Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei.

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.

Transient expression of a purine-selective nucleoside transporter (SPNTint) in a human cell line (HeLa).

PURPOSE: The goal of this study was to develop a mammalian expression system for the cloned rat intestinal, Na(+)-dependent, purine-selective nucleoside transporter (SPNTint) and to study the interactions of nucleosides and nucleoside analogs with this transporter. METHODS: Lipofection was used to transfect HeLa cells with a mammalian expression vector (pcDNA3) containing the cDNA insert encoding SPNTint. Nucleoside transport activity was measured using [3H]inosine, [3H]uridine, [3H]-dideoxyinosine (ddI), and [3H]-2-chloro-2'-deoxyadenosine (2CdA) as model substrates. RESULTS: Expression of SPNTint was observed between 36 and 90 h post-transfection, with maximal expression at 66 h. At 66 h, Na(+)-stimulated uptake of [3H]inosine in cells transiently transfected with SPNTint was approximately threefold greater than that in cells transfected with empty vector (p < 0.05). The Na(+)-stimulated uptake of both inosine and uridine was saturable (K(m) = 28.1 +/- 7.1 microM and 20.6 +/- 5.6 microM, respectively) in the transfected cells and was significantly inhibited by the naturally occurring nucleosides (1 mM) inosine and uridine and to a lesser extent by thymidine. The nucleoside analogs ddI (IC50 = 46 microM) and 2CdA (IC50 = 13 microM) also significantly inhibited the Na(+)-stimulated uptake of [3H]inosine. A Na(+)-stimulated uptake of [3H]2CdA was observed suggesting that 2CdA is also a permeant of SPNTint. CONCLUSIONS: HeLa cells transiently transfected with SPNTint represent a useful tool to study the kinetics and interactions of drugs with SPNTint.

Mechanism of the inotropic action of inosine on canine myocardium.

The positive inotropic effect of inosine in intact dog hearts was approximately halved by beta-adrenergic blockade with sotalol. The same result was obtained in dogs with chronic denervation of the heart, in which the sympathetic nerve terminals had degenerated. Therefore part of the positive inotropic action of inosine appears to be non-adrenergic. Inosine infusion caused supersensitivity of the positive inotropic effect over a wide range of doses of adrenaline. It is postulated that inosine may act by blocking the desensitising action of cyclic AMP on the reaction between calcium ions and the contractile proteins.

Vasoconstriction after adenosine and inosine in the rat isolated hindlimb abolished by blockade of tryptaminergic mechanisms.

The isolated right hindlimb of the rat was perfused at a fixed flow rate through the femoral artery with heparinized blood from the carotid artery of a donor. Single injections of adenosine (1--300 microgram) induced a biphasic response, a long-lasting vasoconstriction preceded by a transient vasodilatation. Inosine (1--300 microgram) produced only vasoconstriction. After repeated administration of 300 microgram of these substances, the vasoconstriction became less prominent, and finally reverted to vasodilatation. The vasoconstrictor response to these substances (300 microgram) was also diminished or reverted to vasodilatation after pretreatment with reserpine or methysergide. From these results, it is concluded that vasoconstriction after adenosine or inosine may be mediated by 5-hydroxytryptamine released from the peripheral stores and that the intrinsic direct action of these substances on the femoral vascular bed is vasodilator.

Positive inotropic response to inosine in the in situ canine heart.

Effects of inosine on left ventricular contractile force, circumflex blood flow, heart rate, and arterial pressure were investigated in mongrel dogs. Infusion of 50 ml of 10, 25, or 50 mM inosine into the right atrium over 5 min produced arterial blood inosine concentrations of 20-120 microM. Infusion of inosine concentrations of 10 mM or greater produced statistically significant increases in contractile force and circumflex blood flow (P less than 0.05). The increases in contractile force and circumflex blood flow caused by 50 inosine were approximately 40% and 110%, respectively. No statistically significant increases in heart rate or arterial pressure were observed during infusion of inosine at any concentration. Administration of propranolol (2 mg/kg) in no way altered the effects of inosine on contractile force or circumflex blood flow. Thus, the present study suggests that inosine in concentrations which may be produced in the myocardium during stressful conditions causes a substantial effect on the inotropic state of the heart and that the effects of inosine are not mediated through adrenergic mechanisms.

Output of (14C)adenine nucleotides and their derivatives from cerebral tissues. Tetrodotoxin-resistant and calcium ion-requiring components.

1. Neocortical tissues, exposed briefly to [(14)C]adenine and containing over 98% of their (14)C as adenine nucleotides, when superfused with glucose-bicarbonate salines released about 0.1% of their (14)C content/min to the superfusate. 2. Addition of unlabelled adenosine to the superfusing fluid increased the (14)C output three- to four-fold; half-maximal increase was given by about 40mum-adenosine, and reasons are adduced for considering the activity of adenosine kinase to be a major factor in conditioning the (14)C output. Adenosine similarly increased the enhanced (14)C output caused by electrical excitation of the superfused tissue; it brought about only a small increase in tissue glycolysis. 3. Output of (14)C from the [(14)C]adenine-labelled tissues was increased when Ca(2+) was omitted from the superfusing fluids, but electrical stimulation did not then liberate more (14)C. Nevertheless, such tissues still responded to electrical stimulation by increased glycolysis, and their (14)C output again became susceptible to increase by electrical stimulation when Ca(2+) was restored. 4. The six-fold increase in tissue glycolysis caused by electrical excitation was almost completely inhibited by tetrodotoxin at 0.1mum and above, but this was associated with about 50% inhibition only in the output of (14)C from tissues preincubated with [(14)C]adenine. The (14)C-labelled compounds of which output was most inhibited by tetrodotoxin were adenosine, inosine and hypoxanthine whereas output in a nucleotide fraction was little affected.