SSTR2/somatostatin receptor 2

研究人员报道，在豚鼠离体右心房中，seglitide是一种有效的环六肽生长抑素激动剂，作为一种竞争性生长抑素受体拮抗剂，其对SS14、SS25和SS28的pA2值分别为6.50 +/- 0.40、6.24 +/- 0.08和6.09 +/- 0.06。Seglitide对碳醇或n6 -环己基腺苷的负性肌力作用几乎没有影响。我们的研究结果表明，豚鼠心房的受体-反应耦合特性使得该制剂中的seglitide具有较低的内在活性，并且特异性地作为生长抑素受体拮抗剂。[1]

口服环肽生长抑素类似物MK-678的降压作用已在高血压糖尿病大鼠模型中得到证实。自发性高血压大鼠服用该药后，血压没有持续下降。低血压的机制似乎独立于对多种激素的影响，包括胰岛素、胰高血糖素、生长激素和肾素-血管紧张素系统的组成部分，包括肾素活性、血浆血管紧张素转换酶和醛固酮。[3]

Histochemistry of sstr2 expression [2]
Six adult male and six female sstr2+/lacZ mice were perfused with 3% PFA and coronal sections through the MS, rPOA, and AH were processed for X-gal histochemistry by washing in TBS followed by X-gal solution [2 mM MgCl2, 4 mM K3Fe(CN)6, 4 mM K4Fe(CN)6, and 4 mg/ml 5-bromo-4-chloro-3-indoyl-β-D-galactosidase in TBS] overnight at room temperature to reveal lacZ-expressing cells within the brain sections. Sections were then processed for GnRH immunocytochemistry using an LR1 antibody and diaminobenzidine chromogen as detailed previously. GnRH neurons located in MS, rPOA, and AH were examined. Two sections from each brain region were selected, and the numbers of single (GnRH) and double-labeled (GnRH plus X-gal staining) neurons were determined. The X-gal expression in GnRH-immunoreactive neurons was calculated as the percentage of total number of GnRH-immunoreactive neurons in each region.

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Brain slice preparation and electrophysiology [2]
Brain slices were acutely prepared as described (18). Mice were decapitated and brains rapidly removed and placed in the ice-cold bicarbonate-buffered artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.2 MgCl2, 11 D-glucose, 1.4 NaH2PO4, and 25 NaHCO3 (pH 7.4 when bubbled with 95% O2 and 5% CO2). Brains were blocked and glued with cyanoacrylate to the chilled stage of a vibratome, and 150- to 200-μm-thick coronal slices containing the rPOA were cut. The slices were placed in oxygenated ACSF for at least 1 h at room temperature. The slices were transferred to the recording chamber, held submerged, and continuously superfused with ACSF at a rate of 4–5 ml/min. The slices were viewed with an upright microscope and fluorescent GnRH neurons identified at ×10 and ×40 objective magnification by brief fluorescence illumination and then viewed and patched under Nomarski differential interference contrast optics. Patch pipettes were pulled from thin-wall borosilicate glass-capillary tubing on a Flaming/Brown Micropipette puller. The pipette solution was passed through a disposable 0.22-μm filter and contained (in mM) 130 KCl, 5 NaCl, 0.4 CaCl2, 1 MgCl2, 10 HEPES, and 1.1 EGTA (pH 7.3 with KOH). Gramicidin was first dissolved in dimethylsulfoxide to a concentration of 2.5–5 mg/ml and then diluted in the pipette solution just before use to a final concentration of 2.5–5 μg/ml and sonicated for 10 min. The gramicidin-perforated patch recordings were performed using an Axopatch 200B amplifier. The tip resistance of the electrode was 4–6 Mohm. In initial experiments, access resistance was monitored and experiments begun when resistance stabilized at 50–90 Mohm. This typically took 15–20 min after gigaseal formation and always corresponded to the resting membrane potential (RMP) of the cell reaching a stable level below −45 mV. In all subsequent cells, experiments were begun when the RMP reached a stable level below −45 mV. Spontaneous rupture of the seal was evident by a sudden overshooting of action potentials above 0 mV. Membrane potential changes were sampled online using a Digidata 1322A interface connected to an IBM PC. Any GnRH neuron that displayed a shift in resting membrane potential of more than 2 mV was considered to have responded. Acquisition and subsequent analysis of the acquired data were performed using the Clampex9 software. Traces were plotted using the Origin7 software. All recordings were made at room temperature.

[1]. Antagonist effects of seglitide (MK 678) at somatostatin receptors in guinea-pig isolated right atria. Br J Pharmacol. 1993 Aug;109(4):898-9.

[2]. Somatostatin inhibition of gonadotropin-releasing hormone neurons in female and male mice. Endocrinology, 2010, 151(7): 3258-3266.

[3]. Blood pressure reduction in hypertensive-diabetic rats by the somatostatin analog MK-678. Life Sci. 1989;45(3):267-74.

Previous studies indicate that somatostatin regulates gonadotropin secretion. We investigated here whether somatostatin has direct effects on GnRH neurons in the adult male and female mice. Dual-labeling immunofluorescence experiments revealed the presence of somatostatin-immunoreactive fibers adjacent to GnRH neurons, and three-dimensional confocal reconstructions demonstrated apparent somatostatin fiber appositions with 50-60% of GnRH neurons located throughout the brain in both male and female mice. Perforated patch-clamp recordings from GnRH-green fluorescent protein neurons revealed that approximately 70% of GnRH neurons responded in a dose-dependent manner to 10-300 nm somatostatin with an acute membrane hyperpolarization and cessation of firing. This effect persisted in the presence of tetrodotoxin and amino acid receptor antagonists, indicating a direct postsynaptic site of action on the GnRH neuron. The identity of the somatostatin receptors underlying this action was assessed using GnRH neuron single-cell RT-PCR. Of the somatostatin receptor subtypes, the sstr2 transcript was the most prevalent and detected in both males and females. The expression of sstr2 by GnRH neurons was confirmed in the sstr2 knockout/LacZ knock-in mouse line. Electrophysiological studies demonstrated that the sstr2-selective agonist seglitide exerted acute hyperpolarizing actions on GnRH neurons identical to those of somatostatin. Together, these studies reveal somatostatin, acting through sstr2, to be one of the most potent inhibitors of electrical excitability of male and female GnRH neurons identified thus far.[2]

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Our RT-PCR and histochemical studies suggest that sstr2 is the most prevalent somatostatin receptor subtype in GnRH neurons in both sexes. Our results are also in good agreement with Todman et al., who demonstrated that 25% of GnRH neurons express sstr2 in female mice using single-cell microarray on GnRH neurons. Here we further investigated whether sstr2 is involved in the somatostatin-induced inhibitory effects. Seglitide, an sstr2-specific agonist, mimicked the somatostatin-induced membrane hyperpolarization and was maintained in the presence of TTX and the amino acid receptor antagonist cocktail, suggesting a direct action on GnRH neurons. These results demonstrate that sstr2 is very likely expressed postsynaptically in the GnRH neuron and can mediate membrane hyperpolarization in response to somatostatin on GnRH neurons. Interestingly, gene profiling experiments with heterozygous sstr2+/lacZ mice and RT-PCR showed that only 10–20% of GnRH neurons transcribe sstr2 or express sstr2 mRNA, whereas single-cell electrophysiological investigations indicate that 50% of GnRH neurons are suppressed by seglitide, a specific antagonist for sstr2 (IC50/Kd, 0.2–1.5 nM). Although the IC50/Kd values of seglitide for the cloned human sstr5 show a relatively wide range (0.06–23 nM, GnRH neurons do not express sstr5, ruling out the activation of these receptors on GnRH neurons after seglitide administration. The discrepancy between the results of the gene profiling (10–20% expression) and electrophysiological (50–70% expression) investigations are most likely explained by differences in detection sensitivity of these methods, with electrophysiological approaches being the most sensitive index of functional receptor expression.[2]

C46H58N8O8

851.02

868.448

C, 64.92; H, 6.87; N, 13.17; O, 15.04

99248-33-6

81377-02-8 (Seglitide)

9854212

Cyclo({Ala(Me)}-Tyr-{d-Trp}-Lys-Val-Phe); cyclo[N-methyl-L-alanyl-L-tyrosyl-D-tryptophyl-L-lysyl-L-valyl-L-phenylalanyl] acetic acid

Cyclo({Ala(Me)}-Y-{d-Trp}-KVF); cyclo[N(Me)Ala-Tyr-D-Trp-Lys-Val-Phe].CH3CO2H

White to off-white solid powder

4.079

282.6

9

10

11

63

1460

6

CC(=O)O.NCCCC[C@@H]1NC(=O)[C@@H](CC2=CNC3=CC=CC=C23)NC(=O)[C@H](CC2C=CC(O)=CC=2)NC(=O)[C@H](C)N(C)C(=O)[C@H](CC2C=CC=CC=2)NC(=O)[C@H](C(C)C)NC1=O

FIKSSPSBVSPVFU-WIKDFEFZSA-N

InChI=1S/C44H56N8O7.C2H4O2/c1-26(2)38-43(58)50-37(23-28-12-6-5-7-13-28)44(59)52(4)27(3)39(54)48-35(22-29-17-19-31(53)20-18-29)41(56)49-36(24-30-25-46-33-15-9-8-14-32(30)33)42(57)47-34(40(55)51-38)16-10-11-21-45;1-2(3)4/h5-9,12-15,17-20,25-27,34-38,46,53H,10-11,16,21-24,45H2,1-4H3,(H,47,57)(H,48,54)(H,49,56)(H,50,58)(H,51,55);1H3,(H,3,4)/t27-,34-,35-,36+,37-,38-;/m0./s1

acetic acid;(3S,6S,9S,12R,15S,18S)-9-(4-aminobutyl)-3-benzyl-15-[(4-hydroxyphenyl)methyl]-12-(1H-indol-3-ylmethyl)-1,18-dimethyl-6-propan-2-yl-1,4,7,10,13,16-hexazacyclooctadecane-2,5,8,11,14,17-hexone

MK 678; MK-678; L-363586; MK 678; 5P57R7IR6J; CHEMBL311695; Seglitide acetate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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