Receptor binding properties of di (1,N6-ethenoadenosine) 5', 5'''-P1, P4-tetraphosphate and its modulatory effect on extracellular glutamate levels in rat striatum.

Our aim was to investigate the neuromodulatory role of diadenosine tetraphosphate (Ap(4)A). Ap(4)A-binding sites were detected in striatum and hippocampus membranes using [(35)S]-ADP beta S as radioligand and Ap(4)A and epsilon-(Ap(4)A), di-ethenoadenosine tetraphosphate, as displacers. Effects of epsilon-(Ap(4)A) on extracellular glutamate levels were studied using intracerebral perfusion. Both areas contain high-affinity binding sites for [(35)S]-ADP beta S with K(d) values in the low nM range. [(35)S]-ADP beta S binding was displaced by Ap(4)A and epsilon-(Ap(4)A). At 1 and 10 microM doses, epsilon-(Ap(4)A) markedly decreased glutamate levels in the striatum. The possibility of Ap(4)A acting as an endogenous modulator of excitatory neurotransmission is discussed.

Multiple roles of the conserved key residue arginine 209 in neuronal nicotinic receptors.

We have examined the role of a highly conserved arginine (R209), which flanks the M1 transmembrane segment of nAChRs, in the biogenesis and function of neuronal nAChRs. Point mutations revealed that, in alphaBgtx-sensitive neuronal alpha7 nAChRs, the conserved arginine is required for the transport of assembled receptors to the cell surface. By contrast, R209 does not play any role in the transport of assembled alpha-Bgtx-insensitive neuronal alpha3beta4 nAChRs to the cell surface. However, a basic residue at this position of alpha3 and beta4 subunits is necessary for either synthesis, folding, or assembly of alpha3beta4 receptors. Moreover, electrophysiological experiments revealed that in alpha3beta4 receptors the conserved arginine of the alpha3 subunit is involved in either coupling agonist binding to the channel or regulating single channel kinetics.

Human diadenosine triphosphate hydrolase: preliminary characterisation and comparison with the Fhit protein, a human tumour suppressor.

Human platelets diadenosine triphosphatase was characterised and compared with the Fhit protein, a human tumour suppressor with diadenosine triphosphatase activity. Both enzymes exhibit similar Km, are similarly activated by Mg2+, Ca2+ and Mn2+, and inhibited by Zn2+ and suramin. However, they are differentially inhibited by Fhit antibodies and exhibit differences in gel-filtration behaviour.

Potent inhibition of specific diadenosine polyphosphate hydrolases by suramin.

The cytosolic enzymes asymmetrical diadenosine tetraphosphate hydrolase (EC 3.6.1.17, Ap4Aase) and diadenosine triphosphate hydrolase (EC 3.6.1.29, Ap3Aase) are inhibited competitively by suramin. Ap4Aase and Ap3Aase were assayed in cytosolic rat brain extracts using fluorogenic analogues of the respective substrates diadenosine tetraphosphate (Ap4A) and diadenosine triphosphate (Ap3A). Ki values for suramin as inhibitor of Ap4Aase and Ap3Aase were 5 x 10(-6) M and 3 x 10(-7) M, respectively. Results indicate that suramin or suramin-like derivatives may be useful tools to investigate diadenosine polyphosphate cleaving enzymes and that the intracellular diadenosine polyphosphate metabolism may be a pharmacological target of suramin with biological and clinical implications.

Acetylcholine receptor subunit homomer formation requires compatibility between amino acid residues of the M1 and M2 transmembrane segments.

The neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha3 and alpha7 have different assembly behavior when expressed in heterologous expression systems: alpha3 subunits require other subunits to assemble functional nAChRs, whereas alpha7 subunits can produce homomeric nAChRs. A previous analysis of alpha7/alpha3 chimeric constructs identified a domain comprising the first putative membrane-spanning segment, M1, as essential to homomeric assembly. The present study dissected further this domain, identifying three amino acid residues, which are located at the most intracellular third of the M1 transmembrane segment, as important in the assembly of homomers. Moreover, formation of homooligomeric complexes seems to require a compatible accommodation between this region and certain residues of the second transmembrane segment, M2. Thus, compatibility between defined domains of the M1 and M2 transmembrane segments appears as a determinant factor governing homomer association of nAChR subunits.

Apolipoprotein E polymorphism influence on lipids, apolipoproteins and Lp(a) in a Spanish population underexpressing apo E4.

The apolipoprotein E (apo E) polymorphism in a Spanish working population of Tenerife (Canary Islands, Spain) was analyzed. The distribution of apo E alleles (epsilon 3, 0.850; epsilon 2, 0.075; epsilon 4, 0.075) and phenotypes (E3/3, 72.6%; E3/4, 13%; E3/2, 11.5%; E4/4, 0.8%; E2/2, 1.5%; E4/2, 0.5%) was significantly different from those of a combined Caucasian population owing to a lower frequency of apo E4. We have also investigated the effect of apo E polymorphism on serum levels of cholesterol, LDL-cholesterol, HDL-cholesterol, Lp(a) and apolipoproteins A-I, B and E. The average effect of E4 (in whole sample and men only, respectively) was to raise serum levels of total cholesterol (by 4.1 mg/dl and 8.3 mg/dl), LDL-cholesterol (by 6.5 mg/dl and 9 mg/dl), and apo B (5.3 mg/dl and 4.5 mg/dl). The average effect of E2 was to lower serum levels of total cholesterol (by 14.8 mg/dl mg/dl and 8.3 mg/dl), LDL-cholesterol (by 20.2 mg/dl and 15.5 mg/dl) and apo B (11.5 mg/dl and 6.5 mg/dl), and to raise apo E (1.14 mg/dl and 3.4 mg/dl and 3.4 mg/dl). We found significantly higher serum triglyceride levels in individuals carrying E4, but no differences were found in serum HDL-cholesterol, apo A-I or Lp(a) by alleles. Data confirm previous reports about an underexpression of apo E4 in societies living in Southern Europe, and its repercussion in a more beneficial lipid profile and relatively low cardiovascular mortality rate in the Mediterranean region.

Use of serum cholesterol/triglyceride ratio to discern for which individuals the Friedewald formula can be used confidently.

The values of low-density lipoprotein cholesterol obtained according to the Friedewald formula (Clin Chem 1972; 18:499-502), or by the De Long transformation (J Am Med Assoc 1986;256:2372-7), were compared with the values obtained when the individual cholesterol/triglyceride ratio of very-low-density lipoprotein was used for estimating the contribution of this lipoprotein to the total cholesterol. We found that these formulas gave the greatest errors for individuals with a low serum cholesterol/triglyceride ratio. We propose criteria for deciding when the numerically calculated value of low-density cholesterol is appropriate, and when it is not.