Why transport matters: an update on carrier proteins in Apicomplexan parasites.

The uptake of solutes across the cell-surface membrane can occur by either an active or passive transport mechanism. Active transport mechanisms are characterized by the uptake of solutes against their concentration gradient at the expense of metabolic energy. Active transport of hexoses occurs in two major tissues in mammals, kidney and intestine, and will not be dealt with in this review. Passive transport can be subdivided into the two categories of simple diffusion or facilitative diffusion. In both cases, the movement of solutes across the cell membrane is driven solely by the concentration gradient between the intracellular and extracellular environment without any metabolic energy being required. Net uptake ceases when the concentration of solutes between the inside and outside of the cell has reached equilibrium. Facilitative diffusion differs from simple diffusion in that the former process is mediated by membrane-bound proteins which exhibit a high degree of specificity and whose activity is competitively inhibited with appropriate analogues. The difference in transport rate between simple and facilitative diffusion is dramatically exemplified by the permeability of d-glucose across synthetic lipid bilayers with a permeability coefficient of 10−9–10−10 cm/sec (Lidgard and Jones, 1975; Jung, 1971a), whereas for the intact erythrocyte, the permeability coefficient is approximately 10−4 cm/sec (Jung, 1971b).

Evaluation of active and passive transport mechanisms in genital tracts of IUD-bearing women with radionuclide hysterosalpingoscintigraphy

Transport characteristics of isorhamnetin across intestinal Caco-2 cell monolayers and the effects of transporters on it

This year marks the 20th anniversary of the publication of a landmark paper by Dr. Michael Matthay and colleagues in the Journal of Applied Physiology that proved to be pivotal in the study of lung edema clearance. To honor this achievement, I am pleased to introduce the newest Highlighted Topics

L‐carnitine, a betaine derivative of β‐hydroxybutyrate, is found in virtually all cells of higher animals and also in some microorganisms and plants. In animals it is synthesized almost exclusively in the liver. Two essential amino acids, i.e., lysine and methionine serve as primary substrates for its biosynthesis. Also required for its synthesis are sufficient amounts of vitamin B6, nicotinic acids, vitamin C and folate. The first discovered ergogenic function of L‐carnitine is the transfer of activated long‐chain fatty acids across the inner mitochondrial membrane into the mitochondrial matrix. For this transfer acyl‐CoA esters are transesterified to form acylcarnitine esters. Thus, in carnitine deficiency fat oxidation and energy production from fatty acids are markedly impaired. Skeletal muscles constitute the main reservoir of carnitine in the body and have a carnitine concentration at least 200 times higher than blood plasma. Uptake of carnitine by skeletal muscles takes place by an active transport mechanism which transports L‐carnitine into muscles probably in the form of an exchange process with γ‐butyrobetain. In young animals including foals, the capacity for biosynthesis of carnitine is not yet fully developed and apparently cannot meet the requirements of sucking animals. Sucking animals depend therefore on an extra supply of carnitine which is usually provided with milk. Additionally, young animals including foals possess a lower concentration of carnitine in blood plasma than adult animals. Besides its role as carrier of activated acyl groups, L‐carnitine functions as a buffer for acetyl groups which may be present in excess in different tissues during ketosis and hypoxic muscular activity. Other functions of L‐carnitine are protection of membrane structures, stabilizing of a physiologic CoA‐SH/acetyl‐CoA ratio and reduction of lactate production. Animal's derived feeds are rich in L‐carnitine whereas plants contain usually very little or no carnitine. carnitine is absorbed from the small intestine by active and passive transport mechanisms. From the increase in renal excretion of L‐carnitine after oral supplementations of 10g/d to horses it has been concluded that the efficiency of absorption of L‐carnitine is rather low (about 5 to 10% of the supplied dose). A further decrease in fractional carnitine absorption was observed when the oral dose of carnitine was increased. L‐carnitine is virtually not degraded in the body and renal excretion of carnitine is comparatively small under normal conditions. The concentration of L‐carnitine in blood plasma of horses varies markedly between animals and between different days. In addition, circadian changes in carnitine concentration in plasma have been reported. Peak concentrations were found during late afternoon, being up to 30% higher than those in the morning. In breeding mares the carnitine concentration in blood plasma declines with onset of lactation. In resting skeletal muscles about 90% of the total carnitine content is present as free carnitine with the remaining part being available as carnitine esters. With increasing exercise intensity a continuing greater proportion of free carnitine (up to 80%) is converted into carnitine esters, mainly into acetylcarnitine. This shift from free to acetylcarnitine is readily reversed within about 30 min after termination of exercise. It appears that acute exercise does not have a marked effect on the content of total carnitine in skeletal muscle whereas training seems to elevate its total concentration in the middle gluteal muscle of 3 to 6 year old horses and to reduce variation of its concentration compared to age‐matched untrained horses. Oral supplementations of 5 to 50 g of L‐carnitine per day to horses elevated the carnitine concentration in blood plasma to about twice its basal concentration. No clear relationship existed, however, between the orally administered dose of carnitine and the increase of L‐carnitine concentration in blood plasma. Oral supplementations of carnitine also tended to elevate the carnitine concentration in milk of mares and in blood plasma of sucking foals. Long‐term oral administration of L‐carnitine (e.g., for months) also appeared to increase the carnitine concentration in skeletal muscles. But information is lacking as to whether such administrations also affect physical performance of the exercising muscle. Oral supplementation of carnitine to horses reduced the resting values of lactate in plasma and appeared to reduce the concentration of non‐esterified fatty acids in plasma during exercise. These effects of carnitine appeared also to be influenced by the amount and type of fat which is contained in the feed. Oral supplementations of carnitine to stallions may improve impaired motility of sperm. Improvements of feed conversion and weight gain in growing horses due to oral supplementations of carnitine have been reported. But these preliminary findings probably require further confirmation. Further studies are also required to better evaluate possible effects of oral supplementations of carnitine on energy metabolism, cardiac functions and physical performance in horses at rest and during exercise, and to perhaps better characterize the conditions under which carnitine may be beneficial to horses.

The cell membrane, the gastrointestinal tract, and the blood-brain barrier (BBB) are good examples of biological barriers that define and protect cells and organs. They impose different levels of restriction, but they also share common features. For instance, they all display a high lipophilic character. For this reason, hydrophilic compounds, like peptides, proteins, or nucleic acids have long been considered as unable to bypass them. However, the discovery of cell-penetrating peptides (CPPs) opened a vast field of research. Nowadays, CPPs, homing peptides, and blood-brain barrier peptide shuttles (BBB-shuttles) are good examples of peptides able to target and to cross various biological barriers. CPPs are a group of peptides able to interact with the plasma membrane and enter the cell. They display some common characteristics like positively charged residues, mainly arginines, and amphipathicity. In this field, our group has been focused on the development of proline rich CPPs and in the analysis of the importance of secondary amphipathicity in the internalization process. Proline has a privileged structure being the only amino acid with a secondary amine and a cyclic side chain. These features constrain its structure and hamper the formation of H-bonds. Taking advantage of this privileged structure, three different families of proline-rich peptides have been developed, namely, a proline-rich dendrimer, the sweet arrow peptide (SAP), and a group of foldamers based on γ-peptides. The structure and the mechanism of internalization of all of them has been evaluated and analyzed. BBB-shuttles are peptides able to cross the BBB and to carry with them compounds that cannot reach the brain parenchyma unaided. These peptides take advantage of the natural transport mechanisms present at the BBB, which are divided in active and passive transport mechanisms. On the one hand, we have developed BBB-shuttles that cross the BBB by a passive transport mechanism, like diketoperazines (DKPs), (N-MePhe)n, or (PhPro)n. On the other hand, we have investigated BBB-shuttles that utilize active transport mechanisms such as SGV, THRre, or MiniAp-4. For the development of both groups, we have explored several approaches, such as the use of peptide libraries, both chemical and phage display, or hit-to-lead optimization processes. In this Account, we describe, in chronologic order, our contribution to the development of peptides able to overcome various biological barriers and our efforts to understand the mechanisms that they display. In addition, the potential use of both CPPs and BBB-shuttles to improve the transport of promising therapeutic compounds is described.

The role of glucocorticoids in the regulation of the postnatal development of ileal active bile salt transport was examined in the rat using the villus technique. Ileal taurocholate uptake was initially by passive transport alone on day 14 and 16 which changed to an active and passive transport mechanism at 18 days which persisted thereafter. The Michaelis-Menten constant (Km, mM) was unchanged between days 18 (0.67 +/- 0.12 mM), 20 (0.84 +/- 0.25 mM), 21 (0.49 +/- 0.05 mM), 28 (0.59 +/- 0.06 mM), and 49 (0.50 +/- 0.05 mM) whereas the apparent maximal velocity nmol/mg(dry wt)/min declined after a peak at 18 days (18.17 +/- 1.92 on day 18, 16.14 +/- 1.89 on day 20, 14.65 +/- 0.52 on day 21, 11.40 +/- 0.35 on day 28, and 10.51 +/- 0.32 on day 49). Adrenalectomy performed in sucklings on day 14 with taurocholate transport studies on day 21 and in adults on day 42 studied on day 49 resulted in reductions in uptake at most study concentrations but no change in the Km (1.33 +/- 0.54 in sucklings and 0.75 +/- 0.14 mM in adults) or apparent maximal velocity [11.78 +/- 2.06 in sucklings and 9.24 +/- 0.65 nmol/mg (dry wt)/min in adults]. Treatment of sucklings with corticosterone (5 mg/100 g body weight) on days 10-13 with study on day 14 and 16 did not produce precocious development of ileal active taurocholate transport; however, corticosterone treatment led to apparent increases in ileal permeability to taurocholate in both sucklings and adults. Glucocorticoids appear to play a minor, if any, role in the physiologic postnatal development of ileal active bile salt transport.

Robust, predictive ex vivo/in vitro models to study intestinal drug absorption by passive and active transport mechanisms are scarce. Membrane transporters can significantly impact drug uptake and transporter-mediated drug–drug interactions can play a pivotal role in determining the drug safety profile. Here, the presence and activity of seven clinically relevant apical/basolateral drug transporters found in human jejunum were tested using ex vivo porcine intestine in a Ussing chamber system. Experiments using known substrates of peptide transporter 1 (PEPT1), organic anion transporting polypeptide (OATP2B1), organic cation transporter 1 (OCT1), P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multi drug resistance-associated protein 2 and 3 (MRP2 and MRP3), in the absence and presence of potent inhibitors, showed that there was a statistically significant change in apparent intestinal permeability Papp,pig (cm/s) in the presence of the corresponding inhibitor. For MRP2, a transporter reportedly present at relatively low concentration, although Papp,pig did not significantly change in the presence of the inhibitor, substrate deposition (QDEP) in the intestinal tissue was significantly increased. The activity of the seven transport proteins was successfully demonstrated and the results provided insight into their apical/basolateral localization. In conclusion, the results suggest that studies using the porcine intestine/Ussing chamber system, which could easily be integrated into the drug development process, might enable the early-stage identification of new molecular entities that are substrates of membrane transporters.

The limited efficacy of deep tumor treatments has been considered the “Achilles’ heel” of anticancer therapy due to multiple biological barriers. Whether passive diffusion or active transport has difficulties completely overcoming these obstacles. Herein, cation‐π interactions are utilized to construct a tumor drug delivery system integrating the merits of both passive and active transport mechanisms. A cation‐π interaction bridged trimetallic supramolecular drug complex (Cπ‐TMSDC) is constructed based on a drug consisting of one cisplatin molecule linked by K+ (Pt‐COOK) and the other drug with a Ru metal complex containing curcumin (Ru‐Cur). The obtained Cπ‐TMSDC further self‐assembles into cation‐π‐based trimetallic supramolecular drug micelles (Cπ‐TMSDMs) with efficient and stable transportation in vivo due to the strong cation‐π interaction formed between K+ and the curcumin unit in the Cπ‐TMSDC. In acidic tumor microenvironment, the cation‐π interaction smartly dissociates, facilitating the quick release of Pt‐COOK outside Cπ‐TMSDMs to rapidly infiltrate the outer cellular layers by passive diffusion. Meanwhile, the dissociated Ru‐Cur from the core layer of the Cπ‐TMSDMs form secondary self‐assemblies to deeply penetrate inside the solid tumor. Therefore, this strategy results in an efficient tumor drug delivery platform with enhanced deep intratumoral penetration, improved therapeutic effects, and reduced systemic toxicity to normal organs.

The isolated anatomical position and blood-labyrinth barrier hampers systemic drug delivery to the mammalian inner ear. Intratympanic placement of drugs and permeation via the round- and oval window are established methods for local pharmaceutical treatment. Mechanisms of drug uptake and pathways for distribution within the inner ear are hard to predict. The complex microanatomy with fluid-filled spaces separated by tight- and leaky barriers compose various compartments that connect via active and passive transport mechanisms. Here we provide a review on the inner ear architecture at light- and electron microscopy level, relevant for drug delivery. Focus is laid on the human inner ear architecture. Some new data add information on the human inner ear fluid spaces generated with high resolution microcomputed tomography at 15 μm resolution. Perilymphatic spaces are connected with the central modiolus by active transport mechanisms of mesothelial cells that provide access to spiral ganglion neurons. Reports on leaky barriers between scala tympani and the so-called cortilymph compartment likely open the best path for hair cell targeting. The complex barrier system of tight junction proteins such as occludins, claudins and tricellulin isolates the endolymphatic space for most drugs. Comparison of relevant differences of barriers, target cells and cell types involved in drug spread between main animal models and humans shall provide some translational aspects for inner ear drug applications.

In response to extracellular stimuli, mitogen-activated protein kinase (MAPK, also known as ERK) translocates from the cytoplasm to the nucleus. MAP kinase kinase (MAPKK, also know as MEK), which possesses a nuclear export signal (NES), acts as a cytoplasmic anchor of MAPK. Here we show evidence that tyrosine (Tyr190 in Xenopus MPK1/ERK2) phosphorylation of MAPK by MAPKK is necessary and sufficient for the dissociation of the MAPKK-MAPK complex, and that the dissociation of the complex is required for the nuclear translocation of MAPK. We then show that nuclear entry of MAPK through a nuclear pore occurs via two distinct mechanisms. Nuclear import of wild-type MAPK (mol. wt 42 kDa) was induced by activation of the MAPK pathway even in the presence of wheat germ agglutinin or dominant-negative Ran, whereas nuclear import of beta-galactosidase (beta-gal)-fused MAPK (mol. wt 160 kDa), which occurred in response to stimuli, was completely blocked by these inhibitors. Moreover, while a dimerization-deficient mutant of MAPK was able to translocate to the nucleus upon stimulation, this mutant MAPK, when fused to beta-gal, became unable to enter the nucleus. These results suggest that monomeric and dimeric forms of MAPK enter the nucleus by passive diffusion and active transport mechanisms, respectively.

Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex (NPC), while most proteins are transported by energy driven transport mechanisms. Active transport of viral proteins is mediated by nuclear localization signals (NLS), which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral proteins. Usually they contain short stretches of lysine or arginine residues. These signals are recognized by the importin super-family (importin α and β) proteins that mediate the transport across the nuclear envelope through Ran-GTP. In contrast, only one class of the leucine-rich nuclear export signal (NES) on viral proteins is known at present. Chromosome region maintenance 1 (CRM1) protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.

In this paper, we present a detailed and comprehensive mathematical model of active and passive ion and water transport in plant roots. Two key features are the explicit consideration of the separate, but interconnected, apoplastic, and symplastic transport pathways for ions and water, and the inclusion of both active and passive ion transport mechanisms. The model is used to investigate the respective roles of the endodermal Casparian strip and suberin lamellae in the salt stress response of plant roots. While it is thought that these barriers influence different transport pathways, it has proven difficult to distinguish their separate functions experimentally. In particular, the specific role of the suberin lamellae has been unclear. A key finding based on our simulations was that the Casparian strip is essential in preventing excessive uptake of Na+ into the plant via apoplastic bypass, with a barrier efficiency that is reflected by a sharp gradient in the steady-state radial distribution of apoplastic Na+ across the barrier. Even more significantly, this function cannot be replaced by the action of membrane transporters. The simulations also demonstrated that the positive effect of the Casparian strip of controlling Na+ uptake, was somewhat offset by its contribution to the osmotic stress component: a more effective barrier increased the detrimental osmotic stress effect. In contrast, the suberin lamellae were found to play a relatively minor, even non-essential, role in the overall response to salt stress, with the presence of the suberin lamellae resulting in only a slight reduction in Na+ uptake. However, perhaps more significantly, the simulations identified a possible role of suberin lamellae in reducing plant energy requirements by acting as a physical barrier to preventing the passive leakage of Na+ into endodermal cells. The model results suggest that more and particular experimental attention should be paid to the properties of the Casparian strip when assessing the salt tolerance of different plant varieties and species. Indeed, the Casparian strip appears to be a more promising target for plant breeding and plant genetic engineering efforts than the suberin lamellae for the goal of improving salt tolerance.

Active HCO-3 secretion in the anterior rectal salt gland of the mosquito larva, Aedes dorsalis, is mediated by a 1:1 Cl-/HCO-3 exchanger. The cellular mechanisms of HCO-3 and Cl- transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO2 and HCO-3 from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (Va) and had little effect on basolateral potential (Vbl). Changes in luminal Cl- concentration altered Va in a rapid, linear manner with a slope of 42.2 mV/decalog a1Cl-. Intracellular Cl- activity was 23.5 mM and was approximately 10 mM lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl- concentration had no effect on Vbl, indicating an electrically silent basolateral Cl- exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated acHCO-3 was 14.4 mM. These results show that under control conditions HCO-3 enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl- enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl- -selective channel, and HCO-3 exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl- exit and HCO-3 entry, and the effects of serosal addition of the Cl-/HCO-3 exchange inhibitor, DIDS, on JCO2net and transepithelial potential (Vte) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl- and HCO-3 movements.

Approximately three-quarters of all chemical elements are metals. Metal ions associate with RNA in solution in a number of interesting ways. The highly water-soluble ionic forms of certain metal ions are abundant in seawater and inside cells and play vital roles in RNA folding and catalysis. In this chapter, we examine the role of divalent ions in RNA folding. The properties of a number of divalent metal ions are summarized, and experiments examining their binding to RNA in solution and crystals are evaluated. We also review the evidence that precisely placed metal ions can promote catalysis by RNA. Since the mechanisms of group I introns and RNase P are the subject of another chapter (Cech, this volume), we focus here on biological and nonbiological examples of catalytic cleavage of the RNA that give products containing 2′, 3′ -cyclic phosphate and 5′ -hydroxyl termini. PROPERTIES OF METAL IONS The concentrations of a number of metal ions in seawater and cells are given in Table 1. Intracellular metal ion concentrations vary widely and are maintained and adjusted by a variety of active and passive transport mechanisms. Inside cells, free metal ion concentrations are much lower than total metal ion concentrations, since intracellular macromolecules bind substantial amounts of metal ions. Table 2 lists certain chemical properties of selected metal ions that are relevant to their interaction with RNA. In aqueous solutions, metal ions form coordination complexes with water or solutes. The coordination number (number of ligands that bind) and coordination geometry (geometric arrangement...