Maximal Efflux Rate Research Articles

Physiologically-Based Pharmacokinetic Modeling Approach to Predict Rifampin-Mediated Intestinal P-Glycoprotein Induction.

Physiologically‐based pharmacokinetic (PBPK) modeling is a powerful tool to quantitatively describe drug disposition profiles in vivo, thereby providing an alternative to predict drug–drug interactions (DDIs) that have not been tested clinically. This study aimed to predict effects of rifampin‐mediated intestinal P‐glycoprotein (Pgp) induction on pharmacokinetics of Pgp substrates via PBPK modeling. First, we selected four Pgp substrates (digoxin, talinolol, quinidine, and dabigatran etexilate) to derive in vitro to in vivo scaling factors for intestinal Pgp kinetics. Assuming unbound Michaelis‐Menten constant (K m) to be intrinsic, we focused on the scaling factors for maximal efflux rate (Jmax) to adequately recover clinically observed results. Next, we predicted rifampin‐mediated fold increases in intestinal Pgp abundances to reasonably recover clinically observed DDI results. The modeling results suggested that threefold to fourfold increases in intestinal Pgp abundances could sufficiently reproduce the DDI results of these Pgp substrates with rifampin. Hence, the obtained fold increases can potentially be applicable to DDI prediction with other Pgp substrates.

Absorption of poorly water soluble drugs subject to apical efflux using phospholipids as solubilizers in the Caco-2 cell model

The purpose of this work was to determine the influence of liposomal solubilization of poorly water soluble drugs exhibiting apical efflux on permeation kinetics and cell toxicity in Caco-2 cells. The HIV-protease inhibitors indinavir and saquinavir were incorporated in phosphatidylcholine liposomes at maximal drug-to-lipid mass ratios and their absorption was determined in Caco-2 cell cultures grown on Transwell inserts using purely aqueous drug solutions as reference. A novel mathematical model was developed to quantitatively delineate the contribution of passive membrane permeation and carrier mediated efflux to transport across the cell monolayer and passive permeability coefficient and maximal efflux rate and affinity constant of the transporter system were determined. Cell toxicity of phospholipids was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) and the lactate dehydrogenase (LDH) assay. Cell integrity was not significantly affected by phospholipid concentrations of up to 150 mg/ml with respect to the used standard tests. Maximum drug concentration was increased 10- and 750-fold for indinavir and saquinavir, respectively, by the use of liposomes. The passive membrane permeability coefficient differed between the two drugs in accordance with their lipophilicity and the affinity for apical efflux transporters was on average 4-fold greater for saquinavir than for indinavir. Liposomal solubilization diminished the passive permeability coefficient of both drugs but the passive apical-to-basal delivery rate was increased by the liposomes compared to the purely aqueous solutions at maximal donor concentrations for at least one of the two drugs. Efflux rate reached a maximum for the liposomal formulations reflecting transporter saturation. Hence, liposomal solubilization considerably increased drug concentration in the media and altered absorption behavior by affecting both the passive diffusion and the carrier mediated efflux components of cell monolayer permeation.

Phosphate absorption and efflux of three ectomycorrhizal fungi as affected by external phosphate, cation and carbohydrate concentrations

A prerequisite for symbiotic phosphate transfer in an ectomycorrhizal (ECM) association is hypothesized to be conditions in the interface between both symbiotic partners, that either promote the release of inorganic phosphate (P) from the Hartig net into the interfacial apoplast and/or decrease the fungal reabsorption from this location. To get more information about conditions, which might be involved in the regulation of P efflux or P reabsorption, the effect of various external conditions on 33P-orthophosphate (33P) uptake or efflux by axenic cultures of the ECM basidiomycetes Hebeloma crustliniforme, Amanita muscaria and Laccaria laccata was analysed. In short-time experiments the following external conditions were analysed: an external supply of (1) P in the preculture, (2) cations (0.1-100 mM K, 0.1-50 mM Na, Mg and Ca), and (3) carbohydrates (0.5-50 mM glucose, fructose or sucrose). The P absorption was generally reduced in cultures previously supplied with an abundant P supply and with increased P concentrations in their tissues. The P uptake was also affected by an external supply of cations, whereas carbohydrates had only a slight effect. Compared to Na, Mg and Ca, the P absorption by H. crustuliniforme and L. laccata was increased by 0.1 mM K in the labelling solution but decreased after a supply of 100 mM K and then did not differ from the other cation treatments. Compared to other cations, an addition of 50 mM Ca led to a decrease of P absorption by A. muscaria, whereas 50 mM Mg increased the P uptake by H. crustuliniforme. The P efflux from the fungi was affected by both the cation and carbohydrate concentration of the bathing solution. High concentrations of the monovalent cations K and Na (5 mM or 50 mM) in the bathing solution increased the P efflux by H. crustuliniforme (only Na) and L. laccata (K and Na), but had little effects on A. muscaria. By contrast, the same concentrations of the divalent cation Mg reduced the P efflux from all fungal basidiomycetes. The P efflux from hyphae of H. crustuliniforme and A. muscaria was also affected by the carbohydrate concentration in the bathing solution. Compared to the hexoses, glucose and fructose, the P efflux by both basidiomycetes was stimulated even by low concentrations of sucrose (0.5 mM) in the bathing solution. The effect of carbohydrates on P efflux especially from hyphae of H. crustuliniforme was clearly concentration-dependent and the maximal efflux rates were observed after a supply of 2.5 mM sucrose or fructose or 5 mM glucose in the bathing solution. A further rise of the carbohydrate concentration led to a progressive decrease of P efflux from hyphae of H. crustuliniforme. The results are discussed with respect to the transfer processes between both symbiotic partners in a mycorrhizal association and a possible regulation of these exchange processes.

Differential effect of structural modification of human dopamine transporter on the inward and outward transport of dopamine

The effect of structural modification of the human dopamine transporter protein on bi-directional transport was explored using site-directed mutagenesis and rotating disk electrode voltammetry. The substrate-induced DA efflux, as inferred from the K m or K i, was dependent on common structural features for uptake of the substrate inducer: reduced by β-hydroxylation, stereoselective to α-methylation, and relatively insensitive to a switch of a single phenolic hydroxyl group between m- and p-positions. The potencies for substrates to compete with external DA for uptake and to induce DA efflux were similar and highly correlated. Despite these similarities, the efflux of internal DA was substantially slower than the uptake of its inducers. Mutation of serine-528 of the hDAT to alanine (S528A) did not change the structure-activity relationships, maximal uptake rates, and the cation dependence for the uptake of external substrates, although it modestly reduced K m or K i of most tested substrates. In contrast, it substantially enhanced substrate-induced DA efflux, with maximal efflux rates doubled for all tested inducers. Simultaneous monitoring of tyramine uptake and resulting DA efflux revealed that S528A accelerated the DA efflux relative to tyramine uptake. Saturation analysis suggested that the mutation significantly enhanced the efflux kinetics of internal DA but it exerted little effect on the uptake kinetics of external DA. These findings suggest that Ser-528 may play a role in stabilizing a hDAT conformation unfavorable for outward transport of internal DA, thereby contributing to the efficiency of the transporter.

Comparison of the kinetics of active efflux of 99mTc-MIBI in cells with P-glycoprotein-mediated and multidrug-resistance protein-associated multidrug-resistance phenotypes.

The overexpression of two membrane glycoproteins, P-glycoprotein and multidrug-resistance protein (MRP1) is a major cause of resistance to chemotherapeutic agents in the treatment of human cancers. Both proteins confer a similar multidrug-resistant (MDR) phenotype. 99mTc-MIBI, a myocardial imaging agent, which is also useful for the detection of a variety of tumours, has been shown to be a substrate for P-glycoprotein and MRP1. It thus may provide additional information about the P-glycoprotein and MRP1 status of tumour cells. In order to obtain information on the substrate specificity of these proteins, we have studied the transport kinetics of Tc-MIBI in two cell lines, K562/ADR and GLC4/ADR, which overexpress P-glycoprotein and MRP1, respectively. The mean active efflux coefficient ka, which is proportional to the ratio of maximal efflux rate VM to the apparent Michaelis-Menten constant Km, used to characterise the efficiency of the active efflux, was very similar being 1.9 +/- 0.6 x 10(-11) s(-1) x cells x ml and 1.3 +/- 0.5 x 10(-11) s(-1) x cells x ml for drug-resistant K562 and GLC4, respectively. These values are 50-100-times lower than for daunorubicin and other anthracycline derivatives, strongly suggesting that the efficiency of both transporters to pump Tc-MIBI is by far less than that to efflux anthracyclines. Our data show that (a) P-glycoprotein and MRP transporter efficiencies to wash out Tc-MIBI are similar, in spite of a different suspected mechanism of its transport and (b) that both transporters are less efficient to pump Tc-MIBI than to pump anthracyclines (the ka parameter is about 100-times lower for TC-MIBI than for anthracycline).

Kinetics of anthracycline efflux from multidrug resistance protein-expressing cancer cells compared with P-glycoprotein-expressing cancer cells.

The multidrug resistance protein (MRP) has been shown to mediate ATP-dependent efflux of anticancer agents of diverse structure, such as daunorubicin (DNR), vincristine and etoposide. Thus, this protein does confer a multidrug resistant phenotype to cancer cells, similar to P-glycoprotein (Pgp). The substrate specificity of both transporter proteins is partly overlapping but is otherwise very distinct; because MRP is a multiple organic anion transporter, it transports certain glutathione conjugates and may be partly dependent on intracellular glutathione levels for the transport of anthracyclines. We have studied the transport kinetics of a series of anthracyclines in MRP and Pgp that overexpress tumor cell lines to obtain information on the substrate specificity of these proteins. The anthracyclines have modifications in the sugar moiety. The mean active efflux coefficient Ka, used to characterize the efficiency of the active efflux, was very similar for DNR and one of its 4'-deoxy-derivatives (eso-DNR) for MRP and Pgp [10-20 x 10(-10)/sec/(cells/ml)]. The permanently neutral derivatives 3'-deamino-3'-hydroxy-doxorubicin (OH-DOX) and 3'-deamino-3'-hydroxy-daunorubicin (OH-DNR) were effluxed by both proteins but had a lower Ka [2 x 10(-10) and 6 x 10(-10)/sec/(cells/ml) (OH-DOX)] and 2 x 10(-10) and 5 x 10(-10)/sec/(cells/ml) (OH-DNR)] for MRP and Pgp. Two anthracyclines, the doxorubicin derivative pirarubicin and 2'-bromo-4'-epi-DNR seemed to have a slightly higher Ka value for Pgp than for MRP. The apparent Michaelis-Menten constants (K(m)) and maximal efflux rates (VM) for the active transport were within a narrow range for both transporters, except for OH-DOX and OH-DNR, which had a lower VM in the case of MRP-mediated transport, suggesting a role of the amino group in the interaction with glutathione. Determination of the Hill coefficient (nH) of the MRP-mediated efflux gave most values close to 2, which suggests cooperativity of the transport of anthracyclines as reported before for Pgp. In conclusion, the transport kinetics of anthracyclines by MRP and Pgp are very similar.

Erythrocyte Na+ -Li+ countertransport in essential hypertension

To examine whether Na(+)-Li+ countertransport (SLC) activity is linked to erythrocyte membrane lipid content. An observational case-control study. The maximal efflux rate of SLC, plasma cholesterol, triglycerides, phospholipids, low- and high-density lipoprotein cholesterol levels and the erythrocyte membrane cholesterol, phospholipids and fatty acids contents were determined both in fasting normolipaemic normotensive subjects and in hypertensive patients. The Li(+)-stimulated Na+ efflux was measured in Li(+)-preloaded erythrocytes. Membrane cholesterol and phospholipids levels were determined by the latroscan technique. Membrane fatty acids were identificated by gas chromatography. Several derived indices were also obtained. Erythrocyte membranes of hypertensive patients showed an increase in cholesterol: phospholipid ratio and a decrease in the total amount of polyunsaturated fatty acids, mainly at the expense of arachidonic acid and docosatetraenoic acid. SLC activity was higher in hypertensive patients and correlated positively with the plasma triglycerides level and negatively with the ratio of C20:4 to C20:3. Our data from untreated normolipaemic hypertensive patients show that a higher SLC activity was accompanied by parameters that indicate a lower membrane fluidity.

Cellular Cholesterol Efflux Mediated by Cyclodextrins: DEMONSTRATION OF KINETIC POOLS AND MECHANISM OF EFFLUX

The efflux of cholesterol from cells in culture to cyclodextrin acceptors has been reported to be substantially more rapid than efflux induced by other known acceptors of cholesterol (Kilsdonk, E. P. C., Yancey, P., Stoudt, G., Bangerter, F. W., Johnson, W. J., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 17250-17256). In this study, we compared the kinetics of cholesterol efflux from cells with 2-hydroxypropyl-beta-cyclodextrins and with discoidal high density lipoprotein (HDL) particles to probe the mechanisms governing the remarkably rapid rates of cyclodextrin-mediated efflux. The rate of cholesterol efflux was enhanced by shaking cells growing in a monolayer and further enhanced by placing cells in suspension to achieve maximal efflux rates. The extent of efflux was dependent on cyclodextrin concentration, and maximal efflux was observed at concentrations >50 mM. For several cell types, biexponential kinetics of cellular cholesterol efflux were observed, indicating the existence of two kinetic pools of cholesterol: a fast pool (half-time (t1/2) approximately 19-23 s) and a slow pool with t1/2 of 15-30 min. Two distinct kinetic pools of cholesterol were also observed with model membranes (large unilamellar cholesterol-containing vesicles), implying that the cellular pools are in the plasma membrane. Cellular cholesterol content was altered by incubating cells with solutions of cyclodextrins complexed with increasing levels of cholesterol. The number of kinetic pools was unaffected by raising the cellular cholesterol content, but the size of the fast pool increased. After depleting cells of the fast pool of cholesterol, this pool was completely restored after a 40-min recovery period. The temperature dependence of cyclodextrin-mediated cholesterol efflux from cells and model membranes was compared; the activation energies were 7 kcal/mol and 2 kcal/mol, respectively. The equivalent activation energy observed with apo-HDL-phospholipid acceptor particles was 20 kcal/mol. It seems that cyclodextrin molecules are substantially more efficient than phospholipid acceptors, because cholesterol molecules desorbing from a membrane surface can diffuse directly into the hydrophobic core of a cyclodextrin molecule without having to desorb completely into the aqueous phase before being sequestered by the acceptor.

Inhibition of Na +/H + exchanger activity by an alkyl-lysophospholipid analogue in a human breast cancer cell line

The mechanisms by which ET-18-OCH3 (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine) and other analogues of alkyl-lysophospholipids exert their antineoplastic effects are not yet fully elucidated. Possible interference with mechanisms involving intracellular pH (pH,) regulation was examined by measuring the effect of ET-18-OCH3 on the activity of the Na +/H + exchanger in the breast cancer-derived cell line MCF-7. When ET-18-OCH3 was added to culture medium at 10 μM (determined as a noncytotoxic but cytostatic concentration), it led to an intracellular acidification (0.15 pH unit). It also decreased the rate of pH i recovery by Na +/H + exchange following artificial acidification. Kinetic parameters of the exchange indicated that this was due to a decrease in the affinity of the exchanger for both transported ions, rather than to a decrease in the number of exchanger proteins in the membrane (same maximal efflux rate for treated and untreated cells). These results suggest that Na +/H + exchanger inhibition and subsequent cytoplasmic acidification participate in the mode of action of ET-18-OCH3, and could be used for modulation of tumor-cell chemosensitivity or their subsequent commitment into programmed cell death.

Lack of elevated drug efflux in adriamycin-resistant immunoblastic B lymphoma cells with mdr1 overexpression

A multidrug-resistant (MDR) subline of the immunoblastic B lymphoma cell line was established by sequentially selecting in increasing concentrations of adriamycin. The adriamycin-resistant cell line (HOB1/ADR) demonstrated resistance to a wide spectrum of chemotherapeutic agents including MDR drugs (Vinca alkaloids and anthracycline), antimicrotubule drug (colchicine), and DNA-damaging agents (cisplatin and mitomycin C). The expression of human mdr1 gene, as analyzed by RT-PCR and Western blotting, revealed a 13–15-fold increase in resistant cells. Unexpectedly, HOB1/ADR cells demonstrated a lack of reduced accumulation and of enhanced efflux of adriamycin. More than 60% adriamycin was effluxed at the same rate in both cell lines within 10 min. In contrast, the initial rate of vincristine accumulation was reduced by 3 fold in this resistant cell line. The maxima level of vincristine accumulation was 50% lower in the resistant cells than the parental cells. The maximal efflux rate was enhanced by 5 fold in the resistant cells. Inhibition of vincristine resistance by verapamil associated with restoration of drug accumulation, suggesting that acquired resistance in these cells is due to P-glycoprotein. These studies demonstrated that immunoblastic B lymphoma cells selected for adriamycin resistance preferentially developed P-glycoprotein-mediated vincristine efflux which plays an important role in vincristine resistance. In contrast, the resistant cells did not elevate adriamycin efflux, suggesting an additional mechanism responsible for adriamycin resistance.

Cellular Cholesterol Efflux Mediated by Cyclodextrins

In this study, we compared cholesterol efflux mediated by either high density lipoproteins (HDL3) or beta-cyclodextrins, cyclic oligosaccharides that are able to dissolve lipids in their hydrophobic core. beta-Cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, and methyl-beta-cyclodextrin at 10 mM induced the release of 50-90% of L-cell [3H]cholesterol after 8 h of incubation, with a major portion of this cholesterol being released in the first 1-2 h of incubation. The cholesterol efflux kinetics are different if cells are incubated with HDL3, which induces a relatively constant rate of release of cholesterol throughout an 8-h incubation. Cholesterol efflux to cyclodextrins was much greater than phospholipid release. To test the hypothesis that maximal efflux rate constants for a particular cell are independent of the type of acceptor, we estimated the maximal rate constants for efflux (Vmax) of cellular cholesterol from L-cells, Fu5AH cells, and GM3468A fibroblasts. The rate constant for HDL3-mediated efflux varied among cell lines in the order Fu5AH > L-cells > fibroblasts. However, these differences were not evident when cyclodextrins were used as cholesterol acceptors. The estimated Vmax values for cyclodextrin-mediated efflux were 3.5-70-fold greater than for HDL3 for the three cell lines. The very high efficiency of cyclodextrins in stimulating cell cholesterol efflux suggests that these compounds can be used in two general ways for studies of atherosclerosis: 1) as research tools to probe mechanisms of cholesterol transport and aspects of membrane structure or 2) as potential pharmacological agents that could modify in vivo cholesterol metabolism and influence the development of the atherosclerotic plaque.

Transport of C4-dicarboxylates by anaerobically grown Escherichia coli. Energetics and mechanism of exchange, uptake and efflux.

Transport activities for uptake, efflux and exchange of C4-dicarboxylates were observed in anaerobically grown Escherichia coli. All three transport modes were only present in strains containing the transcriptional activator FNR of anaerobic respiration, and were repressed by nitrate and O2. The kinetic and energetic parameters of C4-dicarboxylate transport and the mechanism of the uptake, efflux and exchange reactions were analyzed in whole cells and in membrane vesicles. Fumarate/succinate exchange could be characterized as homologous or heterologous 1:1 counter-exchange. The external substrate was determined as divalent fumarate2- (or succinate2-) at pH 6-9, whereas monovalent H-fumarate dominated as the substrate at pH 3-4. The exchange was not inhibited by dissipation of delta p or constituents of it (delta psi or delta pH). We conclude that this transport mode functions as an electroneutral exchange of C4-dicarboxylates. The uptake of C4-dicarboxylates did not depend on internal counter-substrate and resulted in an accumulation of the substrate. Similar to antiport, fumarate was accepted in the divalent form at pH values greater than or equal to 6 and in the monovalent form at pH 3.5-6. The uptake was inhibited by dissipation of delta p or delta psi. Artificially imposed delta pH, delta psi or fumarate gradients were able to drive fumarate uptake. An involvement of Na+ could not be detected. Thus the uptake is likely to operate as an electrophoretic H+/fumarate symport. Independent of the presence of an external counter-substrate, the substrates were secreted from cells or membrane vesicles loaded with succinate or fumarate. The efflux was electrogenic. Energizing the cells or membrane vesicles inhibited efflux, maximal efflux rates were obtained only after dissipation of delta p or delta psi. An imposed K(+)-diffusion potential (outside positive) inhibited succinate excretion. The efflux of succinate from de-energized membrane vesicles generated a delta psi of -70 mV. It is thus suggested that succinate efflux functions as a H+/succinate symport.

Increased activity of the Mg2+/Na+ exchanger in red blood cells from essential hypertensive patients.

Epidemiological, clinical, and experimental evidence suggests a relation between Mg2+ metabolism and essential hypertension. The aim of the present study was the detection of abnormalities of the erythrocyte Mg2+/Na+ exchanger in essential hypertensive patients. We studied 66 untreated essential hypertensive patients and 36 normotensive control subjects. Maximal efflux rates of total Mg2+ efflux and the Na(+)-dependent and Na(+)-independent components of Mg2+ efflux were determined in Mg(2+)-loaded red blood cells. Mg2+/Na+ exchanger was calculated as the Na(+)-dependent component of the Mg2+ efflux. Mean values of Mg2+/Na+ exchanger were clearly elevated in hypertensive subjects with respect to normotensive control subjects [184.7 +/- 15.7 versus 84.4 +/- 6 mumol(L.cell.h)-1; P < .001]. This elevation was due primarily to the increased total Mg2+ efflux [324.2 +/- 21.9 versus 257.9 +/- 17.3 mumol(L.cell.h)-1; P < .05], whereas the Na(+)-independent component was not significantly different between the groups [154.5 +/- 11.8 versus 173.4 +/- 15.5 mumol(L.cell.h)-1; P = NS]. Moreover, total erythrocyte Mg2+ content was slightly reduced in hypertensive patients with respect to normotensive control subjects (1.84 +/- 0.04 versus 2.07 +/- 0.04 mmol/L.cell; P < .001). Using the 99% confidence limits of the normotensive population as the normal range, 30 (45.5%) hypertensive subjects showed values of Mg2+/Na+ exchanger higher than 160 mumol(L.cell.h)-1. The Mg2+/Na+ exchanger was inversely correlated with basal intraerythrocyte Mg2+ content (r = -.323; P = .001). From a clinical point of view, we found a positive correlation between diastolic blood pressure values and Mg2+/Na+ exchanger (r = .246; P < .05) in the sample of essential hypertensive patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic alcohol intake induces reversible disturbances on cellular Na+ metabolism in humans: its relationship with changes in blood pressure.

The effect of chronic alcohol consumption on Na(+)-K+ ATPase, Na(+)-Li+ countertransport, outward Na(+)-K(+)-Cl- cotransport system and the Na+ leak was investigated in red blood cells from 18 normotensive subjects with a daily alcohol intake of more than 150 g. The study was repeated after 3 months of alcohol withdrawal, and results were compared with a group of 20 healthy normotensive teetotalers. Maximal efflux rate (Vmax) and apparent dissociation constant for internal Na+ (KNa) of the Na(+)-K+ pump and the Na(+)-Li+ countertransport were significantly higher in alcohol consumers. A positive correlation between daily alcohol intake and Vmax of both transport systems (p less than 0.05) was observed. These values significantly decreased after alcohol withdrawal. A simultaneous stimulation of the Na(+)-K(+)-Cl- cotransport system after alcohol withdrawal was also observed. Blood pressure values were higher in alcoholics (133.7/82.3) than in abstainers (121.4/75 mmHg) and significantly decreased (128.5/76.9 mmHg) after withdrawal. A positive correlation between the stimulation of the Na(+)-K(+)-Cl- cotransport and the decrease of blood pressure after withdrawal was observed. In conclusion, chronic alcohol intake induces disturbances on red blood cell Na+ metabolism that dissipate with the cessation of drinking. Similar abnormalities also reported in humans and animals with primary hypertension have been associated in the pathogenesis of essential hypertension. Therefore, the pressor effect of chronic alcohol intake could be mediated through these changes in cellular Na+ metabolism.

The effects of manganese, cobalt and calcium on amylase secretion and calcium homeostasis in rat pancreas.

1. Mn(2+) evoked an atropine-resistant secretion of amylase from the isolated pancreas of the young rat. The lowest effective concentration of Mn(2+) was 10(-3)m. The response to 10(-2)m-Mn(2+) was biphasic, an initial peak being followed by a slow sustained rise in amylase output. The maximal effect of 10(-2)m-Mn(2+) was to double the basal rate of amylase secretion after 70 min incubation.2. Co(2+) (10(-2)m) also stimulated amylase secretion. The maximal rate, about three times the basal value, was attained after 20 min incubation. Atropine partially inhibited this effect.3. Ca(2+) (10(-2)m) evoked an atropine-resistant amylase secretion similar in both magnitude and time course to the sustained phase observed with 10(-2)m-Mn(2+).4. Mn(2+) (10(-4)-10(-2)m) also increased the rate of (45)Ca efflux from the gland. Maximal efflux rates were attained after 30 min incubation and thereafter declined to basal values. A small increase was also observed with 10(-2)m-Co(2+), but not with 10(-2)m-Ca(2+). The effect of Co(2+) was almost completely abolished by atropine.5. Reducing the extracellular Ca(2+) concentration from 2.5 x 10(-3) to 10(-5)m did not reduce amylase secretion in response to 10(-2)m-Mn(2+), but secretion was abolished in a Ca(2+)-free medium containing EGTA. The increase in (45)Ca efflux rate evoked by Mn(2+) was inversely related to the extracellular Ca(2+) concentration.6. Mn(2+) (10(-2)m) increased the concentration of cyclic 3',5'-guanosine monophosphate (cyclic GMP) within the pancreas. Also, Mn(2+) accumulated within the cellular pool of the gland. The time course of both these effects was similar to the time course of (45)Ca efflux.7. Mn(2+) displaced Ca(2+) bound to isolated pancreatic microsomal membranes. The cation-binding sites on these membranes probably have a higher affinity for Mn(2+) than Ca(2+).8. We conclude that Mn(2+) stimulates enzyme secretion by displacing membrane-bound Ca(2+), the resulting increase in cytosolic Ca(2+) concentration activating the secretory mechanism.9. Mn(2+) partially inhibited amylase secretion stimulated by optimal doses of either acetylcholine (ACh) or caerulein. Maximal inhibition (about 60%) occurred with 10(-3)m-Mn(2+) (i.e. the lowest concentration required to stimulate secretion in the absence of secretagogues). Decreasing the extracellular Ca(2+) concentration reduced the inhibitory effect of Mn(2+).10. When glands were exposed to ACh and Mn(2+) simultaneously, the time required for inhibitory effects to develop was inversely related to the dose of ACh and the concentration of Mn(2+).11. Mn(2+) did not alter the acceleration of (45)Ca efflux evoked by ACh or by caerulein in a medium containing 2.5 x 10(-3)m-Ca(2+). However, under conditions of Ca(2+) deprivation ACh-stimulated (45)Ca efflux was greatly enhanced.12. Mn(2+) reduced the total amount of Ca(2+) accumulated into the cellular pool of the pancreas after 60 min incubation, but had no effect on the initial, rapid phase of Ca(2+) uptake.13. The effects of Mn(2+) on the relationship between ACh dose, amylase release and the extracellular Ca(2+) concentration suggest that the inhibitory actions of Mn(2+) cannot be explained by a simple, competitive interaction with the stimulant or with extracellular Ca(2+). However, the time course of inhibition is consistent with a requirement for Mn(2+) to accumulate within the acinar cells.14. Mn(2+) partially inhibited amylase secretion stimulated by hyperosmolarity and also increased the (45)Ca efflux rate under these conditions.15. Our results are not consistent with Mn(2+) exerting its inhibitory effect on secretagogue-stimulated enzyme secretion solely by blocking Ca(2+) influx from the extracellular space. We conclude that inhibition probably depends on the ability of Mn(2+) to displace Ca(2+) from binding sites involved in secretion, presumably coupled with a reduced ability of Mn(2+) to replace Ca(2+) in the secretory process.

The Sodium-Alanine Interaction in Rabbit Ileum

The model of the interaction between Na and alanine at the mucosal border of rabbit ileum has been tested further by examining the efflux of alanine from the cells toward the mucosal solution. Alanine efflux shows a tendency toward saturation as cellular alanine concentration increases and is influenced by cellular Na concentration. A decrease in cell Na concentration causes an increase in the apparent Michaelis constant with little change in maximal efflux rate. Studies on strips of mucosa treated with ouabain or cyanide showed that the direction of net alanine transfer between the cells and the medium is determined by the direction of the Na concentration difference. The cells extrude alanine against a concentration difference when cell [Na] exceeds medium [Na] and accumulate alanine when cell [Na] is less than medium [Na]. The observations are consistent with the model previously suggested involving a transport site that combines with and translocates both Na and alanine, and with the concept that the Na concentration difference between mucosal solution and cytoplasm provides at least part of the energy for active transport of alanine.