Nobuyuki Nishi

Open pergolide treatment of tricyclic and heterocyclic antidepressant-resistant depression

Background: Recently, a dopamine hypothesis of depression was put forward, and several studies have demonstrated that direct and indirect dopamine agonists have antidepressant effects. Methods: Using Clinical Global Impressions, we evaluated the efficacy of 4-week treatment of pergolide as an antidepressant adjuvant involving 20 unipolar depressed patients who were refractory to standard treatment with antidepressants. Results: One patients (5%) were very much improved, seven (35%) much improved, four (20%) minimally improved, six (30%) no change or worse, and two (10%) not assessed. There was no significant difference in any clinical factors between the pergolide responder and non-responder group. Limitations: This study was a non-blind open trial, and pergolide was added to tricyclic and heterocyclic antidepressants. Conclusion: Pergolide may be useful as an antidepressant adjuvant, suggesting a potential role for dopamine-2 stimulation in the antidepressant response.

Ultrafast excited-state dynamics in photochromic N-salicylideneaniline studied by femtosecond time-resolved REMPI spectroscopy.

Ultrafast processes in photoexcited N-salicylideneaniline have been investigated with femtosecond time-resolved resonance-enhanced multiphoton ionization spectroscopy. The ion signals via the S(1)(n,pi( *)) state of the enol form as well as the proton-transferred cis-keto form emerge within a few hundred femtoseconds after photoexcitation to the first S(1)(pi,pi( *)) state of the enol form. This reveals that two ultrafast processes, excited-state intramolecular proton transfer (ESIPT) reaction and an internal conversion (IC) to the S(1)(n,pi( *)) state, occur on a time scale less than a few hundred femtoseconds from the S(1)(pi,pi( *)) state of the enol form. The rise time of the transient corresponding to the production of the proton-transferred cis-keto form is within 750 fs when near the red edge of the absorption is excited, indicating that the ESIPT reaction occurs within 750 fs. The decay time of the S(1)(pi,pi( *)) state of the cis-keto form is 8.9 ps by exciting the enol form at 370 nm, but it dramatically decreases to be 1.5-1.6 ps for the excitation at 365-320 nm. The decrease in the decay time has been attributed to the opening of an efficient nonradiative channel; an IC from S(1)(pi,pi( *)) to S(1)(n,pi( *)) of the cis-keto form promotes the production of the trans-keto form as the final photochromic products. The two IC processes may provide opposite effect on the quantum yield of photochromic products: IC in the enol form may substantially reduce the quantum yield, but IC in the cis-keto form increase it.

Pharmacological characterization of metabotropic glutamate receptor‐mediated high‐affinity GTPase activity in rat cerebral cortical membranes

Activation of heterotrimeric guanine nucleotide‐binding regulatory proteins (G‐proteins) functionally coupled to metabotropic glutamate receptors (mGluRs) was assessed by agonist‐induced high‐affinity GTPase (EC3.6.1.‐) activity in rat cerebral cortical membranes. L‐Glutamate (1u2003mM) stimulated high‐affinity GTPase activity to the same extent throughout the incubation period up to 20u2003min, in a Mg2+‐dependent manner. The addition of 1u2003mM L‐glutamate augmented Vmax of the enzyme activity (1670 to 3850u2003pmolu2003mg−1 protein 15u2003min−1) with slight increase in KM value (0.26 to 0.63u2003μM). The high‐affinity GTPase activity was stimulated by the following compounds with a rank order of potency of (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl) glycine (DCG‐IV) > u2003(2S,1′S,2′S)‐2‐(carboxycyclopyropyl)glycine (L‐CCG‐I) > L‐glutamate ≥ 2R,4R‐4‐aminopyrrolidine‐2,4‐dicarboxylate [(2R,4R)‐APDC] > 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylate [(1S,3R)‐ACPD] > (S)‐4‐carboxy‐3‐hydroxyphenylglycine [(S)‐4C3HPG] > (S)‐3‐carboxy‐4‐hydroxyphenylglycine [(S)‐3C4HPG] > ibotenate, but not by L‐(+)‐2‐amino‐4‐phosphonobutyrate (L‐AP4), (RS)‐3,5‐dihydroxyphenylglycine [(RS)‐3,5‐DHPG], quisqualate, or L‐serine‐O‐phosphate (L‐SOP), indicative of involvement of group II mGluRs, in particular mGluR2. (2S)‐α‐Ethylglutamate (EGLU), a presumably selective antagonist against group II mGluRs, inhibited DCG‐IV‐stimulated high‐affinity GTPase activity in a competitive manner with an apparent KB of 220u2003μM. L‐Glutamate‐stimulated activity was eliminated by pretreatment of the membranes with sulfhydryl alkylating agent N‐ethylmaleimide (NEM) at 30–50u2003μM, indicating that G‐proteins of the Gi family are involved. These results indicate that mGluR agonist‐induced high‐affinity GTPase activity in rat cerebral cortical membranes may be used to detect the functional interaction between group II mGluRs, in particular mGluR2, and NEM‐sensitive Gi proteins.

Receptor-mediated and receptor-independent activation of G-proteins in rat brain membranes

High-affinity GTPase activity intrinsic to G-proteins, which serves as an index of G-protein activation elicited through agonist-stimulated receptors as well as by receptor-independent direct G-protein activators like mastoparan, was measured in rat brain membranes. Receptor-mediated high-affinity GTPase activity was detectable preferentially for the Gi subfamily associated with adenylyl cyclase inhibition mediated by group II metabotropic glutamate, pirenzepine-insensitive muscarinic acetylcholine, GABA(B), adenosine A1, dopamine D2-like (striatum), and serotonin 5-HT1A (hippocampus) receptors. The pharmacological characteristics of such receptor-mediated high-affinity GTPase activities were presented. Mastoparan, a tetradecapeptide from wasp venom which has been shown to directly activate Gi and Go, inhibited low-affinity GTP hydrolyzing activity, probably due to its activating effect on nucleoside diphosphokinase (NDPK). When NDPK activity was inhibited completely by UDP, mastoparan stimulated high-affinity GTPase activity in a concentration-dependent manner. There are many compounds other than mastoparan with apparently diverse structural properties which have been shown to directly activate G-proteins. The relevance and possible participation of receptor-independent mode of G-protein activation for some neuropeptides were discussed.

Measurement of receptor-mediated functional activation of G proteins in postmortem human brain membranes

Guanine nucleotide-binding regulatory proteins (G proteins) play a pivotal role in receptor-mediated transmembrane signal transduction, and have been implicated in modes of action of psychotropic drugs as well as in pathogenesis of psychiatric disorders. In the present investigation, functional activation of G proteins coupled with several receptors, in particular with GABAB receptors, was assessed by agonist-induced stimulation of high-affinity GTPase, an enzyme that is intrinsic to alpha subunit of G protein, in postmortem human frontal cortical membranes. High-affinity GTPase activity was stimulated by GABA as well as (+/-)-baclofen, a selective GABAB receptor agonist, with EC50 values of 60-150 and 10-40 microM, respectively, in a Mg(2+)-dependent manner. The (+/-)-baclofen-stimulated response was antagonized by the selective GABAB receptor antagonist, 2-hydroxy-saclofen, in a competitive manner with a KB value of 59 microM. Although the maximal percent increase above basal value (% Emax) for GABAB receptor-mediated high-affinity GTPase activity was varied from subject to subject, % Emax values for both agonists were highly correlated with each other, and replicable and stable in a given subject, indicating that this measure is trustworthy as an index of functional coupling between receptors and G proteins in future studies at the aim of elucidating possible alteration of receptor/G protein interaction in psychiatric disorders. The % Emax values for GABAB receptor-mediated responses were correlated inversely with brain storage duration, which should be critically considered in postmortem studies. The increases in high-affinity GTPase activity stimulated by several agonists other than GABAB receptor agonists seemed too low to quantify for making a comparison in future studies.

Effects of the wasp venom peptide, mastoparan, on GTP hydrolysis in rat brain membranes

The effects of mastoparan, a wasp venom toxin, on GTP hydrolyzing activity were examined in rat brain membranes. Mastoparan inhibited the low‐affinity GTPase activity, defined as the amount of 32Pi released from 0.3 μM [γ‐32P]‐GTP in the presence of 100 μM unlabelled GTP, in a concentration‐dependent manner. This inhibitory effect of mastoparan on low‐affinity GTPase activity was diminished by increasing concentrations of UDP and was completely attenuated at 20 mM, indicating that activation of nucleoside diphosphokinase (NDPK) is inolved in the phenomenon. In the presence of 20 mM UDP, mastoparan stimulated the high‐affinity GTPase activity by increasing the Vmax value without affecting the apparent KM for GTP. Mastoparan‐stimulated high‐affinity GTPase activity was apparent at concentrations higher than 1 μM, in a concentration‐dependent manner, but without saturation even at 100 μM. Mastoparan‐induced high‐affinity GTPase activity showed a characteristic sensitivity to MgCl2, quite different from that seen in L‐glutamate‐stimulated activity, a representative of receptor‐mediated G‐protein activation. There appeared to be a simple additive interaction between mastoparan‐ and L‐glutamate‐stimulated high‐affinity GTPase activities, indicting that distinct pools of G‐proteins are involved in receptor‐independent and receptor‐mediated G‐protein activation. These results suggest that G‐proteins in brain membranes are functionally altered by mastoparan through multiple mechanisms of action and that the mastoparan‐induced, direct G‐protein activating process lacks a synergistic or antagonistic interaction with an agonist‐induced, receptor‐mediated activation of G‐proteins.

Lack of Interfering Effects of Lithium on Receptor/G Protein Coupling in Human Platelet and Rat Brain Membranes

To verify the theory that lithium exerts its multiple effects by altering the receptor-mediated G proteins activation, in vitro effects of lithium on agonist-induced guanosine triphosphate (GTP) hydrolysis were examined. The basal GTP hydrolyzing activity in human platelet membranes was decreased nonselectively by either LiCl or NaCl at millimolar concentrations, whereas (-)-epinephrine-stimulated increase in the activity (an index of alpha (2A)-adrenoceptor coupled Gi2 function) was unaltered. Furthermore, the stimulation of high-affinity GTPase activity induced by dopamine, carbachol, and R-N(6)-phenylisopropyladenosine in rat brain membranes (indices of the functional coupling between dopamine D2-like, pirenzepine-insensitive muscarinic, and adenosine A1 receptors and their respective Gi proteins) was substantially unaltered regardless of whether 0.5 mmol/L adenosine 5-(beta, gamma-imido)triphosphate (i.e., 1.75 mmol/L lithium) was included in the assay mixture or not. These results indicate that lithium does not affect in vitro the receptor-mediated activational process of G proteins, at least not of Gi associated with adenylate cyclase inhibition.

Functional coupling between metabotropic glutamate receptors and G proteins in rat brain membranes

Functional activation of GTP-binding (G) proteins coupled with metabotropic glutamate receptors was evaluated in rat brain membranes. L-Glutamate stimulated the high-affinity GTPase activity in cerebral cortical, hippocampal, and striatal membranes with a mean concentration eliciting a half-maximal response (EC50) of 4.8, 1.6, and 4.9 microM, respectively. The enzyme activity in cerebral cortical membranes was also stimulated by (2S, 1S,2S)-2-(carboxycyclopropyl)glycine (L-CCG-I) with a mean EC50 of 0.90 microM, but not by L-2-amino-4-phosphonobutyrate (L-AP4) up to 10 microM. This method opens up a strategy for investigation of functional coupling between Group II metabotropic glutamate receptors and G proteins in native brain membranes.

Postreceptor Signal-Transduction Systems as Potential Targets of Lithium

All cells have the capacity to receive external information and to transduce it to intracellular signals ending in physiological responses specific to the cell type. The rapid advance in neuroscience has elucidated the molecular mechanisms underlying such transmembrane signaling pathways. The extra-cellular information transmitted by hormones, neurotransmitters, and neuromodulators is recognized by specific protein molecules at the cell surface (receptors), which constitute ion channels themselves or modulate intracellular enzymatic machinery, resulting in alteration of second-messenger levels via guanine nucleotide-binding regulatory (G) proteins. The former type of receptor, the ligand-gated ion channels, also known as ionotropic receptors, includes nicotinic acetylcholine, serotonin3 (5-HT3), γ-aminobutyric acidA (GABAA), and excitatory amino acid receptors, such as N-methyl-D-aspantate (NMDA), α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), and kinate subtypes, that respond to the agonist-induced stimulation within milliseconds. The latter type of receptor, the G-protein-coupled or metabotropic receptors, includes adrenergic, serotonergic (except for 5-HT3 receptors), dopaminergic, muscarinic acetylcholine, opioid, adenosine, GABAB, metabotropic glutamate, and many neuropeptide receptors. This type of receptor couples with second-messenger-generating enzymes, such as adenylyl cyclase, phospholipase C, cyclic guanosine monophosphate (GMP) phosphodiesterase, and phospholipase A2, as well as several ion channels, through the respective G proteins, thus mediating much slower signaling than the ionotropic receptors.