Interaction of beta-lactam antibiotics with H+/peptide cotransporters in rat renal brush-border membranes

Two H+/peptide cotransporters, PEPT1 and PEPT2, are expressed in the kidney, mediating the renal tubular reabsorption of oligopeptides and beta-lactam antibiotics. We examined the interactions of beta-lactam antibiotics with peptide transporters in rat renal brush-border membranes by evaluating the inhibitory potencies of the antibiotics against glycylsarcosine transport. Western blot analysis revealed that PEPT1 and PEPT2 were expressed in the renal brush-border membranes with the apparent molecular masses of 75 and 105 kDa, respectively. Using renal brush-border membrane vesicles, the uphill transport of glycylsarcosine was observed in the presence of an inward H+ gradient and an inside-negative membrane potential. Two transport systems with high affinity (Km of 50 microM) and low affinity (Km of 1.2 mM) appeared kinetically to mediate the glycylsarcosine uptake. The inhibition constants of the antibiotics for glycylsarcosine transport were more closely correlated with those in stable LLC-PK1 cells transfected with rat PEPT2 rather than PEPT1 cDNA. The beta-lactam antibiotics with an alpha-amino group showed trans-stimulation effects on the glycylsarcosine uptake, suggesting that these antibiotics and glycylsarcosine share a common peptide transporter. However, the antibiotics lacking an alpha-amino group failed to show the trans-stimulation effect. It is concluded that amino-beta-lactam antibiotics at therapeutic concentrations interact predominantly with PEPT2 localized in the brush-border membranes of rat kidney.     

Hepatic sinusoidal membrane transport of anionic drugs mediated by anion transporter Npt1

The purpose of our study was to establish the localization of the anion transporter Npt1 in liver and the relevance of Npt1 to carrier-mediated hepatic transport of beta-lactam antibiotics. Immunocytochemical examination of mouse liver with antiserum for Npt1 showed basolateral (sinusoidal) membrane localization. Function of Npt1 was characterized in Xenopus laevis oocytes. Injection of in vitro-transcribed cRNA into oocytes resulted in an increased uptake of [14C]benzylpenicillin (PCG). The Npt1-mediated uptake was saturable with a Michaelis constant (Km) of 0.46 +/- 0.18 mM and a maximum rate (Vmax) of 46.6 +/- 8.5 pmol/60 min/oocyte, and the uptake of [14C]PCG was independent of Na+ and pH, but dependent on chloride ion. Npt1-mediated [14C]PCG uptake was inhibited by several beta-lactam antibiotics and probenecid. Oocytes injected with Npt1-cRNA demonstrated significantly enhanced transport activity for other anionic compounds such as [14C]faropenem, [14C]foscarnet and [3H]mevalonic acid, as well as [14C]PCG, compared with water-injected oocytes. In conclusion, Npt1 is suggested to participate in hepatic sinusoidal membrane transport of organic anions such as beta-lactam antibiotics as well as inorganic anions for the efflux from hepatocyte-to-blood direction.     

Interaction of beta-lactam antibiotics with histidine residue of rat H+/peptide cotransporters, PEPT1 and PEPT2

Peptide transporters mediate the H+-coupled uphill transport of oligopeptides and peptide-like drugs such as beta-lactam antibiotics in the intestinal and renal brush-border membranes. Two H+/peptide cotransporters, PEPT1 and PEPT2, have been cloned and functionally characterized. In this study, we examined the interaction of the dipeptides and beta-lactam antibiotics with the histidine residue of rat PEPT1 and PEPT2 transfected into the renal epithelial cell line LLC-PK1. Diethylpyrocarbonate (DEPC), which is a histidine residue modifier, abolished the glycylsarcosine uptake by both transfectants. The DEPC-induced inhibition of glycylsarcosine uptake via PEPT1 or PEPT2 was attenuated by an excess of dipeptide or aminocephalosporin. In contrast, anionic cephalosporins without an alpha-amino group and bestatin, which is an antineoplastic drug with a beta-amino group, did not attenuate the DEPC-induced inactivation of PEPT1 and PEPT2. The DEPC inactivation of PEPT1 was almost prevented by various charged dipeptides, which suggests that the inability of the drugs without an alpha-amino group to prevent the DEPC inactivation was not due to their ionic charge. These findings suggest that the alpha-amino group of beta-lactam antibiotics interacts with the histidine residue of PEPT1 and PEPT2 and may be involved in the mechanism of substrate recognition by the peptide transporters.     

Expression of rat liver cell membrane transporters for thyroid hormone in Xenopus laevis oocytes

The present study was conducted to explore the possible use of Xenopus laevis oocytes for the expression cloning of cell membrane transporters for iodothyronines. Injection of stage V-VI X. laevis oocytes with 23 ng Wistar rat liver polyadenylated RNA (mRNA) resulted after 3-4 days in a highly significant increase in [125I]T3 (5 nM) uptake from 6.4 +/- 0.8 fmol/oocyte x h in water-injected oocytes to 9.2 +/- 0.65 fmol/oocyte x h (mean +/- SEM; n = 19). In contrast, [125I]T4 (4 nM) uptake was not significantly stimulated by injection of total liver mRNA. T3 uptake induced by liver mRNA was significantly inhibited by replacement of Na+ in the incubation medium by choline+ or by simultaneous incubation with 1 microM unlabeled T3. In contrast, T3 uptake by water-injected oocytes was not Na+ dependent. Fractionation of liver mRNA on a 6-20% sucrose gradient showed that maximal stimulation of T3 uptake was obtained with mRNA of 0.8-2.1 kilobases (kb). In contrast to unfractionated mRNA, the 0.7- to 2.1-kb fraction also significantly stimulated transport of T4, and it was found to induce uptake of T3 sulfate (T3S). Because T3S is a good substrate for type I deiodinase (D1), 2.3 ng rat D1 complementary RNA (cRNA) were injected either alone or together with 23 ng of the 0.8- to 2.1-kb fraction of rat liver mRNA. Compared with water-injected oocytes, injection of D1 cRNA alone did not stimulate uptake of [125I]T3S (1.25 nM). T3S uptake in liver mRNA and D1 cRNA-injected oocytes was similar to that in oocytes injected with mRNA alone, showing that transport of T3S is independent of the metabolic capacity of the oocyte. Furthermore, coinjection of liver mRNA and D1 cRNA strongly increased the production of 125I-, showing that the T3S taken up by the oocyte is indeed transported to the cell interior. In conclusion, injection of rat liver mRNA into X. laevis oocytes resulted in a stimulation of saturable, Na+-dependent T4, T3 and T3S transport, indicating that rat liver contains mRNA(s) coding for plasma membrane transporters for these iodothyronine derivatives.     

Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1

The proton-coupled intestinal dipeptide transporter, PepT1, has 707 amino acids, 12 putative transmembrane domains (TMD), and is of importance in the transport of nutritional di- and tripeptides and structurally related drugs, such as penicillins and cephalosporins. By using a combination of molecular modeling and site-directed mutagenesis, we have identified several key amino acid residues that effect catalytic transport properties of PepT1. Our molecular model of the transporter was examined by dividing it into four sections, parallel to the membrane, starting from the extracellular side. The molecular model revealed a putative transport channel and the approximate locations of several aromatic and charged amino acid residues that were selected as targets for mutagenesis. Wild type or mutagenized human PepT1 cDNA was transfected into human embryonic kidney (HEK293) cells, and the uptake of tritiated glycylsarcosine [3H]-(Gly-Sar) was measured. Michaelis-Menton analysis of the wild-type and mutated transporters revealed the following results for site-directed mutagenesis. Mutation of Tyr-12 or Arg-282 into alanine has only a very modest effect on Gly-Sar uptake. By contrast, mutation of Trp-294 or Glu-595 into alanine reduced Gly-Sar uptake by 80 and 95%, respectively, and mutation of Tyr-167 reduced Gly-Sar uptake to the level of mock-transfected cells. In addition, preliminary data from fluorescence microscopy following the expression of N-terminal-GFP-labeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced Vmax.     

Stoichiometry and kinetics of the high-affinity H+-coupled peptide transporter PepT2

Proton-coupled peptide transporters mediate the absorption of a large variety of di- and tripeptides as well as peptide-like pharmacologically active compounds. We report a kinetic analysis of the rat kidney high-affinity peptide transporter PepT2 expressed in Xenopus oocytes. By use of simultaneous radioactive uptake and current measurements under voltage-clamp condition, the charge to substrate uptake ratio was found to be close to 2 for both D-Phe-L-Ala and D-Phe-L-Glu, indicating that the H+:substrate stoichiometry is 2:1 and 3:1 for neutral and anionic dipeptides, respectively. The higher stoichiometry for anionic peptides suggests that they are transported in the protonated form. For D-Phe-L-Lys, the charge:uptake ratio averaged 2.4 from pooled experiments, suggesting that Phe-Lys crosses the membrane via PepT2 either in its deprotonated (neutral) or its positively charged form, averaging a H+:Phe-Lys stoichiometry of 1.4:1. These findings led to the overall conclusion that PepT2 couples transport of one peptide molecule to two H+. This is in contrast to the low-affinity transporter PepT1 that couples transport of one peptide to one H+. Quinapril inhibited PepT2-mediated currents in presence or in absence of external substrates. Oocytes expressing PepT2 exhibited quinapril-sensitive outward currents. In the absence of external substrate, a quinapril-sensitive proton inward current (proton leak) was also observed which, together with the observed pH-dependent PepT2-specific presteady-state currents (Ipss), indicates that at least one H+ binds to the transporter prior to substrate. PepT2 exhibited Ipss in response to hyperpolarization at pH 6.5-8.0. However, contrary to previous observations on various transporters, 1) no significant currents were observed corresponding to voltage jumps returning from hyperpolarization, and 2) at reduced extracellular pH, no significant Ipss were observed in either direction. Together with observed lower substrate affinities and decreased PepT2-mediated currents at hyperpolarized Vm, our data are consistent with the concept that hyperpolarization exerts inactivation effects on the transporter which are enhanced by low pH. Our studies revealed distinct properties of PepT2, compared with PepT1 and other ion-coupled transporters.     

hPepT1-mediated epithelial transport of bacteria-derived chemotactic peptides enhances neutrophil-epithelial interactions

Intestinal epithelial cells express hPepT1, an apical transporter responsible for the uptake of a broad array of small peptides. As these could conceivably include n-formyl peptides, we examined whether hPepT1 could transport the model n-formylated peptide fMLP and, if so, whether such cellular uptake of fMLP influenced neutrophil-epithelial interactions. fMLP uptake into oocytes was enhanced by hPepT1 expression. In addition, fMLP competitively inhibited uptake of a known hPepT1 substrate (glycylsarcosine) in hPepT1 expressing oocytes. hPepT1 peptide uptake was further examined in a polarized human intestinal epithelial cell line (Caco2-BBE) known to express this transporter. Epithelial monolayers internalized apical fMLP in a fashion that was competitively inhibited by other hPepT1 recognized solutes, but not by related solutes that were not transported by hPepT1. Fluorescence analyses of intracellular pH revealed that fMLP uptake was accompanied by cytosolic acidification, consistent with the known function of hPepT1 as a peptide H+ cotransporter. Lumenal fMLP resulted in directed movement of neutrophils across epithelial monolayers. Solutes that inhibit hPepT1-mediated fMLP transport decreased neutrophil transmigration by approximately 50%. Conversely, conditions that enhanced the rate of hPepT1-mediated fMLP uptake (cytosolic acidification) enhanced neutrophil-transepithelial migration by approximately 70%. We conclude that hPepT1 transports fMLP and uptake of these peptide influences neutrophil-epithelial interactions. These data (a) emphasize the importance of hPepT1 in mediating intestinal inflammation, (b) raise the possibility that modulating hPepT1 activity could influence states of intestinal inflammation, and (c) provide the first evidence of a link between active transepithelial transport and neutrophil-epithelial interactions.     

Molecular and cell biological analyses for intestinal absorption and renal excretion of drugs

Intestinal absorption and renal tubular secretion are transport processes determining the availability and the disposition of drugs in the body. In this review, our studies on the molecular and cell biological analyses of intestinal absorption and renal secretion of drugs are described. We evaluated the transepithelial transport and the cellular accumulation of peptide-like drugs such as beta-lactam antibiotics and bestatin (a dipeptide-like antineoplastic agent) in the human adenocarcinoma colon cell line, Caco-2, as an in vitro model for studying absorption mechanisms of these drugs. We have found that the transcellular transport of these peptide-like drugs is mediated by both the apically- and basolaterally-localized peptide transporters. To characterize molecular aspects of absorption of the peptide-like drugs, we studied cDNA cloning of H+/peptide cotransporters, PEPT1 and PEPT2, expressed in rats. The rat PEPT1 has been shown to mediate the H- coupled uphill transport of beta-lactam antibiotics across the brush-border membranes of the intestinal and renal epithelia. The rat PEPT2 is expressed predominantly in the kidney, but not in the intestine, mediating tubular reabsorption of the peptide-like drugs. We examined the transcellular transport of organic cations across monolayers of the kidney epithelial cell line, LLC-PK1. We have found that LLC-PK1 cells possess the H+/organic cation antiporter and the membrane potential-sensitive organic cation transporter in the apical and basolateral membranes, respectively, thereby tetraethylammonium (TEA) being transported unidirectionally from the basolateral to the apical side of the monolayers. We have isolated a cDNA encoding a rat kidney-specific organic cation transporter, OCT 2, which transports TEA in a H(+)-gradient independent manner, suggesting that OCT2 is localized to the basolateral membranes of renal tubular cells. In addition, a cDNA encoding a novel rat organic anion transporter, OAT-K1, has been cloned. OAT-K1 is expressed exclusively in the renal proximal tubules, and mediates the transport of methotrexate. Analyses of the molecular and cell biological mechanisms for drug absorption and secretion will provide information for the understanding of organ specific drug transport systems and for the development of drug design and/or drug delivery system.     

Overexpression of human intestinal oligopeptide transporter in mammalian cells via adenoviral transduction

PURPOSE: Our goals are to establish an in vitro screening system and to evaluate a new approach in improving oral absorption of peptides and peptide-like drugs by overexpression of the human intestinal oligopeptide transporter (hPepT1). This study characterizes the expression of hPepT1 in human intestinal Caco-2 cells, rat intestinal epithelial cells (IEC-18), and human cervix epithelial cells (Hela) after adenoviral transduction. METHODS: A recombinant replication-deficient adenovirus carrying the hPepT1 gene was made and used as a vector for the expression of hPepT1. The increase in the uptake permeability of cephalexin and Gly-Sar was determined. The effects of time, dose, apical pH, and substrate specificity were evaluated. RESULTS: A significant increase in the uptake permeability of Gly-Sar and cephalexin was found in all three cell lines after viral transduction. The increase of Gly-Sar permeability in Hela. IEC-18, and Caco-2 cells was 85-, 46-, and 15-fold respectively. Immunoblotting using an antibody against hPepT1 detected high levels of a 85-98-kDa protein in all three infected cell lines. Substrate permeability was dependent on time of infection, inward pH gradients, and multiplicity of infection (MOI). Decreased infectivity and lower hPepT1 expression were observed in differentiated Caco-2 cells. The uptake was inhibited by dipeptides and beta-lactam antibiotics but not amino acids. CONCLUSIONS: Adenoviral infected Hela cells displayed a pronounced level of hPepT1 expression with a low background and high specificity to dipeptides. These features make this system a useful tool for screening of potential substrates. The success of overexpression of hPepT1 in Caco-2 and IEC-18 cells may lead to a novel approach in improving oral absorption of peptides and peptidornimetic drugs.     

Characterization of stably transfected kidney epithelial cell line expressing rat H+/peptide cotransporter PEPT1: localization of PEPT1 and transport of beta-lactam antibiotics

We established stably transfected LLC-PK1 cells expressing the rat H+/peptide cotransporter PEPT1 (designated LLC-rPEPT1) and examined membrane localization and uptake by rat PEPT1 of oral beta-lactam antibiotics. The LLC-rPEPT1 cells expressed a novel PEPT1 protein with an apparent molecular mass of 75 kdaltons, which was found in rat intestinal membranes. The cell surface biotinylation of LLC-rPEPT1 cell monolayers grown on membrane filters showed that PEPT1 was localized predominantly on the apical membranes and, to a lesser extent, on the basolateral membranes. The amount of [14C]glycylsarcosine uptake in LLC-rPEPT1 cell monolayers was 3-fold greater from the apical, than from the basolateral side, which suggested that rat PEPT1 expressed on both membranes was functionally active. LLC-rPEPT1 cells grown on plastic dishes transported differently charged oral cephalosporins such as ceftibuten (divalent anion lacking an alpha-amino group) and cephradine (zwitterion with an alpha-amino group) in the presence of an inward H+ gradient, whereas those transfected with the vector alone did not have transport activity. Kinetic analysis revealed that the LLC-rPEPT1 cells had much higher affinity for ceftibuten than for cephradine. Di- and tripeptides and bestatin, a dipeptide-like antineoplastic drug, potently inhibited the uptake of these cephalosporins. These results suggest that the LLC-rPEPT1 cells serve as a useful model with which to analyze the mechanisms involved in membrane targeting and substrate recognition by rat PEPT1.     

Transport of the beta-lactam antibiotic benzylpenicillin and the dipeptide glycylsarcosine by brain capillary endothelial cells in vitro

Peripherally administered beta-lactam antibiotics, which are structural analogs of tripeptides, may cause neurotoxic reactions or induce seizures. Previous in vivo studies provided evidence for brain uptake of these antibiotics. In the present work, we studied the extent and mechanism of the uptake of benzylpenicillin and glycylsarcosine by brain microvessel endothelial cells in vitro, using freshly isolated and cultured porcine brain capillary endothelial cells. Characterization of the cell culture model demonstrated the functional expression of the system transporting the neutral amino acids leucine and phenylalanine. The initial rate of uptake of benzylpenicillin was >3-fold greater than the rate of uptake of the extracellular marker sucrose (ratio, 3.29 +/- 0.37), whereas uptake of glycylsarcosine did not differ from that of sucrose. The differences in cellular uptake correlated with the octanol/buffer partition coefficients for glycylsarcosine and benzylpenicillin (1.16 x 10(-3) for glycylsarcosine and 6.83 x 10(-2) for benzylpenicillin). The concentration-dependent uptake of benzylpenicillin (1-2000 microM) was not saturable and was not sensitive to shifts in pH or temperature. The permeability-surface area product for the uptake of benzylpenicillin at pH 7.4 was determined from these experiments and was found to be 8.1 x 10(-5) ml/sec/g of brain. This value was very close to the value determined in in vivo studies. Uptake of benzylpenicillin and glycylsarcosine was not reduced in the presence of 1 mM ceftibuten or 100 microM probenecid. The findings with cultured cell monolayers were confirmed using freshly isolated endothelial cells. These in vitro data are compatible with benzylpenicillin, but not glycylsarcosine, being able to penetrate endothelial cells. Uptake of benzylpenicillin by brain capillary endothelial cells occurs by a slow nonsaturable process, with no evidence for carrier-mediated transport.     

Transport of L-valine-acyclovir via the oligopeptide transporter in the human intestinal cell line, Caco-2

It has been reported that conjugating acyclovir, a potent antiviral with low oral bioavailability, to L-valine increases its urinary excretion in rats. However, it was also reported that this increase is not found for the D-valine ester, suggesting that a carrier-mediated mechanism is involved in its intestinal absorption. Therefore, mechanisms involved in the transepithelial transport of L-valine-acyclovir were investigated using the intestinal cell line, Caco-2, as a model system for the intestinal epithelium. Only the mucosal-to-serosal transport of acyclovir was increased by conjugation with L-valine (approximately 7-fold), suggesting the involvement of a carrier-mediated mechanism. This conclusion was supported by the finding that this increase was saturable. The mucosal-to-serosal transport of L-valine-acyclovir could be inhibited by L-glycylsarcosine, but not by L-valine, suggesting the involvement of the dipeptide carrier. Also it was found that L-valine-acyclovir inhibits the uptake of cephalexin, a substrate for the oligopeptide transporter. Stability of the esters in either the mucosal or serosal bathing solution is more than 90% after completion of the transport study. However, after transport, the receiver solution contained approximately 90% of acyclovir. Based on these findings it was concluded that absorption of the L-valine ester of acyclovir occurs as a result of uptake by the oligopeptide transporter at the apical cell membrane followed by intracellular hydrolysis of the ester and efflux of acyclovir.     

Identification of thyroid hormone transporters

Thyroid hormone action and metabolism are intracellular events that require transport of the hormone across the plasma membrane. We tested the possible involvement of the Na+/taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide (oatp1) in the hepatic uptake of the prohormone T4, the active hormone T3, and the metabolites rT3 and 3,3'-T2. Xenopus laevis oocytes were injected with 2.3 ng Ntcp or oatp1 cRNA and, after 2-3 days, incubated for 1 h at 25 degrees C with usually 0.1 microM 125I-labeled ligand. Uninjected oocytes showed marked uptake of iodothyronines and this was further increased by Ntcp and oatp1 cRNA, i.e., 1.9- and 2.8-fold for T4, 1.7- and 1.7-fold for T3, 1.8- and 6.0-fold for rT3, and 1.3- and 1.4-fold for 3,3'-T2, respectively. Mostly due to much lower uptake by uninjected oocytes, Ntcp and oatp1 cRNA induced larger, 12- to 76-fold increases in uptake of iodothyronine sulfates. The Ntcp cRNA-induced iodothyronine uptake was completely inhibited in Na+-deplete medium, whereas the oatp1 cRNA-induced uptake was not affected. These results suggest that hepatic uptake of thyroid hormones and their metabolites is mediated at least in part by Ntcp and oatp1.     

Chemiluminescent detection of mRNAs on northern blots with digoxigenin end-labeled oligonucleotides

To establish a simplified, nonradioactive approach for identifying mRNAs on Northern blots, antisense oligonucleotides have been used as probes in combination with chemiluminescence-based detection. Oligonucleotides (approximately 32-mer) were end-labeled with digoxigenin (DIG) and used in conjunction with adamantyl 1,2-dioxetane aryl phosphate substrates (Lumigen PPD and CSPD). Oligonucleotides were designed as probes for several mRNAs in tissues of rats and mice, including the mitochondrial uncoupling protein, lipoprotein lipase, GLUT1, GLUT4, and beta-actin. Uncoupling protein mRNA was detected in total RNA from brown adipose tissue with a 32-mer DIG-labeled oligonucleotide, within 2 min of exposure to film. This mRNA could also be detected when as little as 250 ng of total RNA was applied to the gel, following 4 h exposure to film, and was present only in brown fat. The mRNA for lipoprotein lipase was detectable with a 30-mer DIG-labeled oligonucleotide in 1 micrograms of total RNA from mouse heart, within 2 h of exposure. The mRNA for the GLUT1 glucose transporter was detected in total RNA from rat midbrain using a 32-mer DIG-labeled oligonucleotide, while beta-actin mRNA was detected with a 30-mer oligonucleotide. The mRNA for the insulin-sensitive glucose transporter GLUT4 was detected with a 32-mer DIG-labeled oligonucleotide and found only in those tissues in which glucose uptake is stimulated by insulin. The speed of detection was greater with CSPD and was augmented by exposure of membranes to film at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of hemicholinium-3 sensitive choline uptake in Xenopus laevis oocytes by the second C2 domain of synaptotagmin

A size-fractionated torpedo electric lobe cDNA library was screened for the neuronal choline transporter by functional expression in oocytes. A clone, TLC2B, was isolated that induced a component of choline uptake that was hemicholinium-3 sensitive and inhibited by the substitution of lithium for sodium at low choline concentrations. However, [3H]choline uptake by both injected and non-injected oocytes were characterized by high affinity constants, suggesting that TLC2B could be affecting a native choline transporter. Indeed, hemicholinium-3 sensitive choline uptake could also be induced by preincubation of non-injected oocytes with a protein kinase C inhibitor, H-7. By sequence analysis and immuno-precipitation, the peptide produced by injection of TLC2B cRNA was identified as a soluble 24 kDa C-terminal fragment of the neuronal protein, synaptotagmin. Full length synaptotagmin was, however, ineffective in the functional test. The peptide encoded by TLC2B corresponds to the second protein kinase C-homologous domain of torpedo synaptotagmin, and like other soluble C2 domain peptides, was capable of calcium-dependent translocation to membranes. Its action on choline uptake in oocytes was, however, abolished by the addition of calcium in the presence of a calcium ionophore. These results suggest that the interaction of certain C2 domains, such as the C-terminal domain of synaptotagmin, with more specific targets may be anulled in the presence of calcium due to its absorption to membrane phospholipids.