Potential differences in receptor‐mediated G‐protein activation in postmortem human hippocampal membranes prepared from healthy controls and suicide victims

Abstract

Aim

Postmortem brain studies offer enormous opportunities to study molecular mechanisms associated with suicide. In the present study, conventional [³⁵S]GTPγS binding assay and its version‐up method ([³⁵S]GTPγS binding/immunoprecipitation assay) were applied to postmortem human hippocampal membranes prepared from suicide victims and control subjects.

Methods

By using conventional [³⁵S]GTPγS binding assay, functional activations of G_i/o proteins coupled with multiple GPCRs (5‐HT_1A receptor, α_2A‐adrenoceptor, M₂/M₄ mAChRs, adenosine A₁ receptor, histamine H₃ receptor, group II mGlu, GABA_B receptor, μ‐opioid receptor, δ‐opioid receptor, and NOP receptor) were detected by using 15 different agonists. Furthermore, 5‐HT_2A receptor‐ and M₁ mAChR‐mediated Gα_q/11 activation and adenosine A₁ receptor‐mediated Gα_i‐3 activation were detectable by means of [³⁵S]GTPγS binding/immunoprecipitation assay.

Results

No significant differences in pharmacological parameters of all concentration‐response curves investigated were found between suicide victims and control subjects. Significant correlations were obtained for the maximal percent increases between some distinct signaling pathways.

Conclusion

Although only preliminary and auxiliary results were obtained as to the potential differences between suicide victims and control subjects because of the limited number of subjects as well as unmatched age and postmortem delay, adenosine A₁ receptor‐mediated Gα_i/o activation and 5‐HT_2A receptor‐mediated Gα_q/11 activation appear worth focusing on in the future investigations. This study also indicates the possibility that some distinct signaling pathways are interrelated with each other, for example, functional activations of G_i/o proteins coupled to M₂/M₄ mAChR and 5‐HT_1A receptor, NOP receptor, and GABA_B receptor, and NOP receptor and δ‐opioid receptor.

INTRODUCTION

Suicide is one of the leading causes of death and thus a major public health concern worldwide. Suicide has a strong association with almost all mental disorders and contributes to the excess mortality of the mentally ill patients.^1,2 About 90% of people who commit suicide have some form or presence of psychopathology.³ On the other hand, only a small number of the mentally ill patients attempt suicide and an even smaller number commit suicide. Suicidal behavior is indeed a multi-factorial phenomenon, and although the presence of mental illness is a major risk factor for suicide, many other risk factors, such as psychosocial and genetic ones, are involved. Indeed, genetic studies suggest that genetic predisposition to suicide or suicidal behavior may, at least in part, be independent from the genetic risk for mood or other psychiatric disorders.⁴ Therefore, the neurobiology of suicide needs to be studied independently of the presence of other mental disorders.

Postmortem human brain studies offer enormous opportunities to investigate molecular mechanisms related to suicide. Such studies enable a direct insight into the neurobiological abnormalities associated with suicide, and this advantage is of importance since earlier research works using peripheral tissues, such as platelets, lymphocytes, or even cerebrospinal fluid, raised the question of whether some abnormalities detected in such studies reflect similar changes in the brain or have any relevance to the neurobiology of suicide. To date, postmortem studies of suicidality have provided abundant data leading to an insight into abnormalities in brain functioning associated with suicide on various levels, including gene and protein expression, neuroplasticity, neurotransmission, and so on.^5–7

In recent years, we have been engaged in postmortem brain studies to investigate possible abnormalities in functionality of receptor-mediated signal transduction in psychiatric disorders. Since almost all classical neurotransmitters are known to activate cell-surface metabotropic receptors coupled with heterotrimeric G-proteins, we have utilized conventional receptor-mediated guanosine-5′-O-(3-[³⁵S]thio) triphosphate ([³⁵S]GTPγS) binding assay,^8–10 and its version-up method.^8,11–15 In these studies, samples from the dorsolateral prefrontal cortex (Brodmann's area 9) were utilized because the frontal cortex, including this area, was supposed to function as a network hub controlling mood and cognition and thus to play an important role in psychiatric disorders.^16,17

Besides the dorsolateral prefrontal cortex, the hippocampus is another brain region of interest in performing postmortem studies of suicidality.¹⁸ The hippocampus is a subcortical brain region that is highly sensitive to stress as well as to integration of exteroceptive and interoceptive information,¹⁹ richly endowed with glucocorticoid receptors in humans and a part of the limbic–cortical-hypothalamic circuit implicated in the pathophysiology of suicide. In the present study, we applied conventional [³⁵S]GTPγS binding assay and its version-up method to postmortem human hippocampal membranes to investigate functional coupling between multiple neurotransmitter receptors and G-proteins. Although preliminary and inconclusive because of the limited number of samples, hippocampal membranes prepared from the controls and the suicide victims were used to compare the pharmacological properties of the responses between the two groups.

MATERIALS AND METHODS

Postmortem human hippocampal samples

Human brain samples were collected in accordance with the Ethical Rules for Using Human Tissues for Medical Research in Hungary (HM 34/1999) and the Code of Ethics of the World Medical Association (Declaration of Helsinki). Brains were removed from the skull and frozen with a short postmortem delay (1–12 h) in the Department of Forensic Medicine of Semmelweis University and transferred to the Human Brain Tissue Bank, Semmelweis University (HBTB). Brains were sliced into 1–1.5 cm thick coronal sections, and the hippocampal samples were cut out and stored in the HBTB at −80°C until use. The hippocampus (“hippocampal complex” or “hippocampal formation”),²⁰ includes the dentate gyrus, CA1–CA4 hippocampus areas, and the subiculum,²¹ but not the parasubiculum or any parts of the parahippocampal gyrus.

The activity of the HBTB has been authorized by the Committee of Science and Research Ethic of the Ministry of Health Hungary (ETT TUKEB: 189/KO/02.6008/2002/ETT) and the Semmelweis University Regional Committee of Science and Research Ethics (No. 32/1992/TUKEB). The study reported in the manuscript was performed according to a protocol approved by the Committee of Science and Research Ethics, Semmelweis University (TUKEB 189/2015) and by the Human Studies Ethical Committees of Karolinska Institute and Saitama Medical University. The medical history of the subjects was obtained from clinical records, interviews with family members and relatives, as well as pathological and neuropathological reports. All personal data are stored in strict ethical control, and samples were coded before the analyses of tissue.

The subjects consist of seven controls (5 males/2 females) and seven suicide victims (6 males/1 female). The demographic features of each individual subject are described in Table 1. Unfortunately, age at death and postmortem delay are not matched between the two groups. There are significant differences in both age (p < 0.001) and postmortem delay (p < 0.01) by Student's unpaired t-test. Suicide victims died by hanging (n = 6) or drug overdose (n = 1). Causes of death in control subjects were the following: pulmonary embolism (n = 4), myocardial infarction (n = 1), acute cardiorespiratory insufficiency (n = 1), basal intracranial hemorrhage/brain stem infarction (n = 1). All of the controls and suicide victims were Caucasians of Hungarian ethnicity. Samples were taken for testing HIV, tuberculosis, syphilis, hepatitis, and alcohol.

TABLE 1 Demographic data of the subjects.

Sex	Age (y)	PMD (h)	Cause of death
Control
M	63	3.5	Pulmonary embolism
F	72	1	Pulmonary embolism
M	62	4	Basal intracranial hemorrhage/brain stem infarction
M	67	5	Pulmonary embolism
M	72	5.5	Myocardial infarction
F	79	1	Pulmonary embolism
M	55	2	Acute respiratory and cardiac insufficiency
5M/2F	67.1 ± 7.9	3.1 ± 1.8
Suicide victim
M	32	6	Hanging/Asphyxia
M	53	12	Hanging/Asphyxia
M	35	2	Drug overdose
F	39	12	Hanging/Asphyxia
M	22	8	Hanging/Asphyxia
M	18	8	Hanging/Asphyxia
M	25	8	Hanging/Asphyxia
6M/1F	32.0 ± 11.9	8.0 ± 3.5

Membrane preparation

The hippocampal samples from controls and suicide victims weighed 0.6–3.4 (1.9 ± 0.4) g and 0.6–6.8 (3.3 ± 0.7) g, respectively (N.S.). The block weighing over 1.5 g was cut into several small pieces, and each hippocampal tissue was homogenized in 5 mL of ice-cold TED buffer (5 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol; pH 7.4) containing 10% (w/v) sucrose by 20 strokes with a motor-driven Teflon/glass tissue grinder. All of the following centrifuge procedures were performed at 4°C. Subsequent to centrifugation of the homogenate at 1000 g for 10 min, the supernatant was decanted to another centrifuge tube. The pellet was vortexed in 5 mL of TED/sucrose buffer and centrifuged again at 1000 g for 10 min. The combined supernatant (10 mL) was centrifuged at 9000 g for 20 min and resuspended in 10 mL of TED buffer. After the same procedure was repeated, the homogenate was kept on ice for 30 min, followed by a final centrifugation at 35 000 g for 10 min. The resulting pellet was resuspended in 50 mM Tris–HCl buffer (pH 7.4) to produce the homogenate with a protein concentration of 2.0–3.0 mg/mL. The homogenate was divided into aliquots, which were frozen quickly on fine-grained dry ice and stored at −80°C until the day of the experiment.

Conventional [³⁵S]GTPγS binding assay

The conventional [³⁵S]GTPγS binding assay for G_i/o proteins coupled with multiple receptors was performed according to the methods described previously for postmortem human prefrontal cortical membranes.^8–10 In brief, brain membranes equivalent to 60 μg protein were incubated in duplicate at 30°C for 60 min in 500 μL of 50 mM Tris-HCl buffer (pH 7.4) containing 0.2 nM [³⁵S]GTPγS, 5 mM MgCl₂, 0.1 mM EDTA, 0.2 mM ethylene glycolbis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), 0.2 mM dithiothreitol, 100 mM NaCl, 50 μM GDP, and the compound of interest at the indicated concentration. After the incubation, the homogenate was filtered under vacuum through glass fiber filters (GF/B; Whatman International, Maidstone, UK) using a Brandel cell harvester with 2 × 5 mL washing with ice-cold 50-mM Tris-HCl buffer (pH 7.4). The glass fiber filters were put into scintillation mini-vials, to each of which 4 mL of Emulsifier Scintillator Plus cocktail was added, and the radioactivity was determined with a liquid scintillation spectrometer. The nonspecific binding was defined in the presence of 100 μM unlabeled GTPγS.

[³⁵S]GTPγS binding/immunoprecipitation assay

The [³⁵S]GTPγS binding/immunoprecipitation assay was performed as previously described.^8,11–15 Thawed hippocampal membranes equivalent to 80 μg protein per tube were incubated at room temperature for 60 min in 200 μL of 50 mM Tris-HCl buffer (pH 7.4) containing 2.0 nM [³⁵S]GTPγS, 20 mM MgCl₂, 0.2 mM EGTA, 0.5 mM dithiothreitol, 100 mM NaCl, 10 nM (for Gα_q/11) or 300 μM (for Gα_i-3) GDP, and the compound of interest at the indicated concentration. The tubes were incubated for 30 min after addition of Nonidet P40 substitute (0.3%). Finally, 25 μL of Dynabeads Protein A suspension coated with anti-Gα_q/11 antibody or anti-Gα_i-3 antibody (0.25 μg/ tube) was added, and incubated for 60 min at room temperature with gentle occasional mixing. The Dynabeads Protein A were washed thoroughly with wash buffer (100 mM phosphate buffer containing 0.05% Tween 20, pH 7.4) and resuspended in 100 μL of wash buffer. The suspension was transferred into a scintillation mini-vial, to which 4 mL of Emulsifier Scintillator Plus cocktail were added, and the radioactivity was determined with a liquid scintillation spectrometer. Nonspecific binding was determined in the presence of 1 mM unlabeled GTPγS.

Data analysis

The concentration-dependent increase in the specific [³⁵S]GTPγS binding by an agonist was expressed as a percent increase above the basal unstimulated specific binding and analyzed by means of a nonlinear regression method using GraphPad Prism (GraphPad Software, La Jolla, CA, USA) to produce the concentration eliciting the half-maximal effect (EC₅₀), maximal percent increase (%E_max), and slope factor. The EC₅₀ values were transformed into pEC₅₀ (−logEC₅₀) to be analyzed. Pharmacological parameters between suicide victims and control subjects were analyzed using Student's unpaired t-test. Data are presented as mean ± SEM of the indicated number of independent experiments, each performed in duplicate.

Chemicals and reagents

[³⁵S]GTPγS (NEG030H, 1250 Ci/mmol) was purchased from PerkinElmer (Waltham, MA, USA). Dynabeads Protein A were purchased from Life Technologies (Carlsbad, CA, USA). The rabbit polyclonal antibodies to Gα subtypes (sc-393 for Gα_q/11 and sc-262 for Gα_i-3) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Non-ionic detergent Nonidet P40 substitute was obtained from Roche Diagnostics GmbH (Mannheim, Germany). The following agonists used in the present study were purchased from Sigma-Aldrich (St. Louis, MO, USA): serotonin creatinine sulphate monohydrate (5-HT), (R)-(+)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (R(+)-8-OH-DPAT), 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK-14304), (−)-epinephrine (+)-bitartrate salt ((−)-epinephrine), dopamine hydrochloride (dopamine), carbamoylcholine chloride (carbachol), adenosine, l-glutamic acid monosodium salt hydrate (l-glutamate), [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin acetate salt (DAMGO) and [D-Pen^2,5]-enkephalin hydrate (DPDPE). Histamine dihydrochloride (histamine) was obtained from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). The following four agonists were from Tocris Bioscience (Bristol, UK): (RS)-baclofen (baclofen), endomorphin-1, SNC-80 and nociceptin. GDP, GTPγS, and Tween 20 were obtained from Sigma-Aldrich, and all other chemicals used in this study were of the highest commercially available purity from standard sources.

RESULTS

Agonist-induced G-protein activation determined by conventional [³⁵S]GTPγS binding assay

The concentration-response curves for agonist-induced activation of G-proteins in postmortem human hippocampal membranes are shown in Figures 1–4. Most agonists induced a saturable concentration-dependent increase in specific [³⁵S]GTPγS binding, presumably mediated through one receptor subtype. Several agonists, such as R(+)-8-OH-DPAT, (−)-epinephrine, UK-14304, carbachol, and DAMGO, produced biphasic curves, indicating involvement of two or more receptor subtypes. In these cases, the concentration-response curves were analyzed only with the data using lower concentrations of agonists, for example, from 0.1 nM to 10 μM for R(+)-8-OH-DPAT (Figure 1B). For the concentration–response curves induced by l-glutamate (Figure 3E) and SNC-80 (Figure 4D), the data using higher concentrations of agonists were also omitted since the responses declined below the maximal values at higher concentrations.

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Agonist-induced G-protein activation determined by [³⁵S]GTPγS binding/immunoprecipitation assay

The concentration-response curves for activation of Gα_q/11 elicited by 5-HT and carbachol in postmortem human hippocampal membranes are shown in Figure 5A,B, respectively, and adenosine-induced activation of Gα_i-3 is depicted in Figure 5C. These curves were analyzed on the assumption of a one-site model.

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Comparison of pharmacological parameters between suicide victims and control subjects

The pharmacological parameters, that is, pEC₅₀, %E_max, and slope factors were determined by nonlinear regression analysis for each individual sample, and the means ± SEM of these values were summarized for suicidal victims and controls (Table 2). No significant difference was detected between the two groups for any parameter of any response. An exception was the pEC₅₀ value of adenosine-induced G-protein activation (6.14 ± 0.04 in suicidal victims vs. 5.98 ± 0.02 in controls, p < 0.01), but this significance disappeared when corrected with the Benjamini-Hochberg procedure.

TABLE 2 Comparison of pharmacological parameters between suicide victims and controls.

Agonist	Targeted receptor	Parameter	Suicide victims	Controls	Unpaired t-test
Conventional [35S]GTPγS binding assay (Gαi/o)
5-HT	5-HT1A	%Emax	82.7 ± 17.8	116.7 ± 8.1	N.S.
pEC50	6.78 ± 0.07	6.65 ± 0.05	N.S.
Slope factor	0.94 ± 0.03	0.90 ± 0.03	N.S.
R(+)-8-OH-DPAT (~10−5 M)	5-HT1A	%Emax	59.8 ± 17.5	81.2 ± 8.5	N.S.
pEC50	7.00 ± 0.16	7.06 ± 0.04	N.S.
Slope factor	1.09 ± 0.10	1.01 ± 0.05	N.S.
(−)-Epinephrine (~10−4 M)	α2A-Adrenergic	%Emax	28.3 ± 3.3	22.5 ± 2.5	N.S.
pEC50	5.37 ± 0.10	5.44 ± 0.03	N.S.
Slope factor	1.04 ± 0.19	1.05 ± 0.08	N.S.
UK-14304 (~10−5 M)	α2A-Adrenergic	%Emax	24.3 ± 5.2	14.8 ± 2.3	N.S.
pEC50	6.06 ± 0.32	6.06 ± 0.12	N.S.
Slope factor	0.91 ± 0.11	1.25 ± 0.20	N.S.
Dopamine	Needs to be defined	%Emax	37.7 ± 7.7	36.5 ± 5.5	N.S.
pEC50	4.25 ± 0.11	4.06 ± 0.08	N.S.
Slope factor	1.11 ± 0.13	1.10 ± 0.08	N.S.
Adenosine	Adenosine A1	%Emax	262.7 ± 32.2	255.3 ± 15.9	N.S.
pEC50	6.14 ± 0.04	5.98 ± 0.02	P < 0.01a
Slope factor	0.87 ± 0.02	0.89 ± 0.01	N.S.
Baclofen	GABAB	%Emax	49.5 ± 6.5	33.0 ± 7.9	N.S.
pEC50	4.40 ± 0.03	4.70 ± 0.15	N.S.
Slope factor	1.15 ± 0.06	0.98 ± 0.05	N.S.
Carbachol (~10−3 M)	M2/M4 mAChR	%Emax	20.0 ± 5.5	22.0 ± 2.5	N.S.
pEC50	4.37 ± 0.23	4.55 ± 0.12	N.S.
Slope factor	1.19 ± 0.17	1.10 ± 0.19	N.S.
Histamine	Histamine H3	%Emax	8.8 ± 1.4	10.3 ± 2.0	N.S.
pEC50	6.01 ± 0.13	6.07 ± 1.17	N.S.
Slope factor	1.77 ± 0.30	1.41 ± 0.39	N.S.
l-glutamate (~10−3 M)	Group II mGlu	%Emax	6.4 ± 1.4	5.7 ± 1.6	N.S.
pEC50	5.11 ± 0.08	4.72 ± 0.15	N.S.
Slope factor	1.45 ± 0.46	1.68 ± 0.57	N.S.
DAMGO (~3 × 10−5 M)	μ-Opioid	%Emax	34.7 ± 4.7	38.2 ± 8.6	N.S.
pEC50	5.98 ± 0.07	5.89 ± 0.05	N.S.
Slope factor	0.88 ± 0.07	0.97 ± 0.07	N.S.
Endomorphin-1	μ-Opioid	%Emax	12.4 ± 1.8	14.4 ± 4.2	N.S.
pEC50	6.36 ± 0.07	6.21 ± 0.07	N.S.
Slope factor	1.87 ± 0.28	1.88 ± 0.37	N.S.
DPDPE	δ-Opioid	%Emax	14.5 ± 4.8	13.3 ± 3.5	N.S.
pEC50	5.74 ± 0.04	6.10 ± 0.16	N.S.
Slope factor	1.48 ± 0.34	1.03 ± 0.13	N.S.
SNC-80 (~3 × 10−6 M)	δ-Opioid	%Emax	21.6 ± 5.1	24.5 ± 4.9	N.S.
pEC50	7.01 ± 0.15	6.79 ± 0.11	N.S.
Slope factor	1.60 ± 0.24	1.37 ± 0.29	N.S.
Nociceptin	NOP	%Emax	39.0 ± 8.3	35.1 ± 7.1	N.S.
pEC50	7.06 ± 0.08	6.97 ± 0.09	N.S.
Slope factor	1.02 ± 0.05	1.40 ± 0.18	N.S.
[35S]GTPγS binding/immunoprecipitation assay (Gαq/11)
5-HT	5-HT2A	%Emax	52.1 ± 17.0	98.1 ± 15.2	N.S.
pEC50	6.35 ± 0.37	6.90 ± 0.17	N.S.
Slope factor	0.98 ± 0.13	0.79 ± 0.08	N.S.
Carbachol	M1 mAChR	%Emax	354.5 ± 51.9	417.3 ± 61.4	N.S.
pEC50	4.46 ± 0.30	4.48 ± 0.14	N.S.
Slope factor	0.80 ± 0.03	0.80 ± 0.03	N.S.
[35S]GTPγS binding/immunoprecipitation assay (Gαi-3)
Adenosine	Adenosine A1	%Emax	70.4 ± 3.9	80.8 ± 7.0	N.S.
pEC50	5.95 ± 0.04	5.91 ± 0.07	N.S.
Slope factor	0.91 ± 0.20	0.89 ± 0.16	N.S.

Correlation of the %E_max values

The %E_max values determined for each agonist-induced G-protein activating response were correlated with each other, and the results were summarized in Table 3. This procedure was done on the assumption that significant correlation would be expected if the two responses were derived from a common or somehow highly interconnected signaling pathway. As expected, there was a highly significant correlation between the responses elicited by 5-HT and R(+)-8-OH-DPAT (Figure 6A), both of which are supposed to stimulate the same 5-HT_1A receptors, resulting in activation of the same Gα_i/o proteins. Similarly, the %E_max values of DAMGO- and endomorphin-1-stimulated G-protein activating responses were correlated significantly, probably through the activation of the common Gα_i/o proteins coupled with the common μ-opioid receptors. After adjustment with the Benjamini-Hochberg procedure, several other correlations were still significant, that is, 5-HT versus dopamine, R(+)-8-OH-DPAT versus dopamine, 5-HT versus carbachol, R(+)-8-OH-DPAT versus carbachol, dopamine versus carbachol, baclofen versus nociceptin, DPDPE versus nociceptin, and SNC-80 versus nociceptin.

TABLE 3 Correlation of the %E_max values determined by receptor-mediated [³⁵S]GTPγS binding in human hippocampal membranes.

Agonist	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18
5-HT (1)
R(+)-8-OH-DPAT (2)	0.97***
(−)-Epinephrine (3)	0.22	0.19
UK-14304 (4)	0.25	0.34	0.68*
Dopamine (5)	0.79**	0.89***	0.21	0.42
Adenosine (6)	0.54	0.54	0.44	0.42	0.54
Baclofen (7)	0.05	0.02	0.58	0.33	0.10	0.37
Carbachol (8)	0.79**	0.88***	0.18	0.44	0.93***	0.61*	−0.03
Histamine (9)	0.51	0.46	0.17	0.04	0.32	0.47	0.55	0.34
l-glutamate (10)	0.04	0.02	0.03	0.00	0.04	−0.23	0.63*	−0.17	0.45
DAMGO (11)	0.10	0.04	−0.03	−0.13	0.11	0.34	0.54	0.17	0.57	0.46
Endomorphin-1 (12)	−0.05	−0.07	−0.17	−0.03	0.04	0.29	0.33	0.15	0.38	0.33	0.90***
DPDPE (13)	0.16	0.00	0.69*	0.11	−0.12	0.42	0.72*	−0.11	0.43	0.15	0.34	0.07
SNC-80 (14)	0.34	0.21	0.44	0.02	0.11	0.53	0.70*	0.11	0.52	0.35	0.68*	0.45	0.79**
Nociceptin (15)	0.32	0.23	0.50	0.20	0.15	0.57	0.80**	0.17	0.61*	0.34	0.65*	0.38	0.83**	0.85***
5-HT (Gαq) (16)	0.03	−0.15	0.31	−0.08	−0.50	0.07	0.04	−0.37	0.05	−0.18	−0.09	−0.10	0.57	0.42	0.19
Carbachol (Gαq) (17)	0.35	0.26	0.48	0.40	−0.08	0.30	0.18	−0.05	0.26	0.09	−0.13	−0.09	0.31	0.31	0.16	0.59*
Adenosine (Gαi-3) (18)	0.37	0.28	0.26	−0.01	−0.06	0.29	0.59*	−0.16	0.71*	0.58*	0.38	0.23	0.53	0.58*	0.54	0.42	0.62*

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DISCUSSION

In our previous studies, agonist-induced [³⁵S]GTPγS bindings were pharmacologically characterized in membranes prepared from postmortem human prefrontal cortex, as to G_i/o proteins determined with conventional [³⁵S]GTPγS binding assay,^8–10 and also as to G_q/11 and G_i-3 proteins determined with [³⁵S]GTPγS binding/immunoprecipitation assay.^8,11,12 In the present study, these methods were applied to membranes prepared from postmortem human hippocampus, the brain region that plays a key role in controlling memory, emotion, and mood.

By means of conventional [³⁵S]GTPγS binding assay, agonist-induced G-protein activation responses mediated via multiple receptor subtypes have been reported. In general, this conventional assay using filtration techniques is experimentally more feasible and easier for G_i/o-coupled receptors, especially when using brain membranes.^22,23 In postmortem human prefrontal cortical membranes, indeed, we were able to detect functional activation of G-proteins elicited by varied agonists, and these responses were shown to be mediated through receptor subtypes, that is, 5-HT_1A receptor, α_2A-adrenoceptor, M₂/M₄ muscarinic acetylcholine receptors (mAChRs), adenosine A₁ receptor, histamine H₃ receptor, group II metabolic glutamate receptor (mGlu), GABA_B receptor, μ-opioid receptor, δ-opioid receptor, κ-opioid receptor, and nociceptin/orphanin FQ opioid peptide (NOP) receptor, all of which are canonically coupled to G_i/o proteins.^8–10 To the best of our knowledge, there have been few studies investigating receptor-mediated [³⁵S]GTPγS binding in human hippocampal membranes. Functional activation of G_i/o proteins coupled with μ-opioid receptor,²⁴ 5-HT_1A receptor,²⁵ CB₁ cannabinoid receptor,²⁶ and M₂ mAChR²⁷ in human hippocampal membranes has been reported so far.

In the present study, we tested 15 different agonists for conventional [³⁵S]GTPγS binding assay in postmortem human hippocampal membranes, according to the previous results in postmortem prefrontal cortical membranes.⁹ The concentration-dependent responses elicited by each agonist are essentially identical in both brain regions, with a few exceptions. The biphasic concentration-response curves are obtained for R(+)-8-OH-DPAT, UK-14304, and DAMGO commonly in both brain regions. On the other hand, (−)-epinephrine and carbachol also induced biphasic curves in hippocampal membranes, whereas their response curves were analyzed using a one-site model in prefrontal cortical membranes in our previous study.⁹ Furthermore, the declines of the specific binding below the maximal response at higher concentrations of l-glutamate and SNC-80 were noticed in the present study, but not in the previous report using human prefrontal cortical membranes.⁹ These minor discrepancies may be derived from some regional differences in the neurobiological characteristics of the receptors and signal transduction pathways involved.

Because of the limited sample volume, we were unable to perform experiments using specific antagonists to define the receptor subtypes involved in the responses in the present study. However, such experiments in postmortem human prefrontal cortical membranes⁹ enable us to suppose the receptor subtypes involved in the responses examined in this study. Namely, agonist-induced [³⁵S]GTPγS bindings determined in the present study are probably mediated via the following receptor subtypes coupled with G_i/o proteins: 5-HT_1A receptor (5-HT and R(+)-8-OH-DPAT), α_2A-adrenoceptor ((−)-epinephrine and UK-14304), M₂/M₄ mAChRs (carbachol), adenosine A₁ receptor (adenosine), histamine H₃ receptor (histamine), group II mGlu (l-glutamate), GABA_B receptor (baclofen), μ-opioid receptor (DAMGO and endomorphin-1), δ-opioid receptor (DPDPE and SNC-80), and NOP receptor (nociceptin). As for dopamine-stimulated [³⁵S]GTPγS binding, we concluded tentatively that this response in human prefrontal cortex was mediated by α_2A-adrenoceptor but not by dopamine receptors, based on the experiments using L-741626, SCH-39166, and RX-821002.⁹ It is uncertain whether this is the case also in human hippocampal membranes. As shown in Table 3, the %E_max values of dopamine-stimulated responses were significantly correlated with those of 5-HT- and R(+)-8-OH-DPAT-incuced [³⁵S]GTPγS bindings almost certainly mediated through 5-HT_1A receptor, but not with those of (−)-epinephrine- or UK-14304-induced responses supposedly mediated through α_2A-adrenoceptor. These findings may indicate that dopamine-induced [³⁵S]GTPγS is derived from G_i/o proteins coupled to the 5-HT_1A receptor, but not dopaminergic or adrenergic receptors. It has been actually reported that dopamine directly activates 5-HT_1A receptors expressed in Xenopus oocytes and CHO-K1 cells.²⁸

In addition to the conventional [³⁵S]GTPγS binding assay, we also applied its version-up method, that is, [³⁵S]GTPγS binding/immunoprecipitation assay,^8,11,12 to postmortem human hippocampal membranes. As in human prefrontal cortical membranes, prominent concentration-response curves were obtained in postmortem human hippocampal membranes as to 5-HT_2A receptor- and M₁ mAChR-mediated Gα_q/11 activation as well as adenosine A₁ receptor-mediated Gα_i-3 activation. We are unaware of reports showing receptor-mediated [³⁵S]GTPγS binding to G-protein subtypes other than G_i/o in postmortem human hippocampus.

In order to investigate receptor-mediated G-protein activation of non-G_i/o families, antibody-capture [³⁵S]GTPγS binding scintillation proximity assay²⁹ has been usually utilized in brain membranes. In the present study, however, we adopted the [³⁵S]GTPγS binding/immunoprecipitation assay instead of the antibody-capture [³⁵S]GTPγS binding scintillation proximity assay, based on the reasons as discussed previously.³⁰ Unfortunately, the polyclonal antibodies to each Gα subtype utilized in the present study (sc-393 and sc-262 for Gα_q/11 and Gα_i-3, respectively) are now unavailable. We are advancing experiments to re-establish the [³⁵S]GTPγS binding/immunoprecipitation assay using specific anti-Gα antibodies that are now commercially available.³¹

Another main subject in the present study was to examine some possible abnormalities in functional receptor/G-protein interaction in the hippocampus of suicide victims. However, the number of samples is limited, and thus the results obtained in this study should be considered preliminary and auxiliary. Furthermore, neither age nor postmortem delay was matched between the suicide victims and control subjects. In our previous study in postmortem human prefrontal cortical membranes, some pharmacological parameters of concentration-response curves elicited by several agonists were shown to be affected by these variables, especially age.^8,10,12

No significant difference between suicide victims and control subjects was found for any pharmacological parameter of agonist-induced G-protein activation investigated in the present study, with the pEC₅₀ value for adenosine-stimulated [³⁵S]GTPγS binding to G_i/o proteins as an exception (6.14 ± 0.04 in suicide victims vs. 5.98 ± 0.02 in control subjects, p < 0.01 determined by Student's unpaired t-test). This significance, however, disappeared subsequent to rectification by the Benjamini-Hochberg method. In addition to this response, 5-HT-stimulated Gα_q/11 activation mediated through the 5-HT_2A receptor is of interest. The mean %E_max value of control subjects was 98.1%, whereas almost a half (52.1%) for suicide victims, though there was not a significant difference between both groups owing to the small number of brains and large variability. A significant increase in 5-HT_2A receptor density in the prefrontal cortex of suicide victims has been reported by many, but not all, postmortem studies.^5–7 In the hippocampus, however, Cheetham et al.³² showed a significantly lower number of 5-HT₂ receptor binding sites determined with [³H]ketanserin as a radioligand in the antidepressant-free depressed suicide victims as compared with controls. Autoradiographic analysis of [³H]ketanserin binding in postmortem human brains also revealed that the maximal binding sites were reduced in the suicide group both in the prefrontal cortex and hippocampus, after taking the age dependence into account.³³

Significant correlations in the %E_max values were observed for some pairs of agonists. As analyzed in the postmortem human prefrontal cortex,¹⁰ it appears reasonable to see a significant correlation between the two measures mediated through the common receptor subtype, that is, the 5-HT_1A receptor (5-HT vs. R(+)-8-OH-DPAT) as well as the μ-opioid receptor (DAMGO vs. endomorphin-1). Besides these combinations, there are some pairs showing significant correlations. As mentioned above, the common 5-HT_1A receptor may be activated by 5-HT, R(+)-8-OH-DPAT, and dopamine as well, resulting in a significant correlation between the %E_max values for dopamine and 5-HT and also for dopamine and R(+)-8-OH-DPAT. Carbachol-stimulated G_i/o activation also showed a significant correlation with the response elicited by these three compounds. Another interesting response is nociceptin-induced G_i/o activation through the NOP receptor, the %E_max values of which are significantly correlated with those of the response elicited by baclofen, DPDPE, and SNC-80. These results suggest some interrelationships exist between GPCR-mediated signaling pathways through M₂/M₄ mAChR and 5-HT_1A receptor, NOP receptor and GABA_B receptor, and NOP receptor and δ-opioid receptor in human hippocampus. The molecular mechanisms underlying these possible relations remain unclear, and further investigations are needed.

In conclusion, this study indicates functional activation of G_i/o proteins coupled with multiple GPCRs can be detected in postmortem human hippocampal membranes by using the following varied agonists (and assumed receptor subtype(s) involved described in parenthesis), i.e., 5-HT (5-HT_1A receptor), R(+)-8-OH-DPAT (5-HT_1A receptor), (−)-epinephrine (α_2A-adrenoceptor), UK-14304 (α_2A-adrenoceptor), dopamine (possibly 5-HT_1A receptor), carbachol (M₂/M₄ mAChRs), adenosine (adenosine A₁ receptor), histamine (histamine H₃ receptor), l-glutamate (group II mGlu), baclofen (GABA_B receptor), DAMGO (μ-opioid receptor), endomorphin-1 (μ-opioid receptor), DPDPE (δ-opioid receptor), SNC-80 (δ-opioid receptor), and nociceptin (NOP receptor). Furthermore, 5-HT_2A receptor- and M₁ mAChR-mediated Gα_q/11 activation, and adenosine A₁ receptor-mediated Gα_i-3 activation are detectable by means of [³⁵S]GTPγS binding/immunoprecipitation assay. Comparison of pharmacological parameters derived from the concentration-response curves for these agonists between suicide victims and control subjects provides preliminary and auxiliary results because of the limited number of subjects as well as the unmatched age and postmortem delay. Nevertheless, adenosine A₁ receptor-mediated Gα_i/o activation and 5-HT_2A receptor-mediated Gα_q/11 activation attracted our interest to understand molecular abnormalities in the hippocampus underlying suicidal behavior. Further investigations are warranted using a sufficient number of subjects with demographic factors well controlled. Finally, correlation analysis of the E_max values raises the possibility that some distinct signaling pathways are interrelated with each other, that is, functional activations of G_i/o proteins coupled to M₂/M₄ mAChR and 5-HT_1A receptor, NOP receptor and GABA_B receptor, and NOP receptor and δ-opioid receptor. In the future, it will be of interest to test if such functional activations by GPCR can involve participation of allosteric receptor-receptor interactions, as well as intracellular interactions in the cytoplasm and/or possible intercellular interactions between cells.

FUNDING INFORMATION

This work was supported by a grant from the Saitama Medical University to Yuji Odagaki, by Stiftelsen Olle Engkvist Byggmästare 2021 to Kjell Fuxe and Dasiel Oscar Borroto-Escuela, and by the European Commission under the Sixth Framework Programme (BrainNet Europe II, LSHM-CT-2004-503 039) to Miklós Palkovits.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available as Tables S1–S3.

ETHICS STATEMENT

Human brain samples were collected in accordance with the Ethical Rules for Using Human Tissues for Medical Research in Hungary (HM 34/1999) and the Code of Ethics of the World Medical Association (Declaration of Helsinki). Hippocampal samples were cut out and stored in the Human Brain Tissue Bank, Semmelweis University (HBTB), authorized by the Committee of Science and Research Ethic of the Ministry of Health Hungary (ETT TUKEB: 189/KO/02.6008/2002/ETT) and the Semmelweis University Regional Committee of Science and Research Ethics (No. 32/1992/TUKEB).

Approval of the Research Protocol by an Institutional Reviewer Board: The study reported in the manuscript was performed according to a protocol approved by the Committee of Science and Research Ethics, Semmelweis University (TUKEB 189/2015), and by the Human Studies Ethical Committees of Karolinska Institute and Saitama Medical University.

Informed Consent: N/A.

Registry and the Registration No. of the Study/Trial: N/A.

Animal Studies: N/A.

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