RESEARCH ARTICLE
Regulation of Transmural Transport of Amino Acid/Metal Conjugates by Dietary Calcium in Crustacean Digestive Tract RANIA ABDEL‐MALAK AND GREGORY A. AHEARN* Department of Biology, University of North Florida, Jacksonville, Florida
ABSTRACT
J. Exp. Zool. 321A:135–143, 2014
Effects of luminal Ca2þ and Mn2þ on transmural mucosal to serosal (MS) transport of 3H‐L‐leucine were characterized in the isolated and perfused intestine of the American lobster, Homarus americanus. 3H‐L‐leucine MS transport in the presence of 20 mM Mn2þ was a sigmoidal function of luminal amino acid concentration, following the Hill equation for multisite cooperative, carrier‐ mediated, transport. Luminal Ca2þ was a non‐competitive inhibitor of Mn2þ‐stimulated 3H‐L‐ leucine MS flux. Amino acid transport was hyperbolically stimulated by luminal Ca2þ or Mn2þ. During 20 mM Mn2þ‐stimulation of 3H‐L‐leucine MS flux, addition of 25 mM Ca2þ strongly reduced amino acid transport Jmax, without affecting amino acid binding properties. Hyperbolic luminal Mn2þ stimulation of 20 mM 3H‐L‐leucine MS flux was also strongly inhibited by 25 mM luminal Ca2þ, significantly reducing 20 mM 3H‐L‐leucine Jmax. Increasing the luminal concentration of verapamil, a calcium channel blocker, significantly increased MS transport of 20 mM 3H‐L‐ leucine in the presence of 100 nM Mn2þ by reducing diffusional Ca2þ uptake into intestinal epithelial cells through verapamil‐sensitive channels. A model is proposed supporting the concept of molecular mimicry, whereby 3H‐L‐leucine enters lobster intestinal epithelial cells by one or more amino acid‐specific transporters and by a dipeptide‐like transporter that is capable of binding and transporting peptide molecular mimics (bis‐complexes) between Ca2þ or Mn2þ and 3H‐L‐leucine using the membrane potential as a major driving force for the transport event. According to the model, Ca2þ entry through apical Ca2þ channels regulates the magnitude of the membrane potential and therefore the size of the driving force for bis‐complex uptake. J. Exp. Zool. 321A: 135–143, 2014. © 2013 Wiley Periodicals, Inc. How to cite this article: Abdel‐Malak R, Ahearn GA. 2014. Regulation of transmural transport of amino acid/metal conjugates by dietary calcium in crustacean digestive tract. J. Exp. Zool. 321A:135–143.
Amino acids are transported across digestive organ luminal membranes of most organisms by the combination of secondary active transport processes and facilitated diffusion mechanisms. Because of the chemical diversity of this group of organic molecules, a variety of specific transport proteins have evolved to accommodate charge and structural differences of both essential and non‐essential amino acids (Kilberg et al., '93; Bröer, 2008). In recent years a parallel transport pathway has been described for amino acid accumulation by red blood cells (Aiken et al., '92; Horn et al., '95; Horn and Thomas, '96) and epithelial cells of vertebrate digestive (Tacnet et al., '90, '93; Glover and Hogstrand, 2002a,b;
Grant sponsor: USDA National Institute of Food and Agriculture (via Agriculture and Food Research Initiative Competitive grant); grant number: 2010‐65206‐20617. Correspondence to: Gregory A. Ahearn, Department of Biology, University of North Florida, 4567 St. Johns Bluff Road, South Jacksonville, FL 32224. E‐mail: [email protected] Received 15 July 2013; Revised 3 October 2013; Accepted 15 October 2013 DOI: 10.1002/jez.1843 Published online 19 November 2013 in Wiley Online Library (wileyonlinelibrary.com).
© 2013 WILEY PERIODICALS, INC.
136 Glover et al., 2003; Glover and Wood, 2008a,b) and renal organs (Zalups and Barfuss, 2002; Zalups and Bridges, 2010; Bridges and Zalups, 2010), which involves chelation between amino acids and divalent cations in solution, followed by transmembrane transport of the entire bis‐complex into the cytoplasm of the receiving cells. Several studies over the last 25 years have suggested that crustacean gastrointestinal tract organs possess a range of specific amino acid transport proteins (Ahearn, '88; Ahearn and Clay, '88a; Wright and Ahearn, '97) and also appear to have the ability to transport bis‐complexes between luminal amino acids and dietary metals (Conrad and Ahearn, 2005, 2007; Mullins and Ahearn, 2008; Obi et al., 2011; Simmons et al., 2012). Early studies described an apparent dipeptide transporter in brush border membrane (BBM) vesicles of lobster hepatopancreas (Thamotharan and Ahearn, '96) that displayed a number of physiological characteristics in common with PEPT1, a thoroughly investigated vertebrate peptide transport system (Daniel and Rubio‐Aliaga, 2003; Herrera‐Ruiz and Knipp, 2003; Daniel, 2004; Verri et al., 2010). Experiments with perfused lobster intestine suggested that transmural 14C‐glycylsarcosine transport was unaffected by luminal L‐leucine or L‐histidine alone, but was abolished by the presence of bis‐complexes involving both L‐leucine and zinc or L‐ histidine and zinc (Conrad and Ahearn, 2005). These results suggest that both the dipeptide and bis‐complexes appear to use the same carrier system. Transport of 65Zn2þ across lobster intestine was doubled in the presence of luminal L‐histidine, suggesting that both metal and amino acid act synergistically to enhance their individual absorption as a result of bis‐complex transport (Conrad and Ahearn, 2007). More recent work has indicated that bis‐complex transport appears to be a wide‐spread phenomenon in crustacean digestive organs involving Zn2þ, Cu2þ, and Mn2þ and both L‐histidine and L‐leucine and may play an important quantitative role in essential amino acid and metal absorption in these invertebrate animals (Obi et al., 2011; Simmons et al., 2012). The present investigation examines the interaction between two stimulatory divalent cations, zinc, and calcium, present simultaneously, on transmural transport of 3H‐L‐leucine in lobster intestine. Results suggest potential inhibitory interactions between different bis‐complexes for binding to a shared carrier protein, and regulation of bis‐complex transport rates by modification of the cellular resting potential as a driving force for conjugate transport by cation flow through calcium channels.
MATERIALS AND METHODS Live, male American lobsters, Homarus americanus, were maintained at 15°C in filtered saltwater and fed frozen mussel meat prior to their use in experiments. A physiological saline that approximated the ionic strength and composition of lobster hemolymph was utilized in experiments. The salt concentrations consisted of 420 mM NaCl, 25 mM CaCl2, 10 mM KCl, 8.4 mM J. Exp. Zool.
ABDEL‐MALAK AND AHEARN Na2SO4, and 30 mM HEPES at pH 7.0. To examine in vitro transmural mucosal to serosal (MS) movement of 3H‐L‐leucine across the lobster intestine, the intestine of a live lobster was flushed and ligated onto 18–20 gauge stainless steel needles and attached firmly with surgical thread in a perfusion apparatus that contained 35 mL of the physiological saline (serosal medium; Ahearn and Maginniss, '77; Obi et al., 2011). The same saline was perfused through the intestine and served as the mucosal medium using a peristaltic pump (Instech Laboratories, Inc., Plymouth Meeting, PA, USA) at a flow rate of 0.380 mL/min (Conrad and Ahearn, 2005). Transmural transport of [2,3,4,5‐3H]‐L‐leucine (American Radiochemicals, Inc. (St. Louis, MO, USA), from mucosa to serosa (MS) was measured by perfusing the physiological saline containing a fixed concentration of 3H‐L‐leucine and variable concentrations of calcium or manganese through the intestine for periods of time from 30 to 150 min. Every 5 min during a perfusion procedure, triplicate 200 mL samples of the serosal medium were withdrawn and added to scintillation fluid (Scinti‐Vase) for radioactivity counting (Beckman LS‐6500 liquid scintillation counter, Beckman Coulter Co., USA) followed by addition of an equal volume of saline to the bath to maintain its total volume through the experiment. A given experimental treatment was perfused through an intestine for a period of 30 min. Use of multiple experimental treatments involved the sequential 30 min perfusion of each treatment for periods of time up to 150 min. Regression lines were drawn through the resulting 30 min data obtained from sequential sampling of the serosal medium to obtain the rate of isotope transfer, providing an index of the rate of transmural amino acid flux during the test period. When variable concentrations of calcium or manganese were perfused through an intestine, choline was substituted for either divalent cation. Experiments using the calcium channel blocker, verapamil, involved variable concentrations of the inhibitor added to the perfusate in the presence of 100 nM manganese and the presence or absence of 25 mM calcium. Transmural flux rates of 3H‐L‐leucine were calculated using the diameter and length of the experimental intestine to estimate the intestinal surface area: A ¼ pdl, where “d” is the diameter and “l” is the length of the acquired intestine (Obi et al., 2011). To measure the total radioactive counts of 3H‐L‐leucine, the background counts per minute (cpm) were subtracted from the measured cpm values obtained from the scintillation counter. Specific activities of radioactive samples (e.g., cpm/pmol 3H‐L‐leucine) were used to estimate the amino acid fluxes across the tissue. 3H‐L‐leucine transmural flux rates were expressed as mean nmoles/cm2 min or mean pmoles/cm2 min. Experiments were carried out in triplicate with three animals per experiment, and the resulting flux rates and kinetic constants were collected for statistical analysis. Linear regression and hyperbolic analyses were executed using Sigma Plot 10.0 software (Obi et al., 2011). Statistical differences between treatment means were examined using Student's t‐test
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or ANOVA and were considered statistically different when P < 0.05.
RESULTS Effects of Luminal Mn2þ and Mn2þ þ Ca2þ on the Time Course of 3 H‐L‐Leucine MS Transport Effects of variations in luminal concentration of Mn2þ alone, or in combination with luminal Ca2þ, were examined during transmural MS (mucosal to serosal) transport of 3H‐L‐leucine concentrations ranging from 0.5 to 10 mM. Experiments were performed with a pH of 7.0 on both mucosal and serosal sides of the intestine to lessen possible effects of a proton gradient on the transport system. Figure 1A shows the effect of 20 mM luminal Mn2þ on steady‐state MS transport of 3H‐L‐leucine at varying luminal amino acid concentrations (JL ¼ 0.33 0.02 to 6.95 0.82 nmol/cm2 min; where JL is unidirectional MS flux). Results suggest that over 150 min of perfusion, a gradual increase in amino acid transfer (increase in transport slopes at each amino acid concentration) from lumen to bath occurred. During the transitions between increasing luminal amino acid concentrations vertical transport displacements were observed that likely reflect transient tissue adjustments to the novel substrate concentrations. All steady‐state fluxes (slopes) were significantly different than zero (P < 0.05). Figure 1B shows the effects of increasing mucosal 3H‐L‐leucine concentration (0.50–10 mM), on MS fluxes of 3H‐L‐leucine in saline consisting of 20 mM Mn2þ and 25 mM Ca2þ. At each luminal concentration of amino acid, the MS fluxes measured in the presence of both divalent cations were significantly lower (P < 0.05) than those obtained in the absence of luminal calcium. In the latter experimental condition, slopes (e.g., fluxes) increased from 0.08 0.01 to 2.94 0.71 nmoles/cm2 min with all values significantly different than zero (P < 0.05). Comparison of the results in Figure 1A and B, it is clear that addition of 25 mM luminal calcium to a perfusate containing radiolabeled amino acid and a fixed concentration of Mn2þ (20 mM) led to a significant reduction in the rate of transport compared to values obtained without the addition of calcium. Effects of Luminal Ca2þ and Mn2þ on the Kinetics of 3H‐L‐Leucine MS Transport Transmural MS transport of 3H‐L‐leucine was measured in the presence of 20 mM Mn2þ alone, or with 25 mM Ca2þ in addition to 20 mM Mn2þ, to assess the effects of these divalent cations on the kinetic constants of amino acid transport. As displayed in Figure 2, the transport of 3H‐L‐leucine across lobster intestine was a sigmoidal function of luminal amino acid concentration at each experimental condition used, suggesting the presence of carrier‐ mediated transport for this amino acid following the Hill equation for multisite binding cooperativity:
Figure 1. Time course of MS (mucosal to serosal) steady‐state transport at pH 7.0 of 3H‐L‐leucine concentration (0.50, 1, 2.5, 5, and 10 mM) across representative lobster intestines in the presence of 20 mM manganese with no calcium (A) and in the presence of 25 mM calcium (B). Data are means of three replicate samples at each time interval for each leucine concentration. Lines drawn through the data points at each leucine concentration over the 150‐min time course were drawn by linear regression analysis using Sigma Plot 10.0 and the values presented on the figure represents slopes 1 SEM. Vertical displacements of steady‐state fluxes at each treatment are due to transient adjustments of the tissue to increasing perfused 3H‐L‐leucine concentrations.
J¼
J max ½leucinen K nt þ ½leucinen
ð1Þ
where J is 3H‐L‐leucine transport in nmol/cm2 min, Jmax is maximal 3H‐L‐leucine transport, K nt is an apparent binding affinity of the L‐leucine transporter adjusted to accommodate multisite substrate binding cooperativity, and is described as the [L‐leucine] at 1/2Jmax, [leucine]n is the luminal concentration of J. Exp. Zool.
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Figure 2. Transmural MS steady‐state transport of 3H‐L‐leucine at pH 7.0 as a function of L‐leucine concentration (0.50, 1, 2.5, 5, and 10 mM) in the presence of luminal 20 mM manganese and either the presence or absence of 25 mM calcium on both sides of lobster intestine. Symbols are means 1 SEM of three independent experiments involving different test animals. Sigmoidal curves and resulting kinetic constants (Table 1) were obtained using Sigma Plot 10.0.
amino acid in mM, and n is the Hill coefficient which is an estimate of the approximate transport stoichiometry. The control condition in this experiment included only 20 mM Mn2þ in the perfusate as this metal has previously been shown to stimulate MS transport of amino acids in crustacean intestine (Simmons et al., 2012). Under these conditions the kinetic constants for 3H‐L‐leucine transport across the tissue were K nt ¼ 3.98 0.59 mM, Jmax ¼ 8.52 0.75 nmol/cm2 min, and n ¼ 1.57 0.18. Addition of 25 mM Ca2þ to the incubation medium with 20 mM Mn2þ resulted in a significant decrease in 3H‐ L‐leucine Jmax (P < 0.01), without effect on amino acid transport K nt (P > 0.05). In both treatments, Hill coefficients between 1.5 and 2.0 were obtained and suggest an unchanged transport stoichiometry of 2 Leucine/1 divalent cation. The respective kinetic constants from these two experimental conditions are displayed in
Figure 3. Transmural MS steady‐state transport of 20 mM 3H‐L‐ leucine at pH 7.0 as a function of luminal calcium concentration (0, 0.25, 0.50, 1, and 5 mM). Data were curve‐fit with Sigma Plot 10.0. Symbols are means 1 SEM of three independent experiments (e.g., three animals). Resulting kinetic constants are displayed on the figure.
Table 1. As Figure 2 and Table 1 indicate, calcium only had a significant effect on amino acid transport in the presence of Mn2þ by decreasing the rate of 3H‐L‐leucine transport across the tissue without apparently influencing its binding properties or its transport stoichiometry. Effect of Variable Calcium Concentration on MS Transport of H‐L‐leucine Transport Because calcium was found to inhibit 3H‐L‐leucine MS flux in the presence of the stimulating divalent cation, Mn2þ (Fig. 2 and Table 1), the effect of variable luminal concentrations of calcium on amino acid transport was assessed in the absence of Mn2þ. As displayed in Figure 3, MS transport of 20 mM 3H‐L‐leucine was a hyperbolic function of perfusate calcium concentration and followed a modification of the Michaelis–Menten equation below: 3
Table 1. Effect of 20 mM Mn2þ and 25 mM Ca2þ on the kinetic constants of MS 3H‐L‐leucine transport across lobster intestine. Treatment 20 mM Mn (control) 20 mM Mn2þ þ 25 mM Ca2þ 2þ
L‐leucine
K nt (mM)
Jmax (nmol/cm2 min)
n‐value
3.98 0.59 4.80 0.10
8.52 0.75 4.85 0.07
1.57 0.18 1.81 0.13
concentration range: 0, 0.5, 1, 2.5, 5.0, and 10 mM. Values are means 1 SEM. Signifies value is significantly different from control at P < 0.01.
J. Exp. Zool.
REGULATION OF TRANSMURAL TRANSPORT J¼
J max ½Ca2þ K Ca þ ½Ca2þ
139 ð2Þ
where J is 3H‐L‐leucine transport in pmol/cm2 min, Jmax is maximal transport of 20 mM 3H‐L‐leucine, KCa is an apparent calcium binding constant and is described as the [Ca2þ] at 1/2Jmax of 20 mM 3H‐L‐leucine transport, and [Ca2þ] is the luminal calcium concentration in mM. The kinetic constants for this experiment were KCa ¼ 0.81 0.23 mM Ca2þ and Jmax ¼ 18.74 1.67 pmol/cm2 min. The hyperbolic nature of the data in Figure 3 suggests that calcium stimulates amino acid transport as a result of the cation binding to a saturable co‐transport carrier‐mediated possess with a finite number of binding sites. Figure 3 indicates a small, but significant (P < 0.05) vertical axis intercept at 0 mM calcium, suggesting the possible presence of one or more calcium‐independent transport processes for 3H‐L‐leucine in this tissue. Luminal Ca2þ is a Non‐Competitive Inhibitor of Mn2þ Activation of H‐L‐Leucine MS Transport The effects of variable luminal Mn2þ concentration on 20 mM 3 H‐L‐leucine MS transport in the presence and absence of 25 mM Ca2þ is shown in Figure 4. Amino acid transport in both experimental conditions was a hyperbolic function of luminal Mn2þ and followed Equation (2) with Mn2þ substituting for Ca2þ in the equation. It is clear from Figure 4 that addition of 25 mM Ca2þ to the perfusate containing variable [Mn2þ] significantly (P < 0.01) reduced amino acid transport over the 3
Figure 4. Transmural MS steady‐state transport of 20 mM 3H‐L‐ leucine at pH 7.0 across lobster intestine as a function of luminal manganese concentration (0, 25, 50, 100, and 250 nM), in the presence and absence of 25 mM calcium. Data were curve‐fit with Sigma Plot 10.0 and resulting kinetic constants are displayed in Table 2. Symbols are means 1 SEM of three independent experiments (e.g., three animals).
Table 2. Effect of 25 mM Ca2þ on kinetic constants of Mn2þ‐ stimulated 20 mM 3H‐L‐leucine MS transport across lobster intestine.
Treatment 20 mM Mn2þ (control) 20 mM Mn2þ þ 25 mM Ca2þ
KMn (nM)
Jmax (pmol/cm2 min)
23.48 5.68 28.93 10.10
14.03 0.88 5.45 0.56
Manganese concentration range: 0, 25, 50, 100, and 250 nM. Values are means 1 SEM. Signifies value is significantly different from control at P < 0.01.
range of Mn2þ concentrations used. Kinetic constants for 20 mM 3 H‐L‐leucine MS transport in the presence and absence of luminal calcium are displayed in Table 2 and indicate that the effect of adding calcium was to significantly (P < 0.01) reduce MS amino acid transport Jmax, without significantly (P > 0.05) affecting the binding properties of Mn2þ to the transport system. This result suggests that calcium was acting as a non‐ competitive inhibitor of Mn2þ activation of amino acid transport. As in Figure 3, at 0 mM Mn2þ, a significant vertical axis intercept was obtained for both experimental conditions, suggesting the possible presence of undescribed manganese‐ independent transport mechanisms. Calcium Regulates Mn2þ‐Stimulated 3H‐L‐Leucine MS Transport Through calcium Channels Because luminal calcium affected the rate of Mn2þ‐stimulated 3H‐ L‐leucine transport across lobster intestine rather than the binding properties of Mn2þ or amino acid to a shared carrier system, an experiment was conducted to clarify the nature of the reduction in Mn2þ‐enhanced L‐leucine MS transport. Varying concentrations of the calcium channel blocker, verapamil (0–1,000 mM), were perfused through lobster intestine with 20 mM 3H‐L‐leucine and 100 nM Mn2þ with and without the addition of 25 mM Ca2þ for 30 min intervals as described in Figure 1. 3H‐L‐leucine transport rates over five 30‐min incubation periods (one period per [verapamil]) were measured and the results are displayed in Figure 5. The data in this figure show a gradual, but significant, increase in 20 mM 3H‐L‐leucine MS transport across lobster intestine as perfusion concentrations of verapamil were elevated, both in the presence and absence of luminal Ca2þ (25 mM calcium: P < 0.01, F ¼ 17.1, df ¼ 14; 0 mM calcium: P < 0.038, F ¼ 3.9, df ¼ 14; ANOVA). Results in Figure 5 suggest that Mn2þ and/or Ca2þ in the intestinal lumen may enter epithelial apical calcium channels partially depolarizing the membrane potential and decrease its apparent driving force for Mn2þ‐coupled 3H‐L‐leucine transport. Increasing verapamil concentrations, as provided in this study, apparently block increasing numbers of these channels, reducing the depolarizing effect of the luminal divalent cations J. Exp. Zool.
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Figure 5. Transmural MS steady‐state 30‐min transport of 20 mM 3 H‐L‐leucine at pH 7.0 across lobster intestine as a function of luminal verapamil concentration (0, 100, 250, 500, and 1,000 mM) in the presence of 100 nM luminal manganese and either 0 or 25 mM calcium on both sides of perfused intestine. Columns are means 1 SEM of three independent experiments (e.g., three animals).
and allowing greater carrier‐mediated transmural MS transport as a result. Therefore, the apparent non‐competitive inhibition of calcium on Mn2þ‐stimulated 3H‐L‐leucine MS flux in Figure 4 may have been due to calcium depolarization of the apical membrane potential. Results in Figure 5 also show that 100 nM Mn2þ by itself appears capable of this depolarizing effect on its own co‐transport with 3H‐L‐leucine, but because its concentration was much lower than that of calcium in the present set of experiments, its regulatory role would presumably be less.
DISCUSSION In the crustacean digestive tract, amino acids are transported from lumen to blood by first passing across the epithelial apical membrane into the cytoplasm by a wide variety of specific amino acid transport proteins (Ahearn, '88; Ahearn and Clay, '88a; Kilberg et al., '93; Wright and Ahearn, '97; Bröer, 2008) that are either secondary active transport systems, using the transmembrane electrochemical gradient to power their accumulation in the cytosol, or facilitated diffusion processes employing their concentration gradient to enter the cells. Less is known about amino acid efflux from epithelium to the blood across gut basolateral membranes in these arthropods, but if the vertebrate paradigm is followed, diffusion using a variety of carrier proteins on this cellular pole likely accounts for transfer to the circulation. From what is currently known from recent investigations using crustacean hepatopancreatic BBM vesicles and perfused intestine, J. Exp. Zool.
ABDEL‐MALAK AND AHEARN a significant transepithelial transport of amino acids occurs from lumen to blood across the digestive tract epithelia, providing both essential and non‐essential substrates for protein metabolism (Wright and Ahearn, '97; Conrad and Ahearn, 2005, 2007; Mullins and Ahearn, 2008; Obi et al., 2011; Simmons et al., 2012). In recent years a parallel pathway for amino acid absorption across crustacean intestine has been described that involves the apparent transport of amino acid/metal chelates by dipeptide transport proteins and which may significantly supplement the net movements of these organic solutes by amino acid‐specific transport systems (Conrad and Ahearn, 2005, 2007; Mullins and Ahearn, 2008; Obi et al., 2011). Formation of amino acid/metal chelates, called bis‐complexes, and their transport across gastrointestinal membranes was first described for pig intestine (Tacnet et al., '90, '93) and later found to take place in fish digestive tracts (Glover and Hogstrand, 2002a,b; Glover et al., 2003; Glover and Wood, 2008a,b). In both of these vertebrate groups zinc and copper were found to bind to L‐histidine, L‐cysteine, and other amino acids in solution and be transported into the gut epithelium by an undisclosed transport mechanism that appeared distinct from specific amino acid transporters. At the time it was not known how widespread this phenomenon was, how many amino acids might use it, how many different metal cations might form such bis‐complexes, how the transport rates of these chelates might be regulated, and what the quantitative importance such a transport system might have for total net absorption of essential amino acids. Conrad and Ahearn (2005) showed that transmural mucosa to serosa (MS) 3H‐L‐histidine transport across perfused lobster intestine was increased by a factor of 3 by addition of luminal zinc. This enhanced amino acid transport, across the gut in the presence of metal was eliminated by addition of mucosal L‐leucine. Zinc‐stimulated 3H‐L‐histidine transport was similarly inhibited by perfusion with the dipeptide, glycylsarcosine. This study proposed that all three organic solutes (L‐histidine, L‐leucine, and glycylsarcosine) share a common carrier that normally transport dipeptides, but would also accept amino acid/metal bis‐complexes as well. In other experiments, transmural MS flux of 65Zn2þ was doubled when luminal L‐histidine was present, but was significantly abolished by addition of Cu2þ to the perfusate (Conrad and Ahearn, 2005, 2007). These results were interpreted as indicating that both Zn2þ and Cu2þ are able to form bis‐complexes with L‐ histidine and complete for a shared transport system. Subsequent studies with lobster intestine, using 3H‐leucine, indicated that the metals, Zn2þ, Cu2þ, and Mn2þ, are all able to stimulate amino acid MS transport in a hyperbolic fashion with the transport system having the highest apparent binding affinity for Mn2þ (Mullins and Ahearn, 2008; Obi et al., 2011). The use of several divalent metals Zn2þ, Cu2þ, Mn2þ, Cd2þ, and Co2þ, as stimulatory co‐ transporters of amino acids, was extended to both 3H‐leucine and 3 H‐L‐histidine in the hepatopancreas of the marine penaeid shrimp, Litopenaeus setiferus (Simmons et al., 2012), suggesting that
REGULATION OF TRANSMURAL TRANSPORT metal‐enhanced amino acid absorption across crustacean gut may be a general process among digestive organs of decapod crustaceans. These studies proposed that a peptide transport protein, like PEPT1, may be able to bind, and subsequently transport, bis‐complexes involving two amino acids bonded to a divalent cation (e.g., Leu‐Zn‐Leu) and appearing like a dipeptide as a result of molecular mimicry. Molecular mimicry has been suggested to account for the movement of one solute (the mimic) that chemically resembles another (the model) by transporters that typically are restricted in their specificity to the model, but will accept, under certain conditions, other molecular forms that would normally not be accommodated. Work with mammalian intestine and renal proximal tubular cells has described the transport of mercuric complexes with cysteine, glutathione, and other substrates (Zalups and Barfuss, 2002; Bridges and Zalups, 2010; Zalups and Bridges, 2010). These authors suggest that a number of known transport systems, that normally transport different model substrates, will also accept metal complexes involving mercury as a result of molecular mimicry. Some of the specific transporters that were identified as potential systems involved with such mimicry were the Naþ‐independent System bo,þ and a variety of Naþ‐dependent processes as well (Zalups and Bridges, 2010). Furthermore, these authors suggested that mercuric complexes involving dipeptides (e.g., Cys‐Gly‐Hg‐Cys‐Gly) might be able to utilize a peptide transporter that would normally be restricted to dipeptide and tripeptide substrates (Zalups and Bridges, 2010). Additional transporters that were suggested as potential transport sites of metallic complexes with amino acids and other substrates included members of the Organic Anion Exchanger family (e.g., OAT1 and/or OAT3) (Zalups and Barfuss, 2002; Zalups and Bridges, 2010). Therefore, it appears that a significant number of different identified membrane carrier systems may be involved in the movement of metal/organic solute complexes across cell membranes as a result of molecular mimicry. Results of the present investigation support previous studies with crustacean digestive tract (Conrad and Ahearn, 2005, 2007; Mullins and Ahearn, 2008; Obi et al., 2011) and buttress the notion that molecular mimicry appears to occur in crustacean intestine and hepatopancreas and that this process may make a significant contribution to net transmembrane and transcellular transport of essential dietary elements such as amino acids and metals. Figures 3 and 4 suggest that both calcium and manganese are able to support carrier‐mediated 3H‐L‐leucine MS transport by lobster intestine. These results are similar to those obtained for zinc and copper stimulation of 3H‐L‐leucine transport by the same lobster tissue (Obi et al., 2011), and for both manganese and zinc stimulation of 3H‐L‐histidine transport by shrimp (L. setiferus) hepatopancreatic BBM vesicles (Simmons et al., 2012). It, therefore, appears that in crustaceans, a number of different divalent cations are able to interact with amino acids in solution to form bis‐complexes that are transported into and through
141 epithelial cells of both the hepatopancreas and intestine of these animals. Previous experiments with lobster intestine using the dipeptide, glycylsarcosine, have shown that the MS transport of bis‐complexes involving zinc and 3H‐L‐leucine is significantly reduced compared to control conditions lacking the dipeptide (Conrad and Ahearn, 2005; Obi et al., 2011). These data suggest that both the dipeptide and amino acid/metal bis‐complexes appear to use the same transport system such as a PEPT1 dipeptide transporter. In the present study, calcium was a non‐competitive inhibitor of manganese‐stimulated 3H‐L‐leucine MS transport, exerting a significant (P < 0.01) effect on the maximal rate of amino acid transport, but a non‐significant (P > 0.05) effect on manganese binding properties (Fig. 4 and Table 2). Figure 5 shows that addition of the calcium channel blocker, verapamil, to the perfusate containing 3H‐L‐leucine and manganese, with or without calcium, resulted in an increase in amino acid MS transport as the inhibitor concentration was elevated. These results suggest that the non‐ competitive inhibitory effect of luminal calcium on 3H‐L‐leucine MS transport, in the presence of manganese, may have been due to calcium entry into intestinal epithelial cells via verapamil‐sensitive calcium channels, depolarizing the membrane potential, and reducing membrane‐potential‐dependent uptake of bis‐complexes between 3H‐L‐leucine and Mn2þ. Therefore, in vivo, calcium flow through apical calcium channels may regulate the rate of bis‐ complex transport via PEPT1, or other transporters involving divalent cations and amino acids. Figure 6 is a tentative working model of proposed calcium regulation of bis‐complex transport involving a divalent cation and dietary amino acids and an apparent dipeptide transporter. This figure also shows the presence of an independent amino acid‐ specific L‐leucine transporter, which has been previously described (Ahearn and Clay, '88b) on the digestive system apical membrane and which operates in parallel with molecular mimicry occurring by an apparent dipeptide transporter. All divalent cations studied to date (e.g., Zn2þ, Cu2þ, Mn2þ, and Ca2þ) appear capable of forming bis‐complexes with several amino acids in the crustacean intestinal lumen and sharing a common membrane transporter with each other and with the dipeptide, glycylsarcosine. In the present study, Hill coefficients of approximately 2.0 between L‐leucine and Mn2þ (Fig. 2 and Table 1) provide kinetic stoichiometric evidence for transport of 2 amino acids with a single metal ion over the amino acid concentration range used. The apparent binding affinity constants (KCa and KMn) for calcium and manganese are very different, with the membrane exhibiting several orders of magnitude greater apparent binding affinity for Mn2þ than for Ca2þ (Fig. 3 and Table 2). This highly significant difference between the binding properties of the two cations is likely the reason why 25 mM calcium did not appear to be an apparent competitive inhibitor of manganese binding to the transport system in Figure 4 and Table 2, although this might be more apparent at higher calcium concentrations. Figure 6 shows that luminal calcium J. Exp. Zool.
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Figure 6. Proposed transport processes of intestinal epithelial cells of lobster, displaying three different brush border membrane (BBM) transport proteins: (1) Dipeptide/bis‐complex transporter; (2) Amino acid transporter; and (3) Verapamil‐inhibited calcium channel that may accept Ca2þ, Mn2þ, and possibly other divalent cations. Luminal amino acids may be transported into cells by #1 as molecular mimics of dipeptides (e.g., bis‐complexes), and/or by #2 which does not accept bis‐complexes. The proposed calcium channel allows the diffusional entry of divalent cations into the cytoplasm, depolarizing the membrane potential, lowering the electrochemical potential across the membrane, and reducing the uptake of bis‐complexes involving amino acids. The cytoplasmic fates of intracellular bis‐complexes currently are unknown, but it is likely that steady‐state efflux of radiolabeled cytoplasmic amino acids to the serosal medium may occur either as bis‐complexes or as free amino acids.
appears to regulate the transport rates of bis‐complexes across intestine apical membranes by using channels whose openings can presumably be controlled and thereby control calcium depolarization of the apical membrane potential. Figure 5 indicates that luminal Mn2þ alone was apparently able to enter calcium channels, in the absence of calcium, and, to a lesser extent, influence the membrane potential driving force for bis‐complex uptake. Factors affecting the extent of calcium channel opening and its quantitative participation in bis‐complex transport through influences on the magnitude of the trans‐apical membrane potential are still unclear.
LITERATURE CITED Ahearn GA. 1988. Nutrient absorption by the invertebrate gut. In: Gilles R, editor. Advances in environmental and comparative physiology. vol 2. Berlin: Springer‐Verlag. p 91–129. Ahearn GA, Clay LP. 1988a. Sodium‐coupled sugar and amino acid transport in an acidic microenvironment. Comp Biochem Physiol 90A:627–634. J. Exp. Zool.
ABDEL‐MALAK AND AHEARN Ahearn GA, Clay LP. 1988b. Electroneutral Na‐2Cl—leucine cotransport by lobster hepatopancreatic brush border membrane vesicles. J Exp Biol 136:363–381. Ahearn GA, Maginniss LA. 1977. Kinetics of glucose transport by the perfused mid‐gut of the freshwater prawn, Macrobrachium rosenbegii. J Physiol (Lond) 271:319–336. Aiken SP, Horn NM, Saunders NR. 1992. Effect of amino acids on zinc transport in rat erythrocytes. J Physiol 445:69–80. Bridges CC, Zalups RK. 2010. Ionic and molecular mimicry and the transport of metals. In: Zalups RK, Koropatnick J, editors. Cellular and molecular biology of metals. Boca Raton, FL: CRC Press. p 241–294. Bröer S. 2008. Amino acid transport across mammalian intestinal and renal epithelia. Physiol Rev 88:249–286. Conrad EM, Ahearn GA. 2005. 3H‐L‐histidine and 65Zn2þ are cotransported by dipeptide transport system in intestine of lobster Homarus americanus. J Exp Biol 208:287–296. Conrad EM, Ahearn GA. 2007. Transepithelial transport of zinc and L‐histidine across perfused intestine of American lobster, Homarus americanus. J Comp Physiol B 177:297–307. Daniel H. 2004. Molecular and integrative physiology of intestinal peptide transport. Ann Rev Physiol 66:361–384. Daniel H, Rubio‐Aliaga I. 2003. An update on renal peptide transporters. Am J Physiol Renal Physiol 284:F885–F892. Glover CN, Hogstrand C. 2002a. In vivo characterization of intestinal zinc uptake in freshwater rainbow trout. J Exp Biol 205:141–150. Glover CN, Hogstrand C. 2002b. Amino acid modulation of in vivo intestinal zinc absorption in freshwater rainbow trout. J Exp Biol 205:151–158. Glover CN, Wood CM. 2008a. Absorption of copper and copper‐ histidine complexes across the apical surface of freshwater rainbow trout intestine. J Comp Physiol B 178:101–109. Glover CN, Wood CM. 2008b. Histidine absorption across apical surfaces of freshwater rainbow trout intestine: mechanistic characterization and the influence of copper. J Membrane Biol 221:87–95. Glover CN, Bury NR, Hogstrand C. 2003. Zinc uptake across the apical membrane of freshwater rainbow trout intestine is mediated by high affinity, low affinity, and histidine‐facilitated pathways. Biochim Biophys Acta 1614:211–219. Herrera‐Ruiz D, Knipp GT. 2003. Current perspectives on established and putative mammalian oligopeptide transporters. J Pharmaceut Sci 92:691–714. Horn NM, Thomas A. 1996. Interactions between the histidine stimulation of cadmium and zinc influx into human erythrocytes. J Physiol 496:711–718. Horn NM, Thomas AL, Tompkins JD. 1995. The effect of histidine and cysteine on zinc influx into rat and human erythrocytes. J Physiol 489:73–80. Kilberg MS, Stevens BR, Novak DA. 1993. Recent advances in mammalian amino acid transport. Ann Rev Nutrition 13:137– 165.
REGULATION OF TRANSMURAL TRANSPORT Mullins A, Ahearn GA. 2008. Zinc dependent L‐leucine uptake in Homarus americanus midgut. In: Morris S, Voosloo A, editors. Molecules to migration: the pressures of life, 4th CPB meeting in Africa: Mara 2008. Bologna: Medimond. p 83–90. Obi I, Wells A, Ortega P, et al. 2011. 3H‐L‐leucine transport by the promiscuous crustacean dipeptide‐like cotransporter. J Exp Zool 315:465–475. Simmons T, Mozo J, Wilson J, Ahearn GA. 2012. Cation‐dependent nutrient transport in shrimp digestive tract. J Comp Physiol B 182:209–216. Tacnet F, Watkins DW, Ripoche P. 1990. Studies of zinc transport into brush‐border membrane vesicles isolated from pig small intestine. Biochim Biophys Acta 1024:323–330. Tacnet F, Lautheir F, Ripoche P. 1993. Mechanisms of zinc transport into pig small intestine brush‐border membrane vesicles. J Physiol 465:57–72.
143 Thamotharan M, Ahearn GA. 1996. Dipeptide transport by crustacean hepatopancreatic brush border membrane vesicles. J Exp Biol 199:635–641. Verri T, Romano A, Barca A, et al. 2010. Transport of di‐and tripeptides in teleost fish intestine. Aquculture Res 41:641–653. Wright SH, Ahearn GA. 1997. Nutrient absorption in invertebrates. In: Dantzler WH, editor. Handbook of physiology: section 13: comparative physiology. vol II. New York: Oxford University Press. p 1137–1206. Zalups RK, Barfuss DW. 2002. Renal organic anion transport system: a mechanism for the basolateral uptake of mercury‐thiol conjugates along the Pars Recta of the proximal tubule. Toxicol Appl Pharmacol 182:234–243. Zalups RK, Bridges CC. 2010. Molecular and cellular biology of mercury in the kidneys. In: Zalups RK, Koropatnick J, editors. Cellular and molecular biology of metals. Boca Raton, FL: CRC Press. p 35–77.
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Regulation of Transmural Transport of Amino Acid/Metal Conjugates by Dietary Calcium in Crustacean Digestive Tract RANIA ABDEL‐MALAK AND GREGORY A. AHEARN* Department of Biology, University of North Florida, Jacksonville, Florida
ABSTRACT
J. Exp. Zool. 321A:135–143, 2014
Effects of luminal Ca2þ and Mn2þ on transmural mucosal to serosal (MS) transport of 3H‐L‐leucine were characterized in the isolated and perfused intestine of the American lobster, Homarus americanus. 3H‐L‐leucine MS transport in the presence of 20 mM Mn2þ was a sigmoidal function of luminal amino acid concentration, following the Hill equation for multisite cooperative, carrier‐ mediated, transport. Luminal Ca2þ was a non‐competitive inhibitor of Mn2þ‐stimulated 3H‐L‐ leucine MS flux. Amino acid transport was hyperbolically stimulated by luminal Ca2þ or Mn2þ. During 20 mM Mn2þ‐stimulation of 3H‐L‐leucine MS flux, addition of 25 mM Ca2þ strongly reduced amino acid transport Jmax, without affecting amino acid binding properties. Hyperbolic luminal Mn2þ stimulation of 20 mM 3H‐L‐leucine MS flux was also strongly inhibited by 25 mM luminal Ca2þ, significantly reducing 20 mM 3H‐L‐leucine Jmax. Increasing the luminal concentration of verapamil, a calcium channel blocker, significantly increased MS transport of 20 mM 3H‐L‐ leucine in the presence of 100 nM Mn2þ by reducing diffusional Ca2þ uptake into intestinal epithelial cells through verapamil‐sensitive channels. A model is proposed supporting the concept of molecular mimicry, whereby 3H‐L‐leucine enters lobster intestinal epithelial cells by one or more amino acid‐specific transporters and by a dipeptide‐like transporter that is capable of binding and transporting peptide molecular mimics (bis‐complexes) between Ca2þ or Mn2þ and 3H‐L‐leucine using the membrane potential as a major driving force for the transport event. According to the model, Ca2þ entry through apical Ca2þ channels regulates the magnitude of the membrane potential and therefore the size of the driving force for bis‐complex uptake. J. Exp. Zool. 321A: 135–143, 2014. © 2013 Wiley Periodicals, Inc. How to cite this article: Abdel‐Malak R, Ahearn GA. 2014. Regulation of transmural transport of amino acid/metal conjugates by dietary calcium in crustacean digestive tract. J. Exp. Zool. 321A:135–143.
Amino acids are transported across digestive organ luminal membranes of most organisms by the combination of secondary active transport processes and facilitated diffusion mechanisms. Because of the chemical diversity of this group of organic molecules, a variety of specific transport proteins have evolved to accommodate charge and structural differences of both essential and non‐essential amino acids (Kilberg et al., '93; Bröer, 2008). In recent years a parallel transport pathway has been described for amino acid accumulation by red blood cells (Aiken et al., '92; Horn et al., '95; Horn and Thomas, '96) and epithelial cells of vertebrate digestive (Tacnet et al., '90, '93; Glover and Hogstrand, 2002a,b;
Grant sponsor: USDA National Institute of Food and Agriculture (via Agriculture and Food Research Initiative Competitive grant); grant number: 2010‐65206‐20617. Correspondence to: Gregory A. Ahearn, Department of Biology, University of North Florida, 4567 St. Johns Bluff Road, South Jacksonville, FL 32224. E‐mail: [email protected] Received 15 July 2013; Revised 3 October 2013; Accepted 15 October 2013 DOI: 10.1002/jez.1843 Published online 19 November 2013 in Wiley Online Library (wileyonlinelibrary.com).
© 2013 WILEY PERIODICALS, INC.
136 Glover et al., 2003; Glover and Wood, 2008a,b) and renal organs (Zalups and Barfuss, 2002; Zalups and Bridges, 2010; Bridges and Zalups, 2010), which involves chelation between amino acids and divalent cations in solution, followed by transmembrane transport of the entire bis‐complex into the cytoplasm of the receiving cells. Several studies over the last 25 years have suggested that crustacean gastrointestinal tract organs possess a range of specific amino acid transport proteins (Ahearn, '88; Ahearn and Clay, '88a; Wright and Ahearn, '97) and also appear to have the ability to transport bis‐complexes between luminal amino acids and dietary metals (Conrad and Ahearn, 2005, 2007; Mullins and Ahearn, 2008; Obi et al., 2011; Simmons et al., 2012). Early studies described an apparent dipeptide transporter in brush border membrane (BBM) vesicles of lobster hepatopancreas (Thamotharan and Ahearn, '96) that displayed a number of physiological characteristics in common with PEPT1, a thoroughly investigated vertebrate peptide transport system (Daniel and Rubio‐Aliaga, 2003; Herrera‐Ruiz and Knipp, 2003; Daniel, 2004; Verri et al., 2010). Experiments with perfused lobster intestine suggested that transmural 14C‐glycylsarcosine transport was unaffected by luminal L‐leucine or L‐histidine alone, but was abolished by the presence of bis‐complexes involving both L‐leucine and zinc or L‐ histidine and zinc (Conrad and Ahearn, 2005). These results suggest that both the dipeptide and bis‐complexes appear to use the same carrier system. Transport of 65Zn2þ across lobster intestine was doubled in the presence of luminal L‐histidine, suggesting that both metal and amino acid act synergistically to enhance their individual absorption as a result of bis‐complex transport (Conrad and Ahearn, 2007). More recent work has indicated that bis‐complex transport appears to be a wide‐spread phenomenon in crustacean digestive organs involving Zn2þ, Cu2þ, and Mn2þ and both L‐histidine and L‐leucine and may play an important quantitative role in essential amino acid and metal absorption in these invertebrate animals (Obi et al., 2011; Simmons et al., 2012). The present investigation examines the interaction between two stimulatory divalent cations, zinc, and calcium, present simultaneously, on transmural transport of 3H‐L‐leucine in lobster intestine. Results suggest potential inhibitory interactions between different bis‐complexes for binding to a shared carrier protein, and regulation of bis‐complex transport rates by modification of the cellular resting potential as a driving force for conjugate transport by cation flow through calcium channels.
MATERIALS AND METHODS Live, male American lobsters, Homarus americanus, were maintained at 15°C in filtered saltwater and fed frozen mussel meat prior to their use in experiments. A physiological saline that approximated the ionic strength and composition of lobster hemolymph was utilized in experiments. The salt concentrations consisted of 420 mM NaCl, 25 mM CaCl2, 10 mM KCl, 8.4 mM J. Exp. Zool.
ABDEL‐MALAK AND AHEARN Na2SO4, and 30 mM HEPES at pH 7.0. To examine in vitro transmural mucosal to serosal (MS) movement of 3H‐L‐leucine across the lobster intestine, the intestine of a live lobster was flushed and ligated onto 18–20 gauge stainless steel needles and attached firmly with surgical thread in a perfusion apparatus that contained 35 mL of the physiological saline (serosal medium; Ahearn and Maginniss, '77; Obi et al., 2011). The same saline was perfused through the intestine and served as the mucosal medium using a peristaltic pump (Instech Laboratories, Inc., Plymouth Meeting, PA, USA) at a flow rate of 0.380 mL/min (Conrad and Ahearn, 2005). Transmural transport of [2,3,4,5‐3H]‐L‐leucine (American Radiochemicals, Inc. (St. Louis, MO, USA), from mucosa to serosa (MS) was measured by perfusing the physiological saline containing a fixed concentration of 3H‐L‐leucine and variable concentrations of calcium or manganese through the intestine for periods of time from 30 to 150 min. Every 5 min during a perfusion procedure, triplicate 200 mL samples of the serosal medium were withdrawn and added to scintillation fluid (Scinti‐Vase) for radioactivity counting (Beckman LS‐6500 liquid scintillation counter, Beckman Coulter Co., USA) followed by addition of an equal volume of saline to the bath to maintain its total volume through the experiment. A given experimental treatment was perfused through an intestine for a period of 30 min. Use of multiple experimental treatments involved the sequential 30 min perfusion of each treatment for periods of time up to 150 min. Regression lines were drawn through the resulting 30 min data obtained from sequential sampling of the serosal medium to obtain the rate of isotope transfer, providing an index of the rate of transmural amino acid flux during the test period. When variable concentrations of calcium or manganese were perfused through an intestine, choline was substituted for either divalent cation. Experiments using the calcium channel blocker, verapamil, involved variable concentrations of the inhibitor added to the perfusate in the presence of 100 nM manganese and the presence or absence of 25 mM calcium. Transmural flux rates of 3H‐L‐leucine were calculated using the diameter and length of the experimental intestine to estimate the intestinal surface area: A ¼ pdl, where “d” is the diameter and “l” is the length of the acquired intestine (Obi et al., 2011). To measure the total radioactive counts of 3H‐L‐leucine, the background counts per minute (cpm) were subtracted from the measured cpm values obtained from the scintillation counter. Specific activities of radioactive samples (e.g., cpm/pmol 3H‐L‐leucine) were used to estimate the amino acid fluxes across the tissue. 3H‐L‐leucine transmural flux rates were expressed as mean nmoles/cm2 min or mean pmoles/cm2 min. Experiments were carried out in triplicate with three animals per experiment, and the resulting flux rates and kinetic constants were collected for statistical analysis. Linear regression and hyperbolic analyses were executed using Sigma Plot 10.0 software (Obi et al., 2011). Statistical differences between treatment means were examined using Student's t‐test
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or ANOVA and were considered statistically different when P < 0.05.
RESULTS Effects of Luminal Mn2þ and Mn2þ þ Ca2þ on the Time Course of 3 H‐L‐Leucine MS Transport Effects of variations in luminal concentration of Mn2þ alone, or in combination with luminal Ca2þ, were examined during transmural MS (mucosal to serosal) transport of 3H‐L‐leucine concentrations ranging from 0.5 to 10 mM. Experiments were performed with a pH of 7.0 on both mucosal and serosal sides of the intestine to lessen possible effects of a proton gradient on the transport system. Figure 1A shows the effect of 20 mM luminal Mn2þ on steady‐state MS transport of 3H‐L‐leucine at varying luminal amino acid concentrations (JL ¼ 0.33 0.02 to 6.95 0.82 nmol/cm2 min; where JL is unidirectional MS flux). Results suggest that over 150 min of perfusion, a gradual increase in amino acid transfer (increase in transport slopes at each amino acid concentration) from lumen to bath occurred. During the transitions between increasing luminal amino acid concentrations vertical transport displacements were observed that likely reflect transient tissue adjustments to the novel substrate concentrations. All steady‐state fluxes (slopes) were significantly different than zero (P < 0.05). Figure 1B shows the effects of increasing mucosal 3H‐L‐leucine concentration (0.50–10 mM), on MS fluxes of 3H‐L‐leucine in saline consisting of 20 mM Mn2þ and 25 mM Ca2þ. At each luminal concentration of amino acid, the MS fluxes measured in the presence of both divalent cations were significantly lower (P < 0.05) than those obtained in the absence of luminal calcium. In the latter experimental condition, slopes (e.g., fluxes) increased from 0.08 0.01 to 2.94 0.71 nmoles/cm2 min with all values significantly different than zero (P < 0.05). Comparison of the results in Figure 1A and B, it is clear that addition of 25 mM luminal calcium to a perfusate containing radiolabeled amino acid and a fixed concentration of Mn2þ (20 mM) led to a significant reduction in the rate of transport compared to values obtained without the addition of calcium. Effects of Luminal Ca2þ and Mn2þ on the Kinetics of 3H‐L‐Leucine MS Transport Transmural MS transport of 3H‐L‐leucine was measured in the presence of 20 mM Mn2þ alone, or with 25 mM Ca2þ in addition to 20 mM Mn2þ, to assess the effects of these divalent cations on the kinetic constants of amino acid transport. As displayed in Figure 2, the transport of 3H‐L‐leucine across lobster intestine was a sigmoidal function of luminal amino acid concentration at each experimental condition used, suggesting the presence of carrier‐ mediated transport for this amino acid following the Hill equation for multisite binding cooperativity:
Figure 1. Time course of MS (mucosal to serosal) steady‐state transport at pH 7.0 of 3H‐L‐leucine concentration (0.50, 1, 2.5, 5, and 10 mM) across representative lobster intestines in the presence of 20 mM manganese with no calcium (A) and in the presence of 25 mM calcium (B). Data are means of three replicate samples at each time interval for each leucine concentration. Lines drawn through the data points at each leucine concentration over the 150‐min time course were drawn by linear regression analysis using Sigma Plot 10.0 and the values presented on the figure represents slopes 1 SEM. Vertical displacements of steady‐state fluxes at each treatment are due to transient adjustments of the tissue to increasing perfused 3H‐L‐leucine concentrations.
J¼
J max ½leucinen K nt þ ½leucinen
ð1Þ
where J is 3H‐L‐leucine transport in nmol/cm2 min, Jmax is maximal 3H‐L‐leucine transport, K nt is an apparent binding affinity of the L‐leucine transporter adjusted to accommodate multisite substrate binding cooperativity, and is described as the [L‐leucine] at 1/2Jmax, [leucine]n is the luminal concentration of J. Exp. Zool.
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ABDEL‐MALAK AND AHEARN
Figure 2. Transmural MS steady‐state transport of 3H‐L‐leucine at pH 7.0 as a function of L‐leucine concentration (0.50, 1, 2.5, 5, and 10 mM) in the presence of luminal 20 mM manganese and either the presence or absence of 25 mM calcium on both sides of lobster intestine. Symbols are means 1 SEM of three independent experiments involving different test animals. Sigmoidal curves and resulting kinetic constants (Table 1) were obtained using Sigma Plot 10.0.
amino acid in mM, and n is the Hill coefficient which is an estimate of the approximate transport stoichiometry. The control condition in this experiment included only 20 mM Mn2þ in the perfusate as this metal has previously been shown to stimulate MS transport of amino acids in crustacean intestine (Simmons et al., 2012). Under these conditions the kinetic constants for 3H‐L‐leucine transport across the tissue were K nt ¼ 3.98 0.59 mM, Jmax ¼ 8.52 0.75 nmol/cm2 min, and n ¼ 1.57 0.18. Addition of 25 mM Ca2þ to the incubation medium with 20 mM Mn2þ resulted in a significant decrease in 3H‐ L‐leucine Jmax (P < 0.01), without effect on amino acid transport K nt (P > 0.05). In both treatments, Hill coefficients between 1.5 and 2.0 were obtained and suggest an unchanged transport stoichiometry of 2 Leucine/1 divalent cation. The respective kinetic constants from these two experimental conditions are displayed in
Figure 3. Transmural MS steady‐state transport of 20 mM 3H‐L‐ leucine at pH 7.0 as a function of luminal calcium concentration (0, 0.25, 0.50, 1, and 5 mM). Data were curve‐fit with Sigma Plot 10.0. Symbols are means 1 SEM of three independent experiments (e.g., three animals). Resulting kinetic constants are displayed on the figure.
Table 1. As Figure 2 and Table 1 indicate, calcium only had a significant effect on amino acid transport in the presence of Mn2þ by decreasing the rate of 3H‐L‐leucine transport across the tissue without apparently influencing its binding properties or its transport stoichiometry. Effect of Variable Calcium Concentration on MS Transport of H‐L‐leucine Transport Because calcium was found to inhibit 3H‐L‐leucine MS flux in the presence of the stimulating divalent cation, Mn2þ (Fig. 2 and Table 1), the effect of variable luminal concentrations of calcium on amino acid transport was assessed in the absence of Mn2þ. As displayed in Figure 3, MS transport of 20 mM 3H‐L‐leucine was a hyperbolic function of perfusate calcium concentration and followed a modification of the Michaelis–Menten equation below: 3
Table 1. Effect of 20 mM Mn2þ and 25 mM Ca2þ on the kinetic constants of MS 3H‐L‐leucine transport across lobster intestine. Treatment 20 mM Mn (control) 20 mM Mn2þ þ 25 mM Ca2þ 2þ
L‐leucine
K nt (mM)
Jmax (nmol/cm2 min)
n‐value
3.98 0.59 4.80 0.10
8.52 0.75 4.85 0.07
1.57 0.18 1.81 0.13
concentration range: 0, 0.5, 1, 2.5, 5.0, and 10 mM. Values are means 1 SEM. Signifies value is significantly different from control at P < 0.01.
J. Exp. Zool.
REGULATION OF TRANSMURAL TRANSPORT J¼
J max ½Ca2þ K Ca þ ½Ca2þ
139 ð2Þ
where J is 3H‐L‐leucine transport in pmol/cm2 min, Jmax is maximal transport of 20 mM 3H‐L‐leucine, KCa is an apparent calcium binding constant and is described as the [Ca2þ] at 1/2Jmax of 20 mM 3H‐L‐leucine transport, and [Ca2þ] is the luminal calcium concentration in mM. The kinetic constants for this experiment were KCa ¼ 0.81 0.23 mM Ca2þ and Jmax ¼ 18.74 1.67 pmol/cm2 min. The hyperbolic nature of the data in Figure 3 suggests that calcium stimulates amino acid transport as a result of the cation binding to a saturable co‐transport carrier‐mediated possess with a finite number of binding sites. Figure 3 indicates a small, but significant (P < 0.05) vertical axis intercept at 0 mM calcium, suggesting the possible presence of one or more calcium‐independent transport processes for 3H‐L‐leucine in this tissue. Luminal Ca2þ is a Non‐Competitive Inhibitor of Mn2þ Activation of H‐L‐Leucine MS Transport The effects of variable luminal Mn2þ concentration on 20 mM 3 H‐L‐leucine MS transport in the presence and absence of 25 mM Ca2þ is shown in Figure 4. Amino acid transport in both experimental conditions was a hyperbolic function of luminal Mn2þ and followed Equation (2) with Mn2þ substituting for Ca2þ in the equation. It is clear from Figure 4 that addition of 25 mM Ca2þ to the perfusate containing variable [Mn2þ] significantly (P < 0.01) reduced amino acid transport over the 3
Figure 4. Transmural MS steady‐state transport of 20 mM 3H‐L‐ leucine at pH 7.0 across lobster intestine as a function of luminal manganese concentration (0, 25, 50, 100, and 250 nM), in the presence and absence of 25 mM calcium. Data were curve‐fit with Sigma Plot 10.0 and resulting kinetic constants are displayed in Table 2. Symbols are means 1 SEM of three independent experiments (e.g., three animals).
Table 2. Effect of 25 mM Ca2þ on kinetic constants of Mn2þ‐ stimulated 20 mM 3H‐L‐leucine MS transport across lobster intestine.
Treatment 20 mM Mn2þ (control) 20 mM Mn2þ þ 25 mM Ca2þ
KMn (nM)
Jmax (pmol/cm2 min)
23.48 5.68 28.93 10.10
14.03 0.88 5.45 0.56
Manganese concentration range: 0, 25, 50, 100, and 250 nM. Values are means 1 SEM. Signifies value is significantly different from control at P < 0.01.
range of Mn2þ concentrations used. Kinetic constants for 20 mM 3 H‐L‐leucine MS transport in the presence and absence of luminal calcium are displayed in Table 2 and indicate that the effect of adding calcium was to significantly (P < 0.01) reduce MS amino acid transport Jmax, without significantly (P > 0.05) affecting the binding properties of Mn2þ to the transport system. This result suggests that calcium was acting as a non‐ competitive inhibitor of Mn2þ activation of amino acid transport. As in Figure 3, at 0 mM Mn2þ, a significant vertical axis intercept was obtained for both experimental conditions, suggesting the possible presence of undescribed manganese‐ independent transport mechanisms. Calcium Regulates Mn2þ‐Stimulated 3H‐L‐Leucine MS Transport Through calcium Channels Because luminal calcium affected the rate of Mn2þ‐stimulated 3H‐ L‐leucine transport across lobster intestine rather than the binding properties of Mn2þ or amino acid to a shared carrier system, an experiment was conducted to clarify the nature of the reduction in Mn2þ‐enhanced L‐leucine MS transport. Varying concentrations of the calcium channel blocker, verapamil (0–1,000 mM), were perfused through lobster intestine with 20 mM 3H‐L‐leucine and 100 nM Mn2þ with and without the addition of 25 mM Ca2þ for 30 min intervals as described in Figure 1. 3H‐L‐leucine transport rates over five 30‐min incubation periods (one period per [verapamil]) were measured and the results are displayed in Figure 5. The data in this figure show a gradual, but significant, increase in 20 mM 3H‐L‐leucine MS transport across lobster intestine as perfusion concentrations of verapamil were elevated, both in the presence and absence of luminal Ca2þ (25 mM calcium: P < 0.01, F ¼ 17.1, df ¼ 14; 0 mM calcium: P < 0.038, F ¼ 3.9, df ¼ 14; ANOVA). Results in Figure 5 suggest that Mn2þ and/or Ca2þ in the intestinal lumen may enter epithelial apical calcium channels partially depolarizing the membrane potential and decrease its apparent driving force for Mn2þ‐coupled 3H‐L‐leucine transport. Increasing verapamil concentrations, as provided in this study, apparently block increasing numbers of these channels, reducing the depolarizing effect of the luminal divalent cations J. Exp. Zool.
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Figure 5. Transmural MS steady‐state 30‐min transport of 20 mM 3 H‐L‐leucine at pH 7.0 across lobster intestine as a function of luminal verapamil concentration (0, 100, 250, 500, and 1,000 mM) in the presence of 100 nM luminal manganese and either 0 or 25 mM calcium on both sides of perfused intestine. Columns are means 1 SEM of three independent experiments (e.g., three animals).
and allowing greater carrier‐mediated transmural MS transport as a result. Therefore, the apparent non‐competitive inhibition of calcium on Mn2þ‐stimulated 3H‐L‐leucine MS flux in Figure 4 may have been due to calcium depolarization of the apical membrane potential. Results in Figure 5 also show that 100 nM Mn2þ by itself appears capable of this depolarizing effect on its own co‐transport with 3H‐L‐leucine, but because its concentration was much lower than that of calcium in the present set of experiments, its regulatory role would presumably be less.
DISCUSSION In the crustacean digestive tract, amino acids are transported from lumen to blood by first passing across the epithelial apical membrane into the cytoplasm by a wide variety of specific amino acid transport proteins (Ahearn, '88; Ahearn and Clay, '88a; Kilberg et al., '93; Wright and Ahearn, '97; Bröer, 2008) that are either secondary active transport systems, using the transmembrane electrochemical gradient to power their accumulation in the cytosol, or facilitated diffusion processes employing their concentration gradient to enter the cells. Less is known about amino acid efflux from epithelium to the blood across gut basolateral membranes in these arthropods, but if the vertebrate paradigm is followed, diffusion using a variety of carrier proteins on this cellular pole likely accounts for transfer to the circulation. From what is currently known from recent investigations using crustacean hepatopancreatic BBM vesicles and perfused intestine, J. Exp. Zool.
ABDEL‐MALAK AND AHEARN a significant transepithelial transport of amino acids occurs from lumen to blood across the digestive tract epithelia, providing both essential and non‐essential substrates for protein metabolism (Wright and Ahearn, '97; Conrad and Ahearn, 2005, 2007; Mullins and Ahearn, 2008; Obi et al., 2011; Simmons et al., 2012). In recent years a parallel pathway for amino acid absorption across crustacean intestine has been described that involves the apparent transport of amino acid/metal chelates by dipeptide transport proteins and which may significantly supplement the net movements of these organic solutes by amino acid‐specific transport systems (Conrad and Ahearn, 2005, 2007; Mullins and Ahearn, 2008; Obi et al., 2011). Formation of amino acid/metal chelates, called bis‐complexes, and their transport across gastrointestinal membranes was first described for pig intestine (Tacnet et al., '90, '93) and later found to take place in fish digestive tracts (Glover and Hogstrand, 2002a,b; Glover et al., 2003; Glover and Wood, 2008a,b). In both of these vertebrate groups zinc and copper were found to bind to L‐histidine, L‐cysteine, and other amino acids in solution and be transported into the gut epithelium by an undisclosed transport mechanism that appeared distinct from specific amino acid transporters. At the time it was not known how widespread this phenomenon was, how many amino acids might use it, how many different metal cations might form such bis‐complexes, how the transport rates of these chelates might be regulated, and what the quantitative importance such a transport system might have for total net absorption of essential amino acids. Conrad and Ahearn (2005) showed that transmural mucosa to serosa (MS) 3H‐L‐histidine transport across perfused lobster intestine was increased by a factor of 3 by addition of luminal zinc. This enhanced amino acid transport, across the gut in the presence of metal was eliminated by addition of mucosal L‐leucine. Zinc‐stimulated 3H‐L‐histidine transport was similarly inhibited by perfusion with the dipeptide, glycylsarcosine. This study proposed that all three organic solutes (L‐histidine, L‐leucine, and glycylsarcosine) share a common carrier that normally transport dipeptides, but would also accept amino acid/metal bis‐complexes as well. In other experiments, transmural MS flux of 65Zn2þ was doubled when luminal L‐histidine was present, but was significantly abolished by addition of Cu2þ to the perfusate (Conrad and Ahearn, 2005, 2007). These results were interpreted as indicating that both Zn2þ and Cu2þ are able to form bis‐complexes with L‐ histidine and complete for a shared transport system. Subsequent studies with lobster intestine, using 3H‐leucine, indicated that the metals, Zn2þ, Cu2þ, and Mn2þ, are all able to stimulate amino acid MS transport in a hyperbolic fashion with the transport system having the highest apparent binding affinity for Mn2þ (Mullins and Ahearn, 2008; Obi et al., 2011). The use of several divalent metals Zn2þ, Cu2þ, Mn2þ, Cd2þ, and Co2þ, as stimulatory co‐ transporters of amino acids, was extended to both 3H‐leucine and 3 H‐L‐histidine in the hepatopancreas of the marine penaeid shrimp, Litopenaeus setiferus (Simmons et al., 2012), suggesting that
REGULATION OF TRANSMURAL TRANSPORT metal‐enhanced amino acid absorption across crustacean gut may be a general process among digestive organs of decapod crustaceans. These studies proposed that a peptide transport protein, like PEPT1, may be able to bind, and subsequently transport, bis‐complexes involving two amino acids bonded to a divalent cation (e.g., Leu‐Zn‐Leu) and appearing like a dipeptide as a result of molecular mimicry. Molecular mimicry has been suggested to account for the movement of one solute (the mimic) that chemically resembles another (the model) by transporters that typically are restricted in their specificity to the model, but will accept, under certain conditions, other molecular forms that would normally not be accommodated. Work with mammalian intestine and renal proximal tubular cells has described the transport of mercuric complexes with cysteine, glutathione, and other substrates (Zalups and Barfuss, 2002; Bridges and Zalups, 2010; Zalups and Bridges, 2010). These authors suggest that a number of known transport systems, that normally transport different model substrates, will also accept metal complexes involving mercury as a result of molecular mimicry. Some of the specific transporters that were identified as potential systems involved with such mimicry were the Naþ‐independent System bo,þ and a variety of Naþ‐dependent processes as well (Zalups and Bridges, 2010). Furthermore, these authors suggested that mercuric complexes involving dipeptides (e.g., Cys‐Gly‐Hg‐Cys‐Gly) might be able to utilize a peptide transporter that would normally be restricted to dipeptide and tripeptide substrates (Zalups and Bridges, 2010). Additional transporters that were suggested as potential transport sites of metallic complexes with amino acids and other substrates included members of the Organic Anion Exchanger family (e.g., OAT1 and/or OAT3) (Zalups and Barfuss, 2002; Zalups and Bridges, 2010). Therefore, it appears that a significant number of different identified membrane carrier systems may be involved in the movement of metal/organic solute complexes across cell membranes as a result of molecular mimicry. Results of the present investigation support previous studies with crustacean digestive tract (Conrad and Ahearn, 2005, 2007; Mullins and Ahearn, 2008; Obi et al., 2011) and buttress the notion that molecular mimicry appears to occur in crustacean intestine and hepatopancreas and that this process may make a significant contribution to net transmembrane and transcellular transport of essential dietary elements such as amino acids and metals. Figures 3 and 4 suggest that both calcium and manganese are able to support carrier‐mediated 3H‐L‐leucine MS transport by lobster intestine. These results are similar to those obtained for zinc and copper stimulation of 3H‐L‐leucine transport by the same lobster tissue (Obi et al., 2011), and for both manganese and zinc stimulation of 3H‐L‐histidine transport by shrimp (L. setiferus) hepatopancreatic BBM vesicles (Simmons et al., 2012). It, therefore, appears that in crustaceans, a number of different divalent cations are able to interact with amino acids in solution to form bis‐complexes that are transported into and through
141 epithelial cells of both the hepatopancreas and intestine of these animals. Previous experiments with lobster intestine using the dipeptide, glycylsarcosine, have shown that the MS transport of bis‐complexes involving zinc and 3H‐L‐leucine is significantly reduced compared to control conditions lacking the dipeptide (Conrad and Ahearn, 2005; Obi et al., 2011). These data suggest that both the dipeptide and amino acid/metal bis‐complexes appear to use the same transport system such as a PEPT1 dipeptide transporter. In the present study, calcium was a non‐competitive inhibitor of manganese‐stimulated 3H‐L‐leucine MS transport, exerting a significant (P < 0.01) effect on the maximal rate of amino acid transport, but a non‐significant (P > 0.05) effect on manganese binding properties (Fig. 4 and Table 2). Figure 5 shows that addition of the calcium channel blocker, verapamil, to the perfusate containing 3H‐L‐leucine and manganese, with or without calcium, resulted in an increase in amino acid MS transport as the inhibitor concentration was elevated. These results suggest that the non‐ competitive inhibitory effect of luminal calcium on 3H‐L‐leucine MS transport, in the presence of manganese, may have been due to calcium entry into intestinal epithelial cells via verapamil‐sensitive calcium channels, depolarizing the membrane potential, and reducing membrane‐potential‐dependent uptake of bis‐complexes between 3H‐L‐leucine and Mn2þ. Therefore, in vivo, calcium flow through apical calcium channels may regulate the rate of bis‐ complex transport via PEPT1, or other transporters involving divalent cations and amino acids. Figure 6 is a tentative working model of proposed calcium regulation of bis‐complex transport involving a divalent cation and dietary amino acids and an apparent dipeptide transporter. This figure also shows the presence of an independent amino acid‐ specific L‐leucine transporter, which has been previously described (Ahearn and Clay, '88b) on the digestive system apical membrane and which operates in parallel with molecular mimicry occurring by an apparent dipeptide transporter. All divalent cations studied to date (e.g., Zn2þ, Cu2þ, Mn2þ, and Ca2þ) appear capable of forming bis‐complexes with several amino acids in the crustacean intestinal lumen and sharing a common membrane transporter with each other and with the dipeptide, glycylsarcosine. In the present study, Hill coefficients of approximately 2.0 between L‐leucine and Mn2þ (Fig. 2 and Table 1) provide kinetic stoichiometric evidence for transport of 2 amino acids with a single metal ion over the amino acid concentration range used. The apparent binding affinity constants (KCa and KMn) for calcium and manganese are very different, with the membrane exhibiting several orders of magnitude greater apparent binding affinity for Mn2þ than for Ca2þ (Fig. 3 and Table 2). This highly significant difference between the binding properties of the two cations is likely the reason why 25 mM calcium did not appear to be an apparent competitive inhibitor of manganese binding to the transport system in Figure 4 and Table 2, although this might be more apparent at higher calcium concentrations. Figure 6 shows that luminal calcium J. Exp. Zool.
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Figure 6. Proposed transport processes of intestinal epithelial cells of lobster, displaying three different brush border membrane (BBM) transport proteins: (1) Dipeptide/bis‐complex transporter; (2) Amino acid transporter; and (3) Verapamil‐inhibited calcium channel that may accept Ca2þ, Mn2þ, and possibly other divalent cations. Luminal amino acids may be transported into cells by #1 as molecular mimics of dipeptides (e.g., bis‐complexes), and/or by #2 which does not accept bis‐complexes. The proposed calcium channel allows the diffusional entry of divalent cations into the cytoplasm, depolarizing the membrane potential, lowering the electrochemical potential across the membrane, and reducing the uptake of bis‐complexes involving amino acids. The cytoplasmic fates of intracellular bis‐complexes currently are unknown, but it is likely that steady‐state efflux of radiolabeled cytoplasmic amino acids to the serosal medium may occur either as bis‐complexes or as free amino acids.
appears to regulate the transport rates of bis‐complexes across intestine apical membranes by using channels whose openings can presumably be controlled and thereby control calcium depolarization of the apical membrane potential. Figure 5 indicates that luminal Mn2þ alone was apparently able to enter calcium channels, in the absence of calcium, and, to a lesser extent, influence the membrane potential driving force for bis‐complex uptake. Factors affecting the extent of calcium channel opening and its quantitative participation in bis‐complex transport through influences on the magnitude of the trans‐apical membrane potential are still unclear.
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