Orexin receptor

OX2 receptor has an EC50 of 60 nM in the [Ca2+]i transient experiment and an IC50 of 36 nM in the binding assay, indicating that it is a high-affinity receptor for human orexin B. The computed IC50 in the competitive binding test and the EC50 in the [Ca2+]i transient experiment for human orexin-B are 420 nM and 2500 nM, respectively[2]. This indicates that human Orexin B has a much lower affinity for human OX1.

The rat's outer retina contains rod bipolar cells (RBCs), whose activity is modulated by orexin-B. The scotopic electroretinographic b-wave, which is a mirror of RBC activity, was recorded in vivo and its amplitude increased upon intravitreal injection of orexin-B[4].

Whole-cell patch-clamp recording in dissociated RBCs [4]
RBCs were acutely dissociated from retinas by enzymatic and mechanical treatments as described previously (Yu et al., 2006), with minor modifications. In brief, isolated retinas that were digested in 7–8 mg/ml papain containing Hanks' solution, supplied with l-cysteine and bovine serum albumin (0.75 mg/ml for each) for about 30 min at 35-36 °C and were mechanically dissociated by gently triturating with fire-polished Pasteur pipettes. Whole-cell membrane currents were recorded from dissociated RBCs bathed in Ringer's that contained the following (in mM): NaCl 145, KCl 5, CaCl2 2, MgCl2 1, HEPES 10, and glucose 16; pH adjusted to 7.4 with NaOH. These cells were voltage-clamped at −60 mV and the pipettes for current recording were filled with the internal solution containing (in mM): CsCl 125, CaCl2 1, MgCl2 2, EGTA 10, HEPES 10, MgATP 2, NaGTP 0.4, and sodium phosphocreatine 10; adjusted to pH 7.2 with CsOH and to 290–300 mOsmol/L. GABA of 50 μM was applied for 5 s every 2 min and the currents were picked up from RBCs by a patch amplifier. In all whole-cell recording experiments in both slices and isolated cells, fast capacitance was fully cancelled, and cell capacitance was partially cancelled by the circuits of the amplifier as much as possible. Seventy percent of the series resistance of the recording electrode was compensated. All recordings were captured at room temperature (20-25 °C). 

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Calcium imaging [4]
Changes in intracellular calcium concentrations ([Ca2+]i) were assessed using the membrane permeable indicator fura-2 AM. Fura-2 AM was dissolved in 20% Pluronic F-127 (w/v, DMSO) and added to a chamber that contained Ringer's, with a final Fura-2 AM concentration of 2 μM. Isolated RBCs were incubated in the dye solution for 45 min at room temperature and then perfused with dye-free Ringer's for at least 30 min. A high-speed continuously scanning monochromatic light source was used for the excitations at wavelengths of 340 nm and 380 nm. Fluorescence intensities at both wavelengths (F340 and F380) were measured every 1–10 s. Meanwhile, digital fluorescence images were obtained with an inverted microscope furnished with a digital CCD camera. The ratio between the two images was proportional to the [Ca2+]i of the cell under study.

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Orexin-A (OXA) and orexin-B (OXB) are polypeptides derived from the same 130 amino acid long precursor (prepro-orexin) that bind and activate two closely related orphan G protein-coupled receptors OX1-R and OX2-R. These hypothalamic neuropeptides stimulate food intake and energy expenditure and play a significant role in sleep-wakefulness regulation. Present studies aimed to investigate the effects of orexins on proliferative activity and osteocalcin secretion by cultured rat calvarial osteoblast-like (ROB) cells. Conventional RT-PCR methods detected expression of the OX1-R gene in freshly isolated ROB cells and cells cultured for 7, 14 and 21 days. In contrast, at all time points tested, expression of prepro-OX or OX2-R genes was not demonstrated. QPCR revealed the highest expression of OX1-R gene in freshly isolated bone cells and a notably lower one in cultured ROB cells. Exposure of cultured cells to both OXA and OXB stimulated expression of the OX1-R gene. However, this effect was seen at the lowest tested concentration (1x10(-10) M). Exposure of cultured ROB cells to OXA for 48 h did not change osteocalcin concentrations in media analyzed at days 7, 14 and 21 of culture. On the contrary, OXB notably stimulated osteocalcin concentrations in media taken at days 14 and 21 of culture. In contrast, OXA exerted a notable inhibitory effect on the proliferative activity of ROB cells at day 7 of culture, while OXB exerted a similar effect at day 14. Thus, the obtained results suggest that: (i)(ROB) cells are provided with functional OX1-R gene; (ii) in ROB cells expression of this gene seems to be up-regulated by low concentrations of both OXA and OXB; (iii) OXB exerts inhibitory effects on proliferative activity and stimulating effects on osteocalcin secretion by cultured ROB cells; (iv) rat calvarial osteoblasts provided with OX receptor may be a target for circulating orexins. Thus, orexins may be included in the expanding group of neuropeptides involved in the physiological regulation of the major bone cell types[3].

Animal/Disease Models: Male Wistar rats (180 -200 g)[2].
Doses: 3 nM and 30 nM.
Route of Administration: Administered in a 5 mL bolus through a catheter placed in the left lateral ventricle in early light phase.
Experimental Results: Dramatically augmented food intake.

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Intravitreal injection [1]
Rats, dark-adapted for at least 4 h before the experiments, were deeply anesthetized with a mixture of 4% chloral hydrate (0.01 ml/g) and 0.05% atropine (0.02 ml/g) under dim red light illumination (approximately 1.4 lux at the cornea) provided by an LED. Orexin-Bof four doses (dissolved in 2 μL 0.9% saline) were intravitreally injected into the right eyes using a Nanoject II microinjector. The injection yielded final vitreal concentrations of Orexin-B corresponding to 0.1 μM, 0.3 μM, 1 μΜ and 3 μM respectively, given an average vitreal volume of 25 μL in rats. An equal volume of vehicle (0.9% saline) was injected into the contralateral eye as control.

[1]. Orexins: a new family of neuropeptides. Br J Anaesth. 1999 Nov;83(5):695-7.

[2]. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell. 1998 Feb 20;92(4):573-85.

[3]. Orexin B inhibits proliferation and stimulates specialized function of cultured rat calvarial osteoblast-like cells. Int J Mol Med. 2008;22(6):749-755.

[4]. Orexin-B modulates synaptic transmission of rod bipolar cells in rat retina. Neuropharmacology. 2018;133:38-50.

Orexin-A, -B play a crucial role in arousal and feeding by activating two G-protein-coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Orexins, along with orexin receptors, are expressed in retinal neurons, and they have been shown to differentially modulate excitatory AMPA receptors of amacrine and ganglion cells in the inner retina. In this work we report that orexin-B modulates the activity of rod bipolar cells (RBCs) located in the outer retina of rat. Intravitreal injection of orexin-B increased the amplitude of the scotopic electroretinographic b-wave, a reflection of RBC activity, recorded in vivo. Patch clamp recordings in rat retinal slices showed that orexin-B did not change glutamatergic excitatory component of the RBC response driven by photoreceptors. Effects of orexin-B on GABA receptor-mediated synaptic transmission of RBCs were then examined. In retinal slice preparations orexin-B suppressed GABA receptor-mediated inhibitory postsynaptic currents of RBCs in the inner plexiform layer. Furthermore, using whole-cell recordings in isolated RBCs it was shown that orexin-B suppressed GABAC receptor-, but not GABAA receptor-, mediated currents of the RBCs, an effect that was blocked by OX1R and OX2R antagonists. The orexin-B-induced inhibition of GABAC currents was likely mediated by a Gi/o/PC-PLC/Ca2+-independent PKC signaling pathway, as such inhibition was absent when each step of the above-pathway was blocked with GDP-β-S/pertussis toxin (for Gi/o), D609 (for PLC), bisindolylmaleimide IV (for PKC)/rottlerin (for PKCδ), respectively. The orexin-B-induced potentiation of RBC activity may improve visual acuity and contrast sensitivity of the animal during the dark period (wake phase).[4]

C₁₂₈H₂₁₆F₃N₄₅O₃₆S

3050.42

Orexin B, rat, mouse;202801-92-1

RPGPPGLQGRLQRLLQANGNHAAGILTM-NH2

Typically exists as solid at room temperature

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

H2O :~100 mg/mL (~32.78 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)