Transmembrane Transport Systems Research Articles

Harry M. Vars Research Award. A new model to determine in vivo the relationship between amino acid transmembrane transport and protein kinetics in muscle.

The bidirectional transmembrane transport rates of leucine (Leu), valine (Val), phenylalanine (Phe), lysine (Lys), and alanine (Ala) were measured in vivo in the hindlimb muscle of five dogs and related to the rates of protein synthesis and degradation. The compartmental model was based on the systemic continuous infusion of stable isotopic tracers of the amino acids, and the measurement of the enrichment and concentration in the arterial and femoral vein plasma and the intracellular free water in muscle (obtained by biopsy). The transport rate from plasma to tissue (in micromoles per minute) was: Leu, 18.1 +/- 1.8; Val, 26.9 +/- 3.5; Phe, 10.5 +/- 1.6 Lys; 12.2 +/- 1.8; and Ala, 10.7 +/- 3.4. The transport rate from tissue to plasma (in micromoles per minute) was: Leu, 25.5 +/- 2.5; Val, 32.4 +/- 2.8; Phe, 17.0 +/- 2.8; Lys, 24.9 +/- 3.4; Ala, 34.4 +/- 9.0. When the transmembrane transport rate was normalized per unit of amino acid concentration in the source pool, we found that the transport of Leu, Val, and Phe was significantly faster (p less than .05) than the transport of Lys and Ala. The calculated rates of incorporation into hindlimb muscle protein of Phe and Lys (in micromoles per minute) were 4.2 +/- 1.3 and 19.4 +/- 5.3, respectively, and the rates of intracellular appearance from breakdown were 10.7 +/- 1.9 and 32.1 +/- 6.6, respectively. We concluded, therefore, that (1) the transmembrane amino acid transport rate can be measured in vivo in muscle with a relatively noninvasive technique, (2) in the dog hindlimb the equilibration between tissue and plasma free amino acid pool is different for each amino acid depending on the kinetics of the transmembrane transport systems, and (3) the transport rates of amino acids and their rate of appearance from protein breakdown are roughly comparable, suggesting that variations in transport rates could play a role in controlling the rate of protein synthesis.

Effect of a rickets-inducing diet and vitamin D metabolites [25-OH-D 3 and 1,25(OH) 2D 3] on transmembrane transport systems in rat kidney

We previously demonstrated that feeding rats Steenbock and Black's rickets-inducing diet (deficient in vitamin D and with an altered Ca:P ratio) produces remarkable changes in the metabolic picture of the intestinal mucosa, kidney, and liver and in the transmembrane transport systems of D-glucose in intestinal and renal brush-border membrane vesicles. We have now investigated the effect of both 25-hydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 3 on Na +-dependent D-glucose and citrate transport and on calcium, phosphorus, and citrate content in rat serum and kidney. Na +-dependent D-glucose uptake, which decreased in rachitic rat jejunum brush-border membrane vesicles, returned to control values after 25-hydroxyvitamin D 3 or 1,25-dihydroxyvitamin D 3 administration, while in kidney normal D-glucose transport was restored only after 1,25-dihydroxyvitamin D 3 treatment. Na +-dependent citrate uptake was lowered in rachitic rat renal brush-border membrane vesicles and both 25-hydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 3 proved to be ineffective in restoring to normal values. The in vitro addition to vesicle preparations of calcium or phosphate, citrate, 1,25-dihydroxyvitamin D 3, or valinomycin did not show a selective influence on D-glucose and citrate transport.

Evidence for a direct and non-receptor-mediated action of 5HT2 antagonists on transmembrane cation transport systems

Changes in transmembrane sodium fluxes have been reported in normotensive and in hypertensive subjects after ketanserin administration. In this study, the effects of the serotonergic system on transmembrane sodium transport mechanisms have been investigated in vitro. In erythrocytes drawn from ten healthy subjects, we studied the effects of serotonin (5HT) on the Na/K pump, Na/K cotransport, Na/Li countertransport, and passive permeability of Na. No significant changes were found. A direct, non-receptor-mediated action of ketanserin was then suspected, and the effects of two concentrations of ketanserin (5 x 10(-8) and 5 x 10(-7) M) were evaluated in erythrocytes from 12 normal volunteers. Both concentrations of ketanserin significantly decreased the activity of the Na/K pump and increased the activity of Na/Li countertransport. Na/K cotransport and passive permeability were not affected. Indirect evidence of the action of ketanserin on sodium transmembrane fluxes came from other experiments. In the red blood cells taken from five normal subjects and incubated for 2 hours in a plasma pool, we evaluated the changes in intracellular sodium concentration induced by the presence of 5HT or ketanserin. A significant decrease in intracellular sodium concentration occurred only with ketanserin. This study indicates that ketanserin has a direct influence on transmembrane sodium fluxes. If this action were also present in other cells, it might contribute to the actions of the drug at vascular, nervous, and renal tubular levels.

The distribution of plasmodesmata in the phloem ofHevea brasiliensis in relation to laticifer loading

Cell to cell connections, including plasmodesmata and perforations, were examined in the non-conducting secondary phloem ofHevea brasiliensis. Samples were taken from trunks of numerous trees, from several clones, and prepared for thin sectioning and transmission or scanning electron microscopy and as optical sections for fluorescence microscopy. Numerous plasmodesmata were found clustered in primary pit-fields between the ray and axial parenchyma cells. Between the laticifers and adjacent parenchyma sheath cells, structures corresponding to functional plasmodesmata were not observed. But some unusual structural features were occasionally seen in these walls. These observations are discussed in relation to the possible function of the cell types, and to the loss of latex on the tapping ofHevea. It is suggested that the loading of the laticifer might first require a symplastic pathway for the transport of metabolites, at the end of which the assimilates must enter the apoplast. A transmembrane active transport system then transfers the metabolites in the laticifer. The presumable role of parenchyma cells in the loading of laticifers is emphasized.

Photodynamic effects of hematoporphyrin-derivative on enzyme activities of murine L929 fibroblasts

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin-derivative resulted in the inactivation of cytosolic, mitochondrial and lysosomal enzymes and in a decrease in cellular adenosine triphosphate and reduced glutathione concentrations. Comparison of these results with those of previous studies revealed that transmembrane transport systems and DNA repair enzymes are inactivated after much shorter illumination periods than are intracellular enzymes. Although the pattern of photodynamic damage altered by varying the protocol of preincubation with hematoporphyrin-derivative and washing, it appeared that under all experimental conditions the plasma membrane was much more sensitive to photodynamic damage than were the intracellular enzymes. Lysosomal membrane disruption with subsequent detrimental release of lysosomal enzymes has been implicated previously in certain forms of porphyrin-induced photodynamic cell destruction. Cytochemical studies on enzyme localization virtually exclude such a mechanism in hematoporphyrin-derivative-induced cell inactivation in L929 fibroblasts.

Toxic effects of ozone on murine L929 fibroblasts. Damaging action on transmembrane transport systems.

Exposure of murine L929 fibroblasts to ozone caused inactivation of the energy-coupled transport systems for 2-aminoisobutyric acid and Rb+. Ozone induced an increase of the apparent Km for 2-aminoisobutyric acid transport, with a constant Vmax, indicating impairment of the carrier function, rather than a decrease in the number of transport sites. The uptake rate of 2-deoxy-D-glucose, on the other hand, increased after exposure of the cells to ozone. This was caused by an increased Vmax. of facilitated diffusion, without increased after ozone exposure. Together with the K+ leakage described previously, this reflects ozone-induced impairment of the barrier function of the membrane.

Potent in vitro and in vivo antitoxoplasma activity of the lipid-soluble antifolate trimetrexate.

Trimetrexate, a highly lipid-soluble quinazoline antifolate now undergoing trials as an anticancer agent, was found to be a potent inhibitor of the dihydrofolate reductase (DHFR) isolated from Toxoplasma gondii. The concentration required for 50% inhibition of protozoal DHFR was 1.4 nM. As an inhibitor of this enzyme, trimetrexate was almost 600-fold (amount of antifolate required to inhibit catalytic reaction by 50%) and 750-fold (inhibition constant) more potent than pyrimethamine, the DHFR inhibitor currently used to treat toxoplasma infection. When the protozoan was incubated with 1 microM trimetrexate, the drug rapidly reached high intracellular concentrations. Since toxoplasma organisms lack a transmembrane transport system for physiologic folates, host toxicity can be prevented by co-administration of the reduced folate, leucovorin, without reversing the antiprotozoal effect. The effectiveness of trimetrexate against toxoplasma was demonstrated both in vitro and vivo. Proliferation of toxoplasma in murine macrophages in vitro was completely inhibited by exposure of these cells to 10(-7) M trimetrexate for 18 h. When used alone, trimetrexate was able to extend the survival of T. gondii-infected mice.

Photodynamic effects of hematoporphyrin-derivative on transmembrane transport systems of murine L929 fibroblasts

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin-derivative causes deterioration of various membrane functions. Most sensitive to photodynamic inactivation are the energy-coupled transport systems for aminoisobutyric acid and for Rb +. The facilitated diffusion system for 2-deoxy- d-glucose is slightly less sensitive. After longer illumination periods also the membrane barrier function is impaired, as reflected by K + leakage and increased passive Rb + uptake. After still longer illumination periods intermolecular protein crosslinking can be observed. This makes it unlikely that intermolecular protein crosslinking is causally involved in the deterioration of these membrane functions.

Mechanism, regulation and physiological significance of the loop diuretic-sensitive NaCl/KCl symport system in animal cells.

Investigations in numerous laboratories have characterized a salt transport system, present in many animal cell types, which catalyzes the transmembrane transport of NaCl and KCl in a tightly coupled process. The system is inhibited by loop diuretics such as furosemide and bumetanide. This transport system has been designated the loop diuretic-sensitive NaCl/KCl symporter. It has been implicated in transepithelial salt secretion and absorption as well as in cell volume regulation, and it may be defective in patients suffering from essential hypertension. This review serves to evaluate research conducted to date regarding the mechanism, mode of regulation, and physiological significance of the transport system. Ion binding specificities and absolute binding constants for all three naturally occurring ions have been determined in one cell system, the MDCK kidney epithelial cell line. In that same cell line, substrate binding was shown to exhibit apparent cooperativity. although a few reports suggest unidirectional transport of ions via this system under certain conditions, the consensus of reports indicates fully reversible, bidirectional salt transport with the direction of net flux determined by the magnitudes of the gradients of the three transported ions. Growth of cells in media containing a low concentration of K+ (less than 0.25 mM) allows selection of mutants lacking or defective in the symporter. Kinetic analyses with the MDCK cell line have shown that the symporter catalyzes accelerative exchange transport. However, exchange transport of one ion in the absence of one of the other two ionic substrates has not been documented. Comparison with other well-characterized transmembrane transport systems has shown that the characteristics of the NaCl/KCl symporter most resemble those of two-species facilitators (chemiosmotically-coupled symporters) found in prokaryotes and eukaryotes alike. these two-species facilitators consist of a single transmembrane protein and may function by a carrier-type mechanism as originally proposed by Peter Mitchell. A molecular model for the NaCl/KCl symporter is presented and discussed. Activation of symport activity requires ATP and probably occurs by a protein kinase-catalyzed mechanism. In some cell types activation is cyclic AMP dependent. ATP hydrolysis is not stoichiometric with transport. Phosphorylation of an integral membrane protein with an apparent size of 240 000 daltons correlates with activation of transport. It is postulated that this protein is the loop diuretic-sensitive NaCl/KCl symporter.

Membrane specialisation of the extra-junctional sarcolemma of normal and denervated insect muscle

Corrugated areas of sarcolemma were observed in serial transverse sections. Complexes of intramembranous particles that appear after freeze-fracture replication were concluded to represent the same specialisation. These specialisations, up to 14 microns x 2 microns, are orientated parallel to the long axis of the muscle. Intramembranous particles are concentrated along the peaks of the corrugations, and are associated with the P-face. Corresponding pits are found in the E-face. Fourteen and thirty days after sectioning the excitatory motor-nerve supply to the muscle, corrugated areas 0.5-1 microns x 0.5-1 microns are found. Occurring singly or in groups, their orientation with respect to the long axis of the muscle is more variable than those of control muscles. Thin sections reveal no complementary areas on adjacent fibres or intracellular submembrane attachments or specialisations. A structural role is therefore unlikely. Mitochondria are frequently found in close association with these specialisations. Their possible role as receptors or transmembrane transport systems is discussed.

Evidence of a new, preferentially Mg-carrying, transport system besides the fast Na and the slow Ca channels in the excited myocardial sarcolemma membrane

MgCl 2 added to a K-rich (19 m m Ca-deficient (0.4 m m) Tyrode solution in a suitably high concentration (14 to 34 m m Mg) maintains or restores electric excitability, overshoot and propagated action potentials. The experiments reveal the true existence of a special transport system for the Mg entry into the ventricular myocardium that can be distinguished from both the fast Na and the slow Ca channels by means of certain drugs and appropriate electrophysiological techniques. The transmembrane transport systems for Na, Mg, and Ca ions differ with respect to their sensitivities to graded membrane depolarizations. The Na channel is most easily inactivated at a reduced level of membrane potential, whereas the Ca channel is most resistant. The Mg channel has an intermediate position. Ca-mediated action potentials are selectively blocked by specific Ca-antagonists such as verapamil, D 600, nifedipine or diltiazem. However, Mg-carried action potentials are refractory to these inhibitors. Tetrodotoxin and lidocaine are capable of suppressing both the transmembrane Na and Mg influxes without affecting the slow Ca channel. β-Adrenergic catecholamines influence the myocardial Mg and Ca currents in an exactly opposite sense: β-adrenergic substances largely promote myocardial Ca uptake and Ca-dependent action potentials whereas they totally block the transmembrane Mg fluxes into the partially depolarized myocardial fibres and, thereby, inhibit every Mg-dependent biolectric activity. Hence the usual “two-channel concept” that only allowed for a fast Na and a slow Ca inward current, but ignored the existence of a third transmembrane transport system that preferentially carries Mg ions into the ventricular myocardium, has to be considered as an oversimplification. It is suggested that the reciprocal effects of β-adrenergic catecholamines on the inward fluxes of Ca and Mg ions serve to maximize the contractile responses to β-receptor stimulation. The intensification by β-adrenergic catecholamines of the Ca influx simultaneously with a restriction of the transmembrane supply of Ca-antagonistic Mg ions will activate Ca-dependent excitation-contraction coupling of the ventricular myocardium in the most effective manner.

Cation transport alteration associated with plasmid-determined resistance to cadmium in Staphylococcus aureus.

Plasmid-determined resistance to cadmium has only been found with plasmids from Staphylococcus aureus. Resistance to cadmium was associated with a lower accumulation of Cd(2+) ions by the plasmid-bearing resistant cells. Cadmium accumulation by susceptible cells was energy dependent and had those characteristics usually associated with a transmembrane active transport system. There was a specific interrelationship between cadmium accumulation and manganese accumulation and retention. Cd(2+) inhibited the uptake of Mn(2+) and accelerated the loss of intracellular Mn(2+) by the susceptible cells, but was without effect on Mn(2+) transport in resistant S. aureus cells. Under similar conditions, there was no differential effect of Cd(2+) on Mg(2+), Zn(2+), Co(2+), Ni(2+), or Rb(+) accumulation or exchange between the susceptible and the resistant strains.

A Kinetic Study of Calcium Transport by Heart Mitochondria

Initial velocities of energy‐dependent Ca2+ uptake in cardiac mitochondria were measured with the ethyleneglycol bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid/ruthenium red quenching technique. Accurate concentrations of free Ca2+ between 1 and 15μM were generated with ATP as a Ca2+ buffer system. Using the method of iterative approximation, the accurate value of the initial velocity of ATP‐driven 45Ca uptake, and the time (t0.5) for half‐maximal uptake can be calculated.The rate of initial Ca2+ uptake increased in response to the rising free Ca2+ concentrations in the medium (in the presence of 0.6–5.0 mM ATP). A maximal rate was obtained between 10 and 15 μM of free Ca2+. For different ATP concentrations (0.6–5.0 mM), the half‐maximal rate of Ca2+ transport (Km) was observed between 4.1 and 7.4 μM Ca2+ (with a mean Km of 5.6 ± 0.6 μ Ca2+; n= 5). Hill plots of the data yield straight lines with an average slope n, of 1.93 ± 0.29, indicating the existence of two binding sites for Ca2+ in the transmembrane transport system. Keeping the free Ca2+ concentration on a constant level (6 μM), the rate of initial Ca2+ uptake also increased continually with rising ATP concentrations (maximum velocity v= 7.1 nmol of Ca2+× mg protein−1× s−1).From energy‐independent calcium‐ion binding studies with cardiac mitochondria, a dissociation constant (Kd) of 26.8 × 10−6M and a total number of external binding sites (n) of 12.6 nmol of Ca2+/mg of protein was obtained.The data are discussed with regard to the physiological role which cardiac mitochondria may play as a reversible storage site during the contraction relaxation cycle of the heart.