exogenous arachidonate. Moreover, in microsomal fractions of inner medulla, TMB-8 suppressed Ca2+-dependent increases in phospholipase A2 and C activities, an effect which was competitive with Ca2+. Thus, Ca2+ deprivation and TMB-8 act at a step in the immunoreactive prostaglandin E synthetic pathway proximal to cyclooxygenase activity, and probably at the level of Ca2+-dependent acyl hydrolase activity. The results with TMB-8 indicate that an intracellular pool of Ca2+ is involved in expression of the actions of hypertonicity to increase [3H]arachidonate release and immunoreactive prostaglandin E in inner medulla.     

Calcium antagonists and calmodulin inhibitors block cytokinin-induced bud formation in Funaria

The plant hormone cytokinin stimulates nuclear migration followed by an asymmetric cell division in target cells of the protonema of the moss Funaria hygrometrica, leading to bud formation. The role of calcium in this developmental event was investigated by examining the effects of various calcium antagonists on the cytokinin-induced division. Calcium-free medium (buffered with EGTA), the extracellular Ca²⁺ antagonist La³⁺ (lanthanum), and the Ca²⁺ channel inhibitors D 600 and verapamil all block bud formation. These inhibitions are partially reversed by washing the cells or by raising the extracellular [Ca²⁺]. The Ca²⁺ ionophore A23187 partially reversed the effects of D 600 and verapamil. Bud formation is also inhibited by the intracellular Ca²⁺ antagonist TMB-8 (8-diethylamino)ocytl 3,4,5-trimethoxybenzoate HCl), and this inhibition is partially reversed by washing or raising the extracellular [Ca²⁺]. The cross walls of both the filaments and bud initial cells formed during TMB-8 exposure exhibit a distorted morphology. High concentrations of TMB-8 block nuclear migration. The calmodulin inhibitor trifluoperazine stops cytokinin-induced budding more effectively than the related compound chlorpromazine. Low concentrations of these two compounds do not affect nuclear migration; however, the target cell does not enter mitosis. These results support the hypothesis that a rise in intracellular calcium mediates cytokinin-induced bud formation in Funaria. It is concluded that the proposed cytokinin-induced rise in intracellular calcium may be effected in part by the activation of calmodulin. The essential source of Ca²⁺ appears to be extracellular, because blocking Ca²⁺ uptake with Ca²⁺ transport inhibitors can block both nuclear migration and subsequent division.     

Mechanism of activation of lymphocyte Na+/H+ exchange by concanavalin A. A calcium- and protein kinase C-independent pathway

Treatment of thymic lymphocytes with the mitogenic lectin concanavalin A (ConA) increases the intracellular free Ca2+ concentration and stimulates phosphoinositide turnover. ConA also induced a rapid, amiloride-sensitive, Na+-dependent increase in cytosolic pH of 0.13 +/- 0.01, indicative of stimulation of the Na+/H+ antiport. To investigate the mechanism underlying activation of Na+/H+ exchange by ConA, the intracellular free Ca2+ concentration changes induced by this lectin were precluded by loading the cells with Ca2+-buffering agents and suspension in Ca2+-free media. Under these conditions, the ConA-induced cytoplasmic alkalinization proceeded normally. Two approaches were used to assess the role of protein kinase C. First, this enzyme was inhibited by the addition of 1-(5-isoquinolinysulfonyl)-2-methylpiperazine. In the presence of this potent antagonist, stimulation of the antiport by 12-O-tetradecanoylphorbol-13-acetate was greatly inhibited. In contrast, stimulation by ConA was unaffected. Second, protein kinase C was depleted by overnight incubation with phorbol esters. Following this treatment, Na+/H+ exchange was no longer activated by 12-O-tetradecanoyl-13-acetate, but was still stimulated by ConA. These data suggest that a Ca2+- and protein kinase C-independent mechanisms mediates the activation of Na+/H+ exchange by ConA. The possible role of GTP-binding proteins in the activation was also studied. The antiport was not stimulated by either fluoroaluminate or vanadate. Moreover, pretreatment with pertussis toxin failed to inhibit the ConA-induced cytoplasmic alkalinization. In contrast, preincubation with cholera toxin partially inhibited activation. Under these conditions, cholera toxin significantly elevated intracellular cAMP levels. Inhibition was also observed in cells treated with forskolin at concentrations that increased [cAMP]. The data suggest that a novel cAMP-sensitive signaling mechanism not involving Ca2+ and protein kinase C is involved in the stimulation of Na+/H+ exchange by mitogens in T lymphocytes.     

The release of slow-reacting substance of anaphylaxis from guinea pig lung: effects of calcium antagonists

The effects of three calcium antagonists, verapamil, lanthanum, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) were studied on the release of slow-reacting substance of anaphylaxis (SRS-A) from ovalbumin-sensitized chopped guinea pig lung parenchyma in calcium-containing and calcium-free media. The SRS-A levels (mean +/- SEM) obtained from tissues incubated in normal and calcium-free Krebs-bicarbonate buffer were 51 +/- 8 (N = 19) and 21 +/- 4 (N = 14) U/mL, respectively. TMB-8 (0.1-10 microM), a reported intracellular calcium antagonist, reduced antigen-stimulated SRS-A release from lung tissue incubated in calcium-containing, but not calcium-free, medium; A23187-induced SRS-A release from normal guinea pig lung was not significantly altered by TMB-8 at concentrations up to 10 microM. Verapamil and lanthanum consistently reduced SRS-A release only at high concentrations (100 microM and 1mM, respectively). The quantities of SRS-A released from lung tissue incubated in the presence of verapamil in normal medium were similar to those obtained in calcium-free medium. Tissues incubated in the presence of potassium chloride (60 and 100 mM) did not release significant quantities of SRS-A, and release which did occur was not blocked by verapamil, suggesting that antigen-induced SRS-A release is not dependent on membrane depolarization and that verapamil was not exerting inhibition via blockade of voltage-dependent calcium channels. These data suggest that although intracellular calcium is important for the regulation of SRS-A secretion from guinea pig lung tissue, extracellular calcium is necessary for optimal release of SRS-A.     

Differential calcium effects on prostaglandin D2 generation and histamine release from isolated rat peritoneal mast cells

We examined the role of Ca2+ mobilization in prostaglandin (PG) D2 generation and histamine release induced by A23187 from rat peritoneal mast cells. Both PGD2 generation and histamine release accompanied with 45Ca uptake were observed above 0.1 microM A23187. Although an increase of PGD2 generation was not exactly correlated with that of Ca2+ uptake, histamine release occurred in proportion to Ca2+ uptake. In contrast to PGD2 generation, below 0.1 microM A23187, about 20% of the total histamine was released without Ca2+ uptake and this response was inhibited by 10 microM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which is an intracellular Ca2+ antagonist. However, TMB-8 had no effect on PGD2 generation. These results suggest that Ca2+ dependency of histamine release is clearly different from that of PGD2 generation, and that histamine release is induced by not only Ca2+ uptake but also intracellular Ca2+ mobilization.     

Effects of TMB-8, an intracellular calcium antagonist, and W-7, a calmodulin antagonist, on prostaglandin output from the guinea-pig uterus

TMB-8, an intracellular Ca2+ antagonist, inhibited the A23187-induced increase in outputs of prostaglandin (PG) F-2 alpha and 6-keto-PGF-1 alpha from the guinea-pig uterus superfused in vitro. The high basal output of PGF-2 alpha from the Day-15 guinea-pig uterus was not inhibited by TMB-8, indicating that a maintained high intracellular free Ca2+ concentration is not necessary for maintaining this high output of PGF-2 alpha. W-7, a calmodulin antagonist, had similar actions except that PGF-2 alpha output from the Day-15 uterus was reduced 20-30 min after the W-7 treatment had stopped. Overall, these findings suggest that, in the guinea-pig, oestradiol acting on a progesterone-primed uterus causes a prolonged stimulation of endometrial phospholipase A-2 in the absence of a maintained high Ca2+ concentration, thus providing a continuous release of arachidonic acid for increased endometrial PGF-2 alpha synthesis during the last third of the oestrous cycle.     

Effects of TMB-8, a calcium antagonist, on transport properties of the isolated canine tracheal epithelium

The effects of the putative intracellular Ca2+ antagonist, TMB-8 (8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate), on the canine tracheal epithelium were examined. Luminal addition reduced rapidly, but reversibly, the transmucosal potential difference and increased the resistance across the open-circuited epithelium. Under short-circuit conditions, the drug reduced stimulation by prostaglandin E2, forskolin, 8-bromo cyclic AMP, prostaglandin F2 alpha and A23187. Inhibition of prostaglandin E2 responses were accompanied by reversal of net Cl- fluxes produced by the agonist. The effects of TMB-8 were unaffected by increasing Ca2+ in the bathing solutions, and were not mimicked by procaine, nitrendipine, calmidazolium, compound 48/80 or trifluoperazine. W7 did, to a limited extent, produce similar responses, though the drug was more toxic, and the effects were irreversible.     

Involvement of calcium and cyclic AMP in controlling ixodid tick salivary fluid secretion.

Catecholamine-stimulated salivary fluid secretion (in vitro) by ixodid ticks is reduced by deletion or lowering the concentration of exogenous bathing medium Ca++. The Ca++ antagonist, verapamil, reversibly inhibits dopamine-stimulated secretion. Ionophore A-23187 is unable to induce glands to secrete. Studies in which labeled and unlabeled Ca++ flux were measured indicate that catecholamines induce release of calcium from intracellular stores during secretion. Cyclic AMP/theophylline-stimulated secretion is inhibited by verapamil, and the exclusion of calcium from the support medium. It is concluded that the primary catecholamine stimulus induces cyclic AMP formation and mobilization of Ca++ (intra- and extracellular). Cyclic AMP and calcium are thought to interact to control secretion within the fluid transporting cells of types II and III alveoli.     

Stimulatory and inhibitory effects of 1,25-dihydroxyvitamin D3 on thymocyte mitogenesis induced by phorbol ester and calcium ionophore.

Mouse medullary thymocytes have specific receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The mitogenic stimulation of these cells by phytohemagglutinin in the presence or absence of the phorbol ester TPA is inhibited by 1,25(OH)2D3. The calcium ionophore A23187 did not reverse the inhibition by 1,25(OH)2D3 of phytohemagglutinin. Stimulation of thymocytes with either TPA or A23187 alone did not result in proliferation. Co-stimulation of the thymocytes with TPA and A23187 induces cell proliferation. 1,25(OH)2D3 markedly enhanced the TPA and A23187-induced cell proliferation even when added 4 h after the initiation of the culture. In contrast, DNA synthesis by thymocytes incubated for 4 h in the presence of TPA and A23187 and then cultured in medium containing 1,25(OH)2D3 but in the absence of both TPA and A23187, was inhibited by 1,25(OH)2D3. The extent of inhibition was comparable to the inhibition of lectin-induced stimulation by the hormone. Using monoclonal antibodies to neutralize IL-2 and block IL-2 receptors we showed that 1,25(OH)2D3 enhanced the IL-2-independent component of the A23187- and TPA-induced mitogenesis. In conclusion: (1) The nature and presence of the mitogenic signal determines whether 1,25(OH)2D3 enhances or inhibits thymocyte stimulation. (2) Both stimulatory and inhibitory actions of 1,25(OH)2D3 seem to take place at points distal to the initial increase in intracellular calcium or activation of protein kinase C.     

Mitogen-stimulated glucose transport in thymocytes. Possible role of Ca++ and antagonism by adenosine 3'':5''-monophosphate

The plant lectin, concanavalin A (Con-A), and the ionophore, A-23187 (specific for divalent cations), stimulated glucose transport in rat thymocytes. Con-A stimulation developed more slowly and was somewhat less extensive than that of stimulation developed more slowly and was somewhat less extensive than that of A-23187. Both responses showed saturation dose dependencies. The two responses were poorly additive, suggesting that A-23187 may saturate regulatory processes shared by the two stimulatory mechanisms. Doses of methylisobutylxanthine (MIX) and prostaglandin E2 which raised adenosine 3':5'-monophosphate (cAMP) levels in these cells also antagonized the Con-A stimulation of glucose transport but did not inhibit basal glucose transport or the A-23187 stimulation. Dibutyryl-cAMP and 8-bromo-cAMP also natagonized Con-A stimulation without inhibiting basal glucose transport. MIX antagonized high Con-A doses about as strongly as it did low Con-A doses, suggesting that MIX did not compete in the Con-A binding step or other process saturable by Con-A. [3H-A1Con-A binding was not affected by MIX. The stimulatory effects of Con-A and A-23187 were reduced by reduction of Ca++ in the medium. Both Con-A and A-23187 enhanced 45Ca++ influx and cellular Ca++ content. The A-23187 dose, which was saturating for glucose transport stimulation, enhanced Ca++ influx and cellular Ca++ content more than did the Con-A dose which was saturating for glucose transport stimulation. The dose fo MIX which specifically antagonized Con-A stimulation of glucose transport proved also to reduce Ca++ influx and cellular Ca++ in the presence of Con-A but not in the presence of A-23187. Thus, glucose transport correlates rather well with cellular Ca++. These results are compatible with the view that Ca++ in a cellular compartment can promote glucose transport, the Con-A's enhancement of Ca++ entry contributes to its stimulation of glucose transport, and the MIX antagonized Con-A action at least partly by reducing Ca++ entry. The action of MIX is apparently mediated by cAMP.     

Relationship between cytosolic Ca2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation.

Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 microM) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.     

Role of Calcium in Phytochrome-Controlled Nyctinastic Movements of Albizzia lophantha Leaflets

The involvement of Ca(2+) on phytochrome-controlled nyctinastic closure in Albizzia lophantha has been studied by testing the effect of the calcium ionophore 6S-[6alpha(2S(*),3S(*)),8beta(R(*)),9beta,11alpha]-5- methyl-amino)-2-[[3,9,11-trimethyl-8-[-1-methyl-2-oxo-2-(1H-pyrrol-2-yl) ethyl]-1,7-dioxaspiro[5.5]-undec-2yl] methyl]-4-benzoxazolecarboxylic acid (A23187) and the intracellular calcium antagonist 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8). An external supply of Ca(2+) or calcium ionophore A23187 to the Albizzia leaflets emulates the effect of red light irradiation and counteracts the inhibitory effect of far red light. The intracellular calcium antagonist TMB-8 supplied to Albizzia leaflets inhibits the effect of red light, but had no effect on far red irradiated plants. This suggests a dependence between phytochrome action and intracellular free Ca(2+). We suggest that calcium acts as a phytochrome messenger on control of ion fluxes that drive turgor changes in pulvinular motor cells.