Molecular mechanisms for nanomolar concentrations of neurosteroids at NR1/NR2B receptors.

Neurosteroids are endogenous steroids acting in the central nervous system. They participate in synaptic plasticity, memory and learning, Alzheimer's disease, and certain drug reward. Some mechanisms behind these effects are thought to be nongenomic, e.g., they modulate the function of the N-methyl-d-aspartate (NMDA) receptor complex. In this study, we used a Chinese hamster ovary cell line stably transfected with NMDA receptor constituents NR1/NR2B, to investigate the effects of nanomolar concentrations of the neurosteroids pregnenolone sulfate (PS) and pregnanolone sulfate (3alpha5betaS) on binding of the radioligand [(3)H]ifenprodil. Neither of the steroids displaced [(3)H]ifenprodil, but both induced a shift in its fit from one to two binding sites. The effects of the neurosteroids were also measured as changes in intracellular calcium ([Ca(2+)](i)) after glutamate stimulation. Although the steroids did not alter the response to glutamate, they influenced the extent of ifenprodil blockade of the receptor: PS increased and 3alpha5betaS decreased this effect. The coincubation of several NMDA receptor ligands in the assay indicated that PS and 3alpha5betaS act via different binding sites from those for glutamate, glycine, and dithiothreitol. Combining the two steroids revealed that they do not share a common binding site. In conclusion, these results substantiate previous evidence of the allosteric modulatory effect induced by PS and 3alpha5betaS on NMDA receptors at nanomolar concentrations. The neurosteroid-mediated modulation of the receptor is also reflected in an altered glutamate stimulated [Ca(2+)](i), in response to ifenprodil.

Neurosteroids alter glutamate-induced changes in neurite morphology of NG108-15 cells.

Activation of the NMDA receptor leads to increased intracellular Ca2+ levels ([Ca2+]i) which induces outgrowth of and morphologic changes in the neurites of the NG108-15 cell line. This effect can be blocked by antagonists for this glutamate receptor subtype (e.g. ifenprodil or AP5). We have previously shown that nanomolar concentrations of various neurosteroids modulate ifenprodil binding to the NMDA receptor. To investigate whether this interaction affects the functioning of the receptor, we studied the effect of 24 and 48 h of pregnenolone sulphate (PS) or pregnanolone sulphate (3alpha5betaS) on glutamate-stimulated NG108-15 cells. Unexpectedly, the neurosteroids themselves had an inhibitory effect on glutamate-induced changes in neurite patterns. This effect was comparable to that of ifenprodil or AP5. Moreover, the effect of combined treatment with 3alpha5betaS and ifenprodil on neurite morphology indicated a functional interaction between the substances. Interestingly, PS induced cell detachment over time, an effect that was further enhanced by ifenprodil. Cell detachment was also seen after 48 h of treatment with 3alpha5betaS; however, the effect was blocked by ifenprodil and weaker than that of PS. The interaction with the NR2B-selective antagonist ifenprodil indicates that this NMDA receptor subunit may be involved in neurosteroid-induced NG108-15 cell detachment.

Brominated cyclodipeptides from the marine sponge Geodia barretti as selective 5-HT ligands.

The brominated cyclodipeptides barettin (cyclo[(6-bromo-8-entryptophan)arginine]) and 8,9-dihydrobarettin (cyclo[(6-bromotryptophan)arginine]) isolated from the marine sponge Geodia barretti have previously been shown to inhibit settlement of barnacle larvae in a dose-dependent manner in concentrations ranging from 0.5 to 25 microM. To further establish the molecular target and mode of action of these compounds, we investigated their affinity to human serotonin receptors. The tryptophan residue in the barettins resembles that of endogenous serotonin [5-hydroxytryptamine]. A selection of human serotonin receptors, including representatives from all subfamilies (1-7), were transfected into HEK-293 cells. Barettin selectively interacted with the serotonin receptors 5-HT2A, 5-HT2C, and 5-HT4 at concentrations close to that of endogenous serotonin, with the corresponding Ki values being 1.93, 0.34, and 1.91 microM, respectively. 8,9-Dihydrobarettin interacted exclusively with the 5-HT2C receptor with a Ki value of 4.63 microM; it failed to show affinity to 5-HT2A and 5-HT4, indicating that the double bond between the tryptophan and arginine residue plays an important role in the interaction with the receptor proteins.

Endomorphin-1 and endomorphin-2 differentially interact with specific binding sites for substance P (SP) aminoterminal SP1-7 in the rat spinal cord.

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) represent two opioid active tetrapeptides with high affinity and selectivity for the mu-opioid (MOP) receptor. Both EM-1 and EM-2 exhibit strong inhibition of pain signals in the central nervous system (CNS). In contrast to these compounds, the undecapeptide substance P (SP) facilitates pain influx in the CNS. SP has been implicated in a number of functions in the central nervous system, including pain processing and reward. Its aminoterminal fragment SP1-7 has been shown to modulate several actions of SP in the CNS, the nociceptive effect included. Although the actions of SP1-7 have been known for long no specific receptor for the SP fragment has yet been cloned. In this study, we demonstrate the presence of specific binding sites for the heptapeptide in the rat spinal cord. The binding affinity for unlabeled SP1-7 to the specific sites for the labeled heptapeptide highly exceeded those of SP and other C- or N-terminal fragments thereof. The NK-1, NK-2 and NK-3 receptor ligands [Sar9, Met(O2)11]SP, R396 and senktide, respectively, showed no or negligible binding. Moreover, both EM-1 and EM-2 were found to interact with SP1-7 binding. However, a significant difference in binding affinity between the two opioid active tetrapeptides was observed. As recorded from replacement curves the affinity of EM-2 was 10 times weaker than that for SP1-7 but about 100 times higher than that of EM-1. Among other Tyr-Pro-containing peptides Tyr-MIF-1 but not Tyr-W-MIF-1 exhibited affinity of similar potency as EM-2. These results strengthen the previously observed differences between EM-1 and EM-2 in various functional studies. Moreover, using a cell line (C6) expressing the MOP receptor it was shown that the labeled SP1-7 did not interact with binding to this receptor and no functional response was seen for the SP heptapeptide on the MOP receptor by means of stimulation in the GTPgammaS assay. This suggests that the identified SP1-7 binding sites, with high affinity also for EM-2, are not identical to the MOP receptor and apparently not to any of the known tachykinin receptors.

Low concentrations of neuroactive steroids alter kinetics of [3H]ifenprodil binding to the NMDA receptor in rat frontal cortex.

The modulatory effects of the two neurosteroids pregnenolone sulphate (PS) and pregnanolone sulphate (3alpha5betaS) on [3H]ifenprodil binding to the N-methyl-D-aspartate (NMDA) receptor in rat frontal cortex were studied. The binding for [3H]ifenprodil itself displayed monophasic kinetics in all experiments. None of the neurosteroids displaced the radioligand from its binding site on the NR2B subunit of the NMDA receptor. However, their continual presence at nanomolar concentrations had significant effects on ligand binding kinetics, interacting through distinct sites in saturation, competition and dissociation experiments. PS at 30 nM enhanced the specific binding to about 150% of that in its absence and enhanced the dissociation rate three-fold indicating a positive modulation of [3H]ifenprodil binding to the NMDA receptor. Furthermore, PS increased Bmax and decreased Kd suggesting that the neurosteroid exposes new [3H]ifenprodil binding sites with altered properties. In contrast, 3alpha5betaS (30 nM) decreased specific [3H]ifenprodil binding to approximately 40% of that determined for the radioligand alone. The presence of 3alpha5betaS at nanomolar concentrations induced biphasic curve fits in saturation, competition as well as dissociation experiments. In conclusion, the present study show that the allosteric modulators PS or 3alpha5betaS change [3H]ifenprodil binding kinetics in a way indicating conformational alteration of its binding site on the NR2B subunit.

AT2-selective angiotensin II analogues containing tyrosine-functionalized 5,5-bicyclic thiazabicycloalkane dipeptide mimetics.

This paper reports the synthesis of two angiotensin II analogues with tyrosine-functionalized 5,5-bicyclic thiazabicycloalkane dipeptide mimetics replacing the Tyr(4)-Ile(5) residues. The preparation of these analogues relies on the synthesis and incorporation of an alpha,alpha-disubstituted chimeric amino acid derivative and on-resin bicyclization to a cysteine residue. The synthesized analogues both displayed high angiotensin AT(2)/AT(1) receptor binding preferences and had AT(2) receptor affinities in the same low nanomolar range as angiotensin II itself. Conformational analysis, using experimental constraints derived from NMR studies, indicated that the Tyr(4) and His(6) residues in one of the angiotensin II analogues were in close proximity to each other.

Effect of 3-5 monocyclizations of angiotensin II and 4-aminoPhe6-Ang II on AT2 receptor affinity.

The endogenous angiotensin II (Ang II) and the synthetic AT(2) selective agonist 4-aminoPhe(6)-Ang II respond very differently to identical cyclizations. Cyclizations of Ang II by thioacetalization, involving the 3 and 5 amino acid residue side chains, provided ligands with almost equipotent binding affinities to Ang II at the AT(2) receptor. In contrast, the same cyclization procedures applied on the AT(2) selective 4-aminoPhe(6)-Ang II delivered significantly less potent AT(2) receptor ligands, although the AT(2)/AT(1) selectivity was still very high. The fact that different structure-activity relationships are observed after imposing conformational restrictions on Ang II and 4-aminoPhe(6)-Ang II, respectively, suggests that the peptides, despite large similarities might adopt quite different backbone conformations when binding to the AT(2) receptor.

Substance P(1-7) affects the expression of dopamine D2 receptor mRNA in male rat brain during morphine withdrawal.

Previous studies have confirmed an important role of the undecapeptide substance P (SP) in opioid reward and dependence. It is further shown that the SP N-terminal metabolite SP(1-7) may attenuate the intensity of opioid withdrawal in mice. In this study we have investigated the effect of the heptapeptide fragment on the expression of the brain dopamine D2 receptor mRNA and on the withdrawal reaction, as well, in morphine-dependent rats. Male Sprague-Dawley rats were randomly distributed into two groups. Guide cannula was implanted and aimed at the lateral ventricle and animals were subsequently made opioid dependent by two daily injections of morphine (10 mg/kg) for 7 days. Half an hour before naloxone challenge (2 mg/kg) one group of rats received an injection of SP(1-7) (28 nmol per rat) and the other, serving as control, was injected with saline through the cannula. Animals were decapitated 4 h following SP(1-7) or saline injections. The results indicated that the level of the dopamine D2 receptor transcript was significantly reduced by SP(1-7) in nucleus accumbens and frontal cortex but not altered in the striatum. In behavioral tests it was found that the heptapeptide attenuated several somatic withdrawal symptoms. The observed reduction in the receptor transcript in nucleus accumbens and frontal cortex is suggested to reflect an increased dopamine activity in these areas, which in turn may counteract the withdrawal reaction.