Quantitative relationships between the measurable transport resistances of membrane carriers: theory and experiment.

A theoretical analysis is made of the possible quantitative relationships between the transport resistances that characterise membrane carrier systems. It is shown that there exist only five possible patterns in which to rank the four transport resistances. Symbolising these as A, B, C and D, the five possible patterns are (i) A = B = C = D; (ii) A = B much greater than C, D; (iii) A = B much greater than C = D; (iv) A = B = 2C = 2D; (v) A = 2B = 2C much greater than D. A survey of the available experimental data shows that pattern (ii) is the most prevalent, pattern (v) is often found and pattern (iii) has been identified. None of the ten transport systems so far analysed experimentally failed to fit one of the predicted patterns.

How the kinetic parameters of the simple carrier are affected by an applied voltage.

The kinetic parameters (K, R00, R12, R21 and Ree) of the simple carrier model are analysed as a function of applied voltage for various cases of charged substrates and carriers. If certain parameters are invariant with voltage, strong conclusions can be made as to the charge on the free carrier. In one particular case (where the parameter R00 is invariant while R12 and R21 display a non-linear dependence on the exponential of the voltage) it may be possible to identify the isomerisation of the carrier-substrate complex.

Multiple controls on the intracellular trapping of uridine.

Uridine uptake by mouse or hamster cells grown in conditions which support good growth is very sensitive to inhibition by cyanide and azide, at concentrations which only slightly reduce overall cellular ATP levels. Iodoacetate, when present alone, reduces uridine uptake only insofar as it reduces cellular ATP levels. At concentrations which by themselves do not affect uridine uptake, iodoacetate greatly reduces the sensitivity of uridine uptake to cyanide or azide. The effect of cyanide is on intracellular trapping of uridine and not on its transport into the cell. The specific effect of cyanide is confined to uridine and not found for the uptake of adenine, thymidine or 2-deoxyglucose. The effect is of rapid onset (within 2 min) and is rapidly reversible (also within 2 min). Phosphorylation of uridine in homogenised cells or in Triton X-100-permeabilised cells is unaffected by cyanide. The data are interpreted in terms of a model in which intracellular trapping of uridine is subject to multiple controls, including one regulated by some factor requiring intact functioning of the mitochondrion. These multiple control systems interact synergistically to affect trapping of uridine by the intact cell.

Zero-trans and infinite-cis uptake of galactose in human erythrocytes.

1. The zero-trans and infinite-cis uptake of galactose into human erythrocytes was measured as a function of galactose concentration at 20 degrees C. 2. A special procedure, the "cis-trans test" has been developed to determine the directionality of an asymmetric transport carrier. 3. Using the "cis-trans test" and results obtained by phloretin inhibition, could show the existance of two sites mediating galactose uptake. The kinetic parameters of the high affinity site are K1 equals 11 mM; V1 equals 16 mmol - cell unit-1-min-1 and of the low affinity site: K2 equals 286 mM; V2 equals 21 mmol-cell unit-2-min-1. 4. The infinite-cis Km, using an intergrated rate equation treatment was 21 mM and that found by a direct preloading procedure was 25 mM. The existence of a high affnity site at the inner side of the membrane was thus confirmed.

Testing the simple carrier using irreversible inhibitors.

1. We analyse the kinetics of irreversible inhibition of the simple carrier. We consider how the rate of inactivation is influenced by the concentrations of permeant on the two sides of the membrane. 2. We consider various kinetic schemes for the simple carrier and show that these are all indistinguishable kinetically, using steady-state transport or inactivation data. We point out the advantages of using the simplest kinetic scheme and the possible pitfalls of using more complicated schemes, including the conventional carrier model. 3. We show that in the absence of information on the transport properties of the system, irreversible inhibition data are ambiguous. Taken together with transport data, however, inactivation data provide new tests for the applicability of the simple carrier. 4. The new tests show that for the simple carrier model to be applicable, the substrate dependencies of transport and of inactivation must be identical in comparable experimental situations. Further, the maximal rates of transport and of stimulation (or inhibition) of inactivation must obey a simple relationship, which we derive. We illustrate the use of these tests with published data on the glucose and choline transport systems of the human red blood cell.

Regulatin of uridine uptake by serum and insulin in density-inhibited 3T3 cells.

Stimulation of nucleoside uptake in quiescent 3T3 cells by insulin and serum is preceded by a substantial lag phase. Our findings point to the length of the lag phase as a major target for regulation. The length of such phase varies markedly with the concentration of insulin (10(-9)-10(-6) M) or serum (0.5-10%) but it is not eliminated by high saturating levels of the activating agents. Further, variations in the temperature at which the stimulation process occurs (24-39 degrees C), addition of compounds like prostaglandin E1 (1-5 mug/ml) or theophylline (0.4 mM) and differences in the age of the cultures primarily affect the length of the lag time while the final uptake rates achieved are remarkably constant. Analysis of the temporal order of the events in the lag phase reveals that there is a discrete temperature-sensitive period located in the early and middle part of the lag, while the prostaglandin E1-sensitive step(s) appear to be toward the end of the lag. The transition from the basal to the stimulated rate of uptake is abrupt. Indeed, the kinetics of activation does not fit a sample exponential law but a high power of an exponential, suggesting that the switching mechanism involves cooperative steps. Since the transition is abrupt, the nucleoside uptake system exists largely in two alternative states either switched off or on. The regulation of the lag period is by the control of the time at which this switch occurs. On the basis of the data presented here, we propose a working hypothesis of uptake stimulation.

Temperature dependence of uridine transport in quiescent and serum-stimulated 3T3 cells.

(1) The kinetics of uptake of uridine into 3T3 cells have been measured as a function of concentration in the temperature range 5-37 degrees C, for both quiescent and serum-stimulated cells. (2) The maximun velocity of uridine uptake is increased some ten-fold by adding serum, but the hald-saturation concentration is not systematically affected in this temperature range. (3) A detailed study of the temperature dependence of the maximum velocity of transport in the range 4-43 degrees C shows that the activation energy of uridine transport is not increased following serum activation. (4) The data suggest that any change in membrane fluidity that might occur as a result of serum activation does not in itself lead to a more rapid rate of turn over of the individual uridine carriers. It would appear, rather, that there is an increase in the number of functional uridine carriers.

Kinetic tests of models for sugar transport in human erythrocytes and a comparison of fresh and cold-stored cells.

We studied the time course of the entry of galactose into human erythrocytes from an external concentration of 500 mM, and analyzed the data by an integrated rate equation treatment. We found evidence for only a single, high-affinity site for sugar at the inner face of the membrane. We studied the effect of pre-loading cells with galactose at various concentrations on the entrance of galactose into the cell from 128 mM, and compared the result we found with a previous report of a similar experiment from 500 mM external sugar. We found no evidence of other than a high affinity for sugar at the inner face of the membrane. The data reject a model in which sugar transport occurs on two asymmetric, oppositely directed carriers. We studied exchange of glucose into and out of the cells as a function of sugar concentration, taking care to minimize metabolism of sugar. We found no evidence for other than a single component for glucose exchange. Our data reject the 'allosteric pore' model for sugar transport. The explanation of the high-affinity site for sugar at the inner membrane face thus remains enigmatic. We find a very significant difference in the kinetics of glucose exchange when we compare freshly drawn and long cold-stored blood. The Km for exchange was almost twice as large for cold-stored as for fresh blood.

Co-operative, competitive and non-competitive interactions between modulators of P-glycoprotein.

We measured the effects of individual modulators and of pairs of modulators of the multidrug resistance pump, P-glycoprotein, on the accumulation of labelled daunomycin into multidrug-resistant P388 leukemia cells at 37 degrees C and developed a kinetic analysis which enables such data to be modelled in terms of co-operative, competitive or non-competitive interaction between pairs of modulators. The modulators verapamil, cyclosporin and trifluoperazine interacted with P-glycoprotein as single molecules, while vinblastine, mefloquine, dipyridamole, tamoxifen and quinidine displayed Hill numbers close to 2, suggesting that pairs of modulator molecules need to act together in order to bring about effective reversal of P-glycoprotein. When the modulators were presented to P-glycoprotein in pairs, we found examples of both competitive and non-competitive behaviour. We interpret these results on a model in which two modulatory sites exit on the MDR pump. To one of these, mefloquine, vinblastine and tamoxifen bind preferentially; to the other, verapamil, dipyridamole, trifluoperazine and quinidine bind (but mefloquine and tamoxifen only weakly if at all). Cyclosporin A can interact with both sites.

Mutually co-operative interactions between modulators of P-glycoprotein.

We measured the effects of combinations of verapamil, vinblastine, mefloquine, and tamoxifen, all being modulators of the multidrug resistance pump, P-glycoprotein, on the accumulation of labelled daunomycin into multidrug-resistant P388 leukemia cells at 37 degrees C. We found that, contrary to our initial expectations (based on Ayesh, Shao and Stein (1996) Biochim. Biophys. Acta 1316, 8), vinblastine, mefloquine, and tamoxifen all appeared to interact with one another synergistically, i.e. by the kinetics of a non-competitive interaction. A simple kinetic analysis showed that pairs of co-operating modulators can give apparent non-competitive behaviour, but refined kinetic analysis enables the two types of interaction to be distinguished. The modulators vinblastine, mefloquine, and tamoxifen thus appear to co-operate with one another in pairs to bring about reversal of P-glycoprotein. This may have important implications for the design of new modulators of P-glycoprotein.

The kinetic dissection of transport from metabolic trapping during substrate uptake by intact cells. Uridine uptake by quiescent and serum-activated Nil 8 hamster cells and their murine sarcoma virus-transformed counterparts.

1. We present a theoretical analysis of the tandem processes of transport and metabolic trapping which together constitute uptake of a substrate by intact cells. 2. Transport is assumed to occur by means of a simple carrier here analysed in its general form. Trapping is assumed to occur by a simple enzymic reaction. 3. We show how to obtain the separate parameters of the steps by analysing uptake data over a range of uptake times and substrate concentrations. 4. We present uptake data for uridine and cytosine-beta-D-arabinoside entering Nil 8 hamster fibroblasts, normal and murine sarcoma virus transformed, in the quiescent condition and after stimulation by added serum. We analyse the data in terms of the theory for tandem processes. 5. Transport is characterised by a system having a high Km and a high V for entry. The data for cytosine-beta-D-arabinoside suggest that the cytosine-beta-D-arabinoside system is not far from a symmetric one. The data for uridine transport do not differ when quiescent and serum-activated cells are compared. Transformed cells transport uridine at half the maximum velocity of normal cells, with or without added serum. 6. Trapping of cytosine-beta-D-arabinoside is insignificant. Trapping of uridine occurs by a system with both V and Km at least an order of magnitude smaller than are these parameters for transport. Trapping of uridine by non-transformed cells activated by serum, has twice the V of such cells in the quiescent state. 7. In the virus-transformed cells, the control of uridine trapping by added serum is lost, along with control of growth by this stimulant.

ATPase activity of P-glycoprotein related to emergence of drug resistance in Ehrlich ascites tumor cell lines.

We have characterized the ATPase activity of a sensitive and five progressively daunorubicin resistant Ehrlich ascites tumor cell lines passaged in mice. For the nine different modulators of drug resistance that we have studied, the ATPase activity first rose with the modulator concentration and then declined. We analyzed the ATPase activity profiles in terms of an activation constant and an inhibition constant for each of the nine drugs and six cell lines. In this series of cell lines, the drug-stimulatable ATPase activity was directly proportional to the amount of P-glycoprotein. Pumping of daunorubicin was also correlated with the amount of P-glycoprotein, except that, for a highly passaged line more daunorubicin was pumped than could be accounted for by the content of P-glycoprotein. Between the 12th and the 36th passage an additional source of resistance emerged, which was not correlated with P-glycoprotein. Pumping of daunorubicin was negatively correlated with the cell volume for the different lines.

Structure-activity relationships of P-glycoprotein interacting drugs: kinetic characterization of their effects on ATPase activity.

We have determined the kinetic parameters for stimulation and inhibition by 34 drugs of the P-glycoprotein ATPase in membranes derived from CR1R12 Chinese hamster ovary cells. The drugs chosen were sets of calmodulin antagonists, steroids, hydrophobic cations, hydrophobic peptides, chemotherapeutic substrates of P-glycoprotein, and some other drugs with lower affinity for P-glycoprotein. We studied how these kinetic parameters correlated with the partition coefficient and the Van der Waals surface area of the drugs. The maximum velocity of ATPase stimulation decreased with surface area and showed a suggestion of a maximum with increasing partition coefficient. The affinity of these drugs for P-glycoprotein showed no significant correlation with partition coefficient but was highly correlated with the surface area suggesting that binding between modulators and P-glycoprotein takes place across a wide interaction surface on the protein.

Competitive, non-competitive and cooperative interactions between substrates of P-glycoprotein as measured by its ATPase activity.

We have studied the interaction between verapamil and other modulators of the P-glycoprotein ATPase from membranes of CR1R12 Chinese hamster ovary cells. Four major categories of interaction were identified. (i) Non-competitive inhibition of verapamil's stimulation of enzyme activity was found with vanadate. (ii) Competitive inhibition of the ATPase was found for the pair verapamil and cyclosporin A. (iii) Allosteric inhibition with an increase in the Hill number for verapamil was found in the cases of daunorubicin, epirubicin, gramicidin S and D, vinblastine, amiodarone, and colchicine. (iv) Cooperative stimulation of verapamil-induced ATPase activity was found with progesterone, diltiazem, amitriptyline, and propranolol. At high levels, progesterone and verapamil mutually enhanced each other's inhibitory action on the ATPase. Our data show that the substrate binding behavior of P-glycoprotein is complex with more than one binding site being present. This information could form the basis for the development of improved modulators of P-glycoprotein.

A high affinity site for sugar transport at the inner face of the human erythrocyte membrane?

A disagreement centering on a method of analysis as to the existence of a high affinity site for glucose transport at the inner face of the human red cell membrane is resolved by using direct fitting methods to confirm the original parameter estimates.

Intra-protein interactions across a fluid membrane as a model for biological transport.

A model is proposed for the mechanism of action of the glucose transport system of the human erythrocyte. The model is based on the possibility of there being interaction through the membrane between superficially disposed protein subunits, these units being embedded within the bimolecular lipid layer, anchored to the aqueous phase, perhaps mobile in the plane of each face of the membrane. The subunits have the ability to bind sugar and, when associated with the symmetrical protein at the opposite face of the membrane, transfer sugar across the membrane. Evidence for the model is presented. The possibility that this model may also be a model for the cell membrane as such is briefly touched upon.

Diffusion within egg lecithin bilayers resembles that within soft polymers.

An analysis is presented of how the permeability coefficient/octanol:water partition coefficient ratio for 33 different chemical substances crossing egg lecithin bilayers depends on the molecular volume of the substances. From this analysis we conclude that bilayers made from egg lecithin behave as soft polymers in their discrimination between permeants of different sizes and shapes.

Synergistic effect of prochlorperazine and dipyridamole on the cellular retention and cytotoxicity of doxorubicin.

Incubation of drug-resistant human tumor cells with a combination of prochlorperazine and dipyridamole has additive/synergistic effect on the cellular retention and cytotoxicity of doxorubicin. In patients administered a fixed dose of doxorubicin and prochlorperazine with escalating doses of dipyridamole, mean plasma levels of dipyridamole and prochlorperazine achieved were as high as 3.01 +/- 0.41 microm and 0.94 +/- 0.09 microm, respectively. Plasma samples from patients were analyzed in an in vitro assay to monitor the effect on the cellular retention of tritium-labeled daunorubicin in MDR1-transfected P388 cells. In 22 of 49 of the plasma samples analyzed, the daunorubicin in efflux blocking activity was one-half or greater than that of cells incubated with 12.5 microM verapamil, a well-known efflux blocker. These observations suggest that a combination of prochlorperazine and dipyridamole may enhance cellular doxorubicin retention by blocking efflux while reducing normal tissue toxicity and unwanted side effects in vivo.

Intractable cancers: the many faces of multidrug resistance and the many targets it presents for therapeutic attack.

Some types of cancer respond far less favorably to treatment than do others. A quantitative estimate of this intuition can be obtained from the SEER (Surveillance, Epidemiology and End-Results) Cancer Statistics Review. Of particular interest, from a drug resistance perspective, are the five-year survival data for patients presenting with tumors that were diagnosed as "distant". A good correlation can be found between those numbers and an estimate of treatment successes obtained from a survey of current literature on chemotherapy applied to cancers originating from these various tissues. These two measures, considered together, define "the axis of intractability", a parameter that characterizes the (possibly) inherent, physiological basis of the tissue-by-tissue intractability of cancers. Exploring the basis of this intractability, it appears that factors other than the classical ABC transporter-based, multidrug resistance systems probably play a major role. An ineffective DNA repair system, coupled to reduced apoptosis, is the basis for the inherent tractability of testicular cancer. For other tissues, important contributions to resistance arise from cell adhesion-mediated drug resistance, which is overcome when cells are released from tissues during anoikis. Making a direct comparison between gene expression in solid tumors and their corresponding cell lines, genes controlling the extracellular matrix and cell-cell communication appear among the genes that are over-expressed in the solid tumors, while genes coding for the protein biosynthesis system are over-expressed in the cell lines. The more tractable cancers are closer to the cell lines in their expression profiles of these sets of genes.