Kinase experiment:

The [35S]-GTPγS binding assay is carried out. Cell membranes (25 ±75 ug) are incubated in Buffer B containing 0.1 mM dithiothreitol (DTT) and 1 uM GDP and drugs in a volume of 0.9 mL for 30 min at 30℃. This preincubation ensures that the agonists tested are at equilibrium when the [35S]-GTPγS (50±150 pM, final concentration) is added (in 100 uL ofBuffer B) to initiate the reaction. The assay mixture is incubated for a further 20 min unless otherwise stated. The assays are terminated by rapid filtration and bound radio-activity determined as described for the radio-ligand binding assays above. The total binding of [35S]-GTPγS is less than 20% of that added[1].

Animal experiment:

Mice[4] Swiss mice (20-25 g) of either sex (total 150) are used. Bromocriptine mesylate is used as dopamine receptor (D2) agonist. Haloperidol is diluted in distilled water which is used for a vehicle of injection. Bromocriptine mesylate is dissolved in one drop of glacial acetic acid and made up to volume in distilled water. Imipramine is dissolved in 0.9% normal saline. Haloperidol (0.1 mg/kg, i.p.) and Bromocriptine mesylate (2 mg/kg, i.p.) are administered for 7 days in groups of mice in Forced Swimming Test (FST) and Tail Suspension Test (TST). Imipramine (10 mg/kg, p.o.) as a standard is administered in positive control groups for 7 days. Rats[5] Adult male Sprague-Dawley rats (N=112, 275-325 g) are used. Two weeks after the 6-OHDA injection, the animals are briefly (

Bromocriptine mesylate is a potent dopamine D2/D3 receptor agonist, which binds D2 dopamine receptor with pKi of 8.05±0.2.
Bromocriptine stimulates [35S]-GTPγS binding at D2 dopamine receptor expressed in CHO cells with pEC50 of 8.15±0.05[1]. Bromocriptine also is a strong inhibitor of brain nitric oxide synthase. The ergot alkaloid Bromocriptine (BKT) is found to act as a strong inhibitor of purified neuronal nitric oxide synthase (NOS) (IC50=10±2 μM) whereas it is poorly active towards inducible macrophage NOS (IC50>100 μM) [2]. Bromocriptine is found to inhibit the activity of at least one human cytochrome P450 enzyme. Bromocriptine is a potent inhibitor of CYP3A4 with a calculated IC50 value for the interaction of 1.69 μM[3].
Bromocriptine mesylate (2 mg/kg, i.p.) is administered for 7 days in groups of mice in forced swimming test (FST) and tail suspension test (TST). Bromocriptine group shows significant anti-immobility action as compared to control. When Bromocriptine administered 30 min after the last dose of 7 days MPE treatment and subjected to FST, this dopaminergic agonist produces significant and dose dependent potentiation of anti-immobility action of MPE (200 mg/kg, p.o.) as compared to MPE treatment alone. Bromocriptine treatment group shows a significant reduction of immobility time as compared to control. Bromocriptine administration after 7 days pretreatment with MPE (100 and 200 mg/kg, p.o.) shows significant and dose dependent potentiation of anti-immobility action of MPE as compared to MPE treatment alone[4]. Intracisternal administration of Bromocriptine decreases significantly the static mechanical allodynia (SMA) score compared to that of sham (saline-injected rats) and its effect lasted for 30 min. Intraperitoneal administration of Bromocriptine induces a significant, dose dependent (0.1 mg and 1 mg/kg) decrease in pain scores in CCI-IoN group when compared to sham and its effect lasted for 6 h. The highest dose induces the highest score decrease (P<0.01). Bromocriptine effect lasts for 20 min. Intraperitoneal administration of Bromocriptine induces a significant dose dependent decrease in SMA score in CCI-IoN+6-OHDA lesioned group compared to that of sham. Its effect lasts for 6 h[5].
Reference:
[1]. Gardner B, et al. Agonist action at D2(long) dopamine receptors: ligand binding and functional assays. Br J Pharmacol. 1998 Jul;124(5):978-84.
[2]. Renodon A, et al. Bromocriptine is a strong inhibitor of brain nitric oxide synthase: possible consequences for the origin of its therapeutic effects.FEBS Lett. 1997 Apr 7;406(1-2):33-6.
[3]. Wynalda MA, et al. Assessment of potential interactions between dopamine receptor agonists and various human cytochrome P450 enzymes using a simple in vitro inhibition screen. Drug Metab Dispos. 1997 Oct;25(10):1211-4.
[4]. Rana DG, et al. Dopamine mediated antidepressant effect of Mucuna pruriens seeds in various experimental models of depression. Ayu. 2014 Jan;35(1):90-7.
[5]. Dieb W, et al. Nigrostriatal dopaminergic depletion increases static orofacial allodynia. J Headache Pain. 2016;17:11.