Mono- and divalent cations modulate the affinities of brain D1 and D2 receptors for dopamine by a mechanism independent of receptor coupling to guanyl nucleotide binding proteins

Summary In order to clarify the question of whether the modulatory effects of cations on dopamine receptor affinities are brought about by shifts in the equilibrium of receptor — G-protein — coupling, it was investigated whether mono- and divalent cations were still able to modulate rat striatal D1 and D2 receptor affinities after selective inactivation of the G-proteins linked to the two receptors. The G_S-protein coupled to the D1 receptor was eliminated by mild thermal inactivation, and the G_i- (or G_o-) protein associated with the D2 receptor by alkylation with a low concentration of N-ethyl-maleimide. Incubation of striatal membranes at 60°C completely abolished the specific binding of³H-GTP. Both treatments resulted in an increase of the IC₅₀-values for dopamine as a displacer of³H-SCH 23390 from D1- and of³H-spiperone from D2 receptors. Concomitantly, the formerly shallow D1 displacement curves became steeper, with their Hill coefficients increasing. This effect was less evident at D2 receptors. Guanosine triphosphate (GTP), which increased the IC₅₀'s of dopamine for both receptors approximately two-fold in control membranes, was without effect in pretreated samples, indicating an effective inactivation of the G-proteins. Na⁺ ions were still able to lower, and Ca²⁺ ions to increase the affinities of D1 and D2 receptors for dopamine after such inactivation of the respective G-proteins. It is concluded that the mechanism underlying the regulation of dopamine receptor affinities by mono- and divalent cations is independent of and superimposed upon the coupling of these receptors to guanyl nucleotide binding proteins.Abbreviations ANOVA Analysis of variance; G-proteins, guanyl nucleotide binding proteins (G_s: stimulatory, G_i: inhibitory); - GTP guanosine-5-triphosphate; Gpp(NH)p, 5-guanylylimidodiphos-phate; - NEM N-ethyl-maleimide     

4-aminoquinoline analogs of chloroquine with shortened side chains retain activity against chloroquine-resistant Plasmodium falciparum.

We have synthesized several 4-aminoquinolines with shortened side chains that retain activity against chloroquine-resistant isolates of Plasmodium falciparum malaria (W. Hofheinz, C. Jaquet, and S. Jolidon, European patent 94116281.0, June 1995). We report here an assessment of the activities of four selected compounds containing ethyl, propyl, and isopropyl side chains. Reasonable in vitro activity (50% inhibitory concentration, < 100 nM) against chloroquine-resistant P. falciparum strains was consistently observed, and the compounds performed well in a variety of plasmodium berghei animal models. However, some potential drawbacks of these compounds became evident upon in-depth testing. In vitro analysis of more than 70 isolates of P. falciparum and studies with a mouse in vivo model suggested a degree of cross-resistance with chloroquine. In addition, pharmacokinetic analysis demonstrated the formation of N-dealkylated metabolites of these compounds. These metabolites are similarly active against chloroquine-susceptible strains but are much less active against chloroquine-resistant strains. Thus, the clinical dosing required for these compounds would probably be greater for chloroquine-resistant strains than for chloroquine-susceptible strains. The clinical potential of these compounds is discussed within the context of chloroquine's low therapeutic ratio and toxicity.     

Ca(2+) requirement for high-affinity gamma-aminobutyric acid (GABA) binding at GABA(B) receptors: involvement of serine 269 of the GABA(B)R1 subunit

The gamma-aminobutyric acid (GABA) receptor type B (GABA(B)R) is constituted of at least two homologous proteins, GABA(B)R1 and GABA(B)R2. These proteins share sequence and structural similarity with metabotropic glutamate and Ca(2+)-sensing receptors, both of which are sensitive to Ca(2+). Using rat brain membranes, we report here that the affinity of GABA and 3-aminopropylphosphinic acid for the GABA(B)R receptor is decreased by a factor >10 in the absence of Ca(2+). Such a large effect of Ca(2+) is not observed with baclofen or the antagonists CGP64213 and CGP56999A. In contrast to baclofen, the potency of GABA in stimulating GTPgammaS binding in rat brain membranes is also decreased by a factor >10 upon Ca(2+) removal. The potency for Ca(2+) in regulating GABA affinity was 37 microM. In cells expressing GABA(B)R1, the potency of GABA, but not of baclofen, in displacing bound (125)I-CGP64213 was similarly decreased in the absence of Ca(2+). To identify residues that are responsible for the Ca(2+) effect, the pharmacological profile and the Ca(2+) sensitivity of a series of GABA(B)R1 mutants were examined. The mutation of Ser269 into Ala was found to decrease the affinity of GABA, but not of baclofen, and the GABA affinity was found not to be affected upon Ca(2+) removal. Finally, the effect of Ca(2+) on the GABA(B) receptor function is no longer observed in cells coexpressing this GABA(B)R1-S269A mutant and the wild-type GABA(B)R2. Taken together, these results show that Ser269, which is conserved in the GABA(B)R1 protein from Caenorhabditis elegans to mammals, is critical for the Ca(2+)-effect on the heteromeric GABA(B) receptor.     

Selected amino acids, dipeptides and arylalkylamine derivatives do not act as allosteric modulators at GABAB receptors

Based on recent reports describing enhancing actions of arylalkylamines (fendiline [N-(3,3-diphenylpropyl)-alpha-methylbenzylamine] and prenylamine [N-(3,3-diphenylpropyl)-alpha-methylphenethylamine]), amino acids (L-phenylalanine, L-leucine and L-isoleucine), and dipeptides (L-Phe-Phe and L-Phe-Leu) on baclofen-induced responses in cortical slices, we have examined whether these compounds might act as positive allosteric modulators at GABA(B) receptors. Unlike the previously described allosteric GABA(B) receptor modulator CGP7930 (2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol), these compounds did not enhance GABA(B) receptor-mediated guanosine 5'-O-(3-thiotriphosphate) [GTP(gamma)35S] binding in native or recombinant cell membrane preparations. Similarly, in a competition binding assay using the antagonist radioligand [3H]CGP62349, CGP7930, but not the other compounds, enhanced the affinities of gamma-aminobutyric acid (GABA) for native GABA(B) receptors from rat brain cortex. Finally, in a cellular assay (Ca(2+) signaling in a recombinant cell line), CGP7930 was again the only compound found to enhance the GABA response. It is concluded that the arylalkylamines, amino acids and dipeptides tested do not act as allosteric modulators at native and recombinant GABA(B) receptors.     

Effect of three anaesthetic techniques on isometric skeletal muscle strength

Background. Our aim was to quantify human involuntary isometricskeletal muscle strength during anaesthesia with propofol, sevoflurane,or spinal anaesthesia using bupivacaine. Methods. Thirty-three healthy patients undergoing anaesthesiafor elective lower limb surgery were investigated. Twenty-twopatients received a general anaesthetic with either propofol(n=12) or sevoflurane (n=10); for the remaining 11 patientsspinal anaesthesia with bupivacaine was used. We used a non-invasivemuscle force assessment system before and during anaesthesiato determine the contractile properties of the ankle dorsiflexormuscles after peroneal nerve stimulation (single, double, triple,and quadruple stimulation). We measured peak torques; contractiontimes; peak rates of torque development and decay; times topeak torque development and decay; half-relaxation times; torquelatencies. Results. Males elicited greater peak torques than females, medians6.3 vs 4.4 Nm, respectively (P=0.0002, Mann-Whitney rank-sumtest). During sevoflurane and propofol anaesthesia, muscle strengthdid not differ from pre-anaesthetic values. During spinal anaesthesia,torques were diminished for single-pulse stimulation from 3.5to 2.0 Nm (P=0.002, Wilcoxon signed rank test), and for double-pulsefrom 7.6 to 5.6 Nm (P=0.02). Peak rates of torque developmentdecreased for single-pulse stimulation from 113 to 53 Nm s^–1and for double pulse from 195 to 105 Nm s^–1. Torque latencieswere increased during spinal anaesthesia. Conclusions. At clinically relevant concentrations, propofoland sevoflurane did not influence involuntary isometric skeletalmuscle strength in adults, whereas spinal anaesthesia reducedstrength by about 20%. Muscle strength assessment using a devicesuch as described here provided reliable results and shouldbe considered for use in other scientific investigations toidentify potential effects of anaesthetic agents. Br J Anaesth 2004; 92: 367–72