Ruth Koren

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.

Uridine transport properties of mammalian cell membranes are not directly involved with growth control or oncogenesis.

A well-studied model for growth control is the serum activation of 3T3 cells (mouse fibroblasts). Uptake of uridine into these cells is one of the earliest events to be activated by serum [2]. At low uridine concentra- tions intracellular metabolic trapping (e.g., phospho- rylation) of uridine is activated rather than transport across the cell membrane [3]. Here we provide a kinetic analysis of the transport step in normal and in SV-40 transformed 3T3 cells. The Michaelis con- stant (Km) for transport in these and several other different cell lines is shown to be some 2 orders of magnitude greater than that in [4]. Our results suggest that no changes in transport of uridine occur in growth control or transformation. 2. Experimental 2.1.

The Relevance of the State of Growth and Transformation of Cells to Their Patterns of Metabolite Uptake

Publisher Summary This chapter discusses the interrelationship between the state of growth or transformation of cells in culture and their pattern of nutrient uptake. Alterations in the cell membrane lead to an increase in transport and, thus, to an increased supply of key nutrients. The increased intracellular concentration of these key nutrients enables malignant cells to proliferate in an uncontrolled manner or at least at a faster rate than their untransformed counterparts. In view of the significant amount of information that has accumulated in the field, it has become possible to test the validity of this hypothesis. The chapter presents the data related to the correlation between uptake rates and the state of growth or transformation of cells. Nutrients enter the cells by mediated, active, or passive transport across the plasma membrane. They are subsequently trapped within the cell by intracellular metabolic reactions, for instance, phosphorylation. The chapter presents the experimental and theoretical criteria that are available for differentiation between the two processes.

The effect of verapamil, lanthanum and local anesthetics on serotonin release from rabbit platelets

The effect of calcium blockers (verapamil, local anesthetics and lanthanum chloride) on serotonin release from rabbit platelets was studied. The following results were obtained: (1) Verapamil and tetracaine (but not lanthanum) caused a time- and dose-dependent release of serotonin. The curves describing the time-course and those describing the concentration dependence of the release were sigmoid, suggesting cooperativity. (2) Thrombin-induced release from the platelets was dependent upon extracellular sodium ions, while no dependence was observed for the drug-induced release. (3) The release by verapamil was partially inhibited by prostaglandin E1 and theophylline which are known to raise intracellular cAMP levels, but was unaffected by the prostaglandin-synthesis inhibitor, indomethacin. (4) Verapamil, tetracaine and lanthanum inhibited thrombin-induced release of serotonin. The curve of dose dependence of the inhibition by verapamil and tetracaine was not sigmoid. The inhibition by verapamil and tetracaine was reversed by extracellular calcium ions, but no effect of this ion on the drug-induced release reaction was observed. It is concluded that the serotonin release induced by some calcium blockers and the inhibition of the thrombin-induced release by the same drugs are two separate phenomena. It is suggested that verapamil and tetracaine-induced release are mediated by exocytotic processes brought about by the interference of the drugs with calcium distribution between the cytosol and storage compartments within the platelet.

Extracellular potassium can simulate the role of serum as an activator of uridine uptake by quiescent hamster cells in culture

Abstract Replacement of ~100 mM of sodium chloride in the extracellular medium of quiescent hamster fibroblasts (Nil 8 and BHK cells) by potassium chloride causes an increase in the rate of uridine uptake. This increase is identical with that achieved by addition of 10% serum to the same cultures. The effects of serum and KCl are not additive. The dependence of the rate of uridine uptake on extracellular KCl concentration is of a sigmoid nature. The time course of the activation process is similar to that of serum activation of uridine uptake in the same cells. The high rate of uridine uptake persists for at least 30 min after return to an extracellular medium containing a high concentration of sodium.

The interaction of ionophore A23187 with the uridine uptake system of quiescent and serum-activated hamster fibroblasts☆

Abstract We have studied the effect of the divalent metal ion ionophore A23187 on uridine uptake by quiescent Nil 8 cells in the presence and absence of dialysed serum and MgCl 2 . In the absence of serum at low (50 μM) Mg 2+ concentration, treatment of the cells with the ionophore caused a dramatic inhibition of uridine uptake. This inhibition is due to an effect on phosphorylation of the nucleoside rather than its transport across the plasma membrane. The inhibition did not occur in the presence of dialysed serum or at high concentrations of MgCl 2 . The protecting effect of Mg 2+ is saturable and the concentration at half-maximum uptake rate is of the same order as the K m for Mg 2+ of the uridine-phosphorylating system as assayed in cell-free extracts. The inhibition is completely reversed by the addition of 10% serum to cultures which were treated with ionophore at low Mg 2+ concentration. This activation occurs with no apparent lag even after 60 min exposure to the ionophore.

Magnesium ions and serum activation of uridine uptake by quiescent hamster fibroblasts

Received 19 July 1979 1. Introduction The uptake of uridine by quiescent cells in culture is one of the earliest processes stimulated by the addition of serum [ 1,2]. It has been shown that the intracellular trapping (phosphorylation) of uridine is activated rather than its transport across the cell membrane [3-51. Uridine kinase is the enzyme responsible for the first and rate limiting step of uridine trapping [3]. This enzyme requires the presence of Mg2+ as its cofactor [6]. Rubin proposed [7] that control of the availability of intracellular Mg” for transphosphorylation reactions and the synthesis of macromolecules has a key role in the cellular response to stimulation by serum. The effect of Mg2+ on the uptake of nutrients by cultures of chick embryo fibroblasts has been demonstrated [8]. McKeehan and Ham [9] have shown that the cellular multiplication rate and cellular survival rate are dependent upon the concentration of divalent metal ions in the growth medium. In this study we show that serum activation of uridine uptake by quiescent NIL 8 hamster fibroblasts is expressed only in the presence of divalent metal ions. MgC12 stimulates uridine uptake in the presence of serum in a saturable manner. Addition of Mg” affects both the time course and extent of activation by serum. 2. Experimental 2.1 . Cell

Water structure and unusual medium effects in the reaction between [Co(NH3)5B]2+ and Fe2+

Abstract The influence of electrolytes, of various alcohols at low concentrations, and of temperature on the rate of the electron transfer between [Co(NH3)5Br]2+ and Fe2+ has been investigated. Specific salt effects are reported. Systems of different electrolyte composition have significantly different energies of activation. Reaction mixtures containing HClO4 as the supporting electrolyte have positive ΔC#p. The rate constant as a function of alcohol concentration passes through a minimum at low temperatures. An isokinetic relationship is found to hold with dΔH#/dΔS# = 298°K. The observed effects are tentatively ascribed to the influence of water structure.

The effect of Verapamil, a calcium antagonist, on the serum-dependent regulation of uridine uptake by cells in culture☆

The stimulation of uridine uptake is one of the early events that occur when quiescent cells are stimulated to grow by adding fresh serum. Serum depletion of cells in their exponential phase of growth results in a decrease of uridine uptake rate, a process which is complete before a significant reduction in DNA synthesis is observed. Elevation of extracellular potassium can stimulate the role of serum as an activator of uridine uptake, and can prevent the inhibition of uptake that results from serum depletion. Verapamil, a known antagonist of calcium movement across biological membranes, inhibits uridine uptake. This inhibition is much stronger in serum- or KCl-activated cells than in serum-depleted or quiescent cells. Verapamil can partially prevent the serum-dependent stimulation of uridine uptake. The effect of Verapamil does not depend on the presence of calcium in the extra-cellular medium. Verapamil may interfere with serum-dependent redistribution of Ca2+ within the cells, and thus uncouple the initial event of binding of serum growth factors to membrane receptors, from the subsequent intracellular response-regulation of uridine uptake rates.

S-substituted derivatives of 6-mercaptopurine ribosides interact both with the transport and metabolic phosphorylation of uridine by virus-transformed hamster fibroblasts

The uptake of uridine by mammalian cells consists of transport of uridine across the plasma membrane followed by its metabolic conversion, mainly by phosphorylation. S-substituted aromatic derivatives of 6-mercaptopurine ribosides are potent inhibitors of the nucleoside uptake systems in human erythrocytes and in mammalian cells in culture and have been studied extensively. We present here a theoretical analysis which enables one to decide whether transport of metabolites, their metabolic trapping within the cell, or both, are susceptible to inhibition. This analysis was applied in the study of the effect of some inhibitors on uridine and cytosine-beta-D-arabinoside uptake by transformed Nil-8 cells. It was found that in Nil-SV cells, both transport and metabolic conversion are susceptible to inhibition by nitrobenzylmercaptoinosine and by dansylaminoethylmercaptoguanosine. Nitrobenzylmercaptoinosine displays inhibition constants of 20 and 7 nM for transport and phosphorylation, respectively, while for dansylaminoethylmercaptoguanosine the inhibition constants are 1.8 and 0.6 microM, respectively, for the same processes. Cytosine-beta-D-arabinoside is a synthetic nucleoside which is not metabolizable in Nil cells. Its uptake properties are determined by the transport mechanism alone. The transport of this nucleoside into Nil-SV cells in inhibited by nitrobenzylmercaptoinosine and the inhibition constant found is approx. 5 times greater than that for uridine.