β adrenergic receptor

In intact rat adipocytes, isoproterenol (300 nM, 3 min) causes an estimated 100% rise in granular cGMP and cilamide-inhibited low-Km cAMP phosphodiesterase (cAMP-PDE) activity [1]. In rat adipocytes, insulin-stimulated glucose transport activity is inhibited by isoproterenol. Isoproterenol, in the absence of adenosine, promotes a time-dependent (t1/2 ~2 min) >50% decrease in GLUT4 accessibility on the surface of insulin-stimulated cells, which is directly correlated with the observed suppression of transport activity [2]. Cyclic AMP levels are raised by isoproterenol (5 nM and 10 μM), cilopamide (10 mM), rolipram, a cyclic GMP elevator, and a cyclic PDE (PDE 4) inhibitor (10 mM). This action can be amplified by 50 nM ANF or 30 nM SNP + 100 nM DMPPO [3]. Gene-specific hybridization of Gs α remained unaltered, whereas isoproterenol enhanced the transcriptional activity of the Gi α-2 gene to 140% of control levels [4]. The iK activation curve shifts negatively by around 10 mV when isoproterenol (20 nM) is added, regardless of whether 300 nM nisoldipine inhibits L-type Ca2+ currents or not [5]. Isoproterenol (20 nM) improved the spontaneous pacing rate of sinoatrial node pacemaker cells in isolated rabbit pacemaker cells by 16% [5].

Isoproterenol (oral, 0.27-0.64 μg/kg) is extensively metabolized in dogs by relatively few reactions [6].

1. In rat aortic rings precontracted with phenylephrine, the beta-adrenoceptor agonist isoprenaline (10 nM to 30 microM) produces greater relaxant effects in preparations with endothelium than in endothelium-denuded preparations. The aim of this study was to determine the mechanisms involved in this effect and in particular investigate the possibility of a synergistic action between adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP). 2. isoprenaline-induced relaxation of rat aortic rings precontracted with phenylephrine was greatly reduced by the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 300 microM) or the soluble guanylate cyclase inhibitors methylene blue (10 microM) or IH-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) but unaffected by indomethacin (10 microM), a cyclo-oxygenase inhibitor. Similarly, in intact rings, the concentration-response curve of forskolin (10 nM to 1 microM) was shifted to the right upon endothelium removal or treatment with methylene blue. 3. In endothelium-denuded rat aortic rings, isoprenaline-induced relaxation was potentiated by the guanylate cyclase activators atrial natriuretic factor (ANF, 1 to 10 nM) and sodium nitroprusside (SNP, 1 to 10 nM), and to a greater extent in the presence of the cyclic GMP-specific phosphodiesterase (PDE 5) inhibitor, 1,3dimethyl-6-(2-propoxy-5-methane sulphonylamidophenyl) pyrazolo [3,4-d] pyrimidin-4-(5H)-one (DMPPO, 30 nM). Relaxation induced by isoprenaline was also potentiated by the cyclic GMP-inhibited PDE (PDE 3) inhibitor cilostamide (100 nM). 4. Intracellular cyclic nucleotide levels were measured either in rat cultured aortic smooth muscle cells or in de-endothelialized aortic rings. In both types of preparation, isoprenaline (5 nM and 10 microM) increased cyclic AMP levels and this effect was potentiated by cilostamide (10 microM), by rolipram, a cyclic AMP-specific PDE (PDE 4) inhibitor (10 microM) and by cyclic GMP-elevating agents (50 nM ANF or 30 nM SNP plus 100 nM DMPPO). In isoprenaline-stimulated conditions, the increase in cyclic AMP induced by rolipram was further potentiated by cilostamide and by cyclic GMP-elevating agents. Cilostamide and cyclic GMP-elevating agents did not potentiate each other, suggesting a similar mechanism of action. 5. We conclude that in vascular smooth muscle (VSM) cells an increase in cyclic GMP levels may inhibit PDE 3 and, thereby, cyclic AMP catabolism. Under physiological conditions of constitutive NO release, and to a greater extent in the presence of the PDE 5 inhibitor DMPPO, cyclic GMP should act synergistically with adenylate cyclase activators to relax VSM.[3]

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Incubation of intact rat fat cells with maximally effective concentrations of insulin (1 nM, 12 min) or isoprenaline (300 nM, 3 min) increased particulate cGMP- and cilostamide-inhibited, low-Km cAMP phosphodiesterase (cAMP-PDE) activity by about 50% and 100%, respectively. In 32P-labeled cells, these agents induced serine 32P-phosphorylation of a 135-kDa particulate protein and, to a variable and lesser extent, a 44-kDa protein, which were selectively immunoprecipitated by anti-cAMP-PDE, as analyzed by SDS/PAGE and autoradiography. In the absence of hormonal stimulation, little phosphorylation was detected (less than 10% of that with the hormones). The two phosphoproteins were identified as cAMP-PDE or a closely related molecule (in the case of the 44-kDa species, perhaps a proteolytic fragment) since (i) amounts of 32P in the immunoprecipitated 135-kDa protein paralleled enzyme inactivation, (ii) prior incubation of the anti-cAMP-PDE with the pure rat or bovine enzyme selectively blocked the immunoprecipitation of the phosphoproteins, (iii) 135- and 44-kDa proteins reacted with the anti-cAMP-PDE on Western immunoblots, and (iv) the two phosphoproteins copurified with cAMP-PDE activity through DEAE-Sephacel chromatography and were isolated by highly selective affinity chromatography on cilostamide-agarose. Thus, in fat cells, catecholamine- and insulin-induced activation of the cAMP-PDE may be mediated via phosphorylation by cAMP-dependent protein kinase and an insulin-activated serine protein kinase, respectively.[1]

Incubation of intact rat fat cells with maximally effective concentrations of insulin (1 nM, 12 min) or isoprenaline (300 nM, 3 min) increased particulate cGMP- and cilostamide-inhibited, low-Km cAMP phosphodiesterase (cAMP-PDE) activity by about 50% and 100%, respectively. In 32P-labeled cells, these agents induced serine 32P-phosphorylation of a 135-kDa particulate protein and, to a variable and lesser extent, a 44-kDa protein, which were selectively immunoprecipitated by anti-cAMP-PDE, as analyzed by SDS/PAGE and autoradiography. In the absence of hormonal stimulation, little phosphorylation was detected (less than 10% of that with the hormones). The two phosphoproteins were identified as cAMP-PDE or a closely related molecule (in the case of the 44-kDa species, perhaps a proteolytic fragment) since (i) amounts of 32P in the immunoprecipitated 135-kDa protein paralleled enzyme inactivation, (ii) prior incubation of the anti-cAMP-PDE with the pure rat or bovine enzyme selectively blocked the immunoprecipitation of the phosphoproteins, (iii) 135- and 44-kDa proteins reacted with the anti-cAMP-PDE on Western immunoblots, and (iv) the two phosphoproteins copurified with cAMP-PDE activity through DEAE-Sephacel chromatography and were isolated by highly selective affinity chromatography on cilostamide-agarose. Thus, in fat cells, catecholamine- and insulin-induced activation of the cAMP-PDE may be mediated via phosphorylation by cAMP-dependent protein kinase and an insulin-activated serine protein kinase, respectively[2].

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 Permeabilized patch whole-cell voltage clamp methods were used to investigate the effects of isoprenaline (ISO) on total delayed rectifier potassium current, iK, in rabbit sino-atrial (SA) node pacemaker cells; total iK is composed of the rapidly activating iKr and the slowly activating iKs, but predominantly iKr in this species. ISO (20 nM) increased the amplitude of total iK and caused a negative shift of approximately 10 mV in the activation curve for iK, both in the absence and in the presence of 300 nM nisoldipine to block the L-type Ca2+ current, iCa,L. The same concentration (20 nM) of ISO increased the spontaneous pacemaker rate of SA node pacemaker cells by 16%. In addition to increasing the amplitude of iK, ISO (20-50 nM) also increased the rate of deactivation of this current. The stimulation of iK by ISO was reversed by 10 microM H-89, a selective protein kinase A inhibitor, but not by 200 nM bisindolymaleimide I, a selective protein kinase C inhibitor. It therefore appears that the mechanisms by which -adrenoceptor agonists increase pacemaking rate in sinoatrial node pacemaker cells include an increase in the rate of deactivation of iK in addition to the well-documented augmentation of iCa,L and the positive shift of the activation curve for the hyperpolarization-activated inward current, if. The observations are also consistent with a role for protein kinase A in the stimulation of iK by ISO in SA node cells[5].

Animal/Disease Models: Dog[1]
Doses: 0.27-0. 64 μg/kg
Route of Administration: Oral
Experimental Results:Most of the radioactivity is excreted unchanged through the urine, and only one-third of the radioactivity in the urine exists in the form of O-methyl metabolites. It shows that almost all plasma radioactivity is bound to isoprenaline, and this metabolite accounts for more than 80% of urine radioactivity. . Indicates that heart rate returns to baseline values when plasma concentrations are high.

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Rats were treated by a 4-day subcutaneous infusion of isoprenaline (2.4 mg/kg per day) or 0.9% NaCl as control. To avoid the influence of developmental expression patterns, adult rats were chosen for all experiments. Signals for Gi alpha-2 and the stimulatory G protein alpha-subunit Gs alpha were specific and due to hybridization of nascent mRNA transcripts. In the isoprenaline group the transcriptional activity of Gi alpha-2 gene increased to 140% of the control value, whereas gene specific hybridization for Gs alpha remained unchanged. These results show that increased Gi alpha-2 mRNA levels after stimulation with isoprenaline are at least partially caused by enhanced transcription of Gi alpha-2 mRNA.[4]

Absorption, Distribution and Excretion
Data on the absorption kinetics of isoproterenol are not readily available. After 48 hours, the recovery rate of isoproterenol in feces was 12.2%–27.0%, and in urine it was 59.1%–106.8%. The main form of drug recovered in urine was conjugated isoproterenol, with free isoproterenol accounting for 6.5%–16.2%, and 3-O-methylisoproterenol and its conjugates accounting for 2.6%–11.4%. In pediatric patients, the volume of distribution was 216±57 mL/kg. In pediatric patients, the clearance rate of isoproterenol was 42.5±5.0 mL/kg/min. Metabolism/Metabolites Isoproterenol is primarily metabolized to glucuronide conjugates. Isoproterenol can also be O-methylated by catechol O-methyltransferase to the metabolite 3-O-methylisoproterenol, which can be further glucuronidated. 1. The metabolism of isoproterenol has been studied in humans and dogs, including intravenous, oral, or duodenal administration. 2. Intravenously administered isoproterenol in humans and dogs is mainly excreted unchanged in urine. Only one-third of the radioactivity in urine is present as the O-methyl metabolite. 3. After oral administration in humans or duodenal administration in dogs, plasma radioactivity is almost entirely present as conjugated isoproterenol, which accounts for more than 80% of urinary radioactivity. 4. Catechol O-methyltransferase may be less important than uptake (2) in limiting the pharmacological effects of isoproterenol. 5. Pharmacological response (increased heart rate) is only correlated with plasma isoproterenol concentration after rapid intravenous injection. In dogs, heart rate returned to baseline even after prolonged infusion or duodenal administration, despite high plasma isoproterenol concentrations. [6]

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Biological half-life
The half-life of intravenously administered isoproterenol is 2.5–5 minutes. The half-life of orally administered isoproterenol is 40 minutes.

Toxicity Summary
The pharmacological effects of isoproterenol are at least partly attributed to the stimulation of intracellular adenylate cyclase by β-adrenergic receptors, an enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). Elevated cAMP levels are associated with bronchial smooth muscle relaxation and inhibition of the release of immediate-type hypersensitivity mediators from cells, particularly mast cells. Protein Binding Isoproterenol has a plasma protein binding rate of 68.8 ± 1.2%, primarily bound to serum albumin.

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The oral LD50 for rats is 2221 mg/kg. Toxicology and Applied Pharmacology, 18(185), 1971 [PMID:5542824]
The intraperitoneal LD50 for rats is 128 mg/kg. Acta Toxicologica Sinica, (5)(40), 1995
The subcutaneous LD50 for rats is 600 μg/kg. Basic and Applied Toxicology, 1(443), 1981
The intravenous LD50 for rats is 26900 μg/kg. Sensory organs and special senses: tearing: eye; behavior: convulsions or effects on the epileptic threshold; lungs, pleura or respiration: respiratory stimulation. Pharmacology and Therapeutics, 7(627), 1979.
The oral LD50 for mice is 1260 mg/kg. Japanese Medicine, -(119), 1990.

[1]. Evidence that insulin and isoprenaline activate the cGMP-inhibited low-Km cAMP phosphodiesterase in rat fat cells by phosphorylation. Proc Natl Acad Sci U S A. 1990 Jan;87(2):533-7.

[2]. Cell surface accessibility of GLUT4 glucose transporters in insulin-stimulated rat adipose cells. Modulation by isoprenaline and adenosine. Biochem J. 1992 Nov 15;288 (Pt 1):325-30.

[3]. Effects of cyclic GMP elevation on isoprenaline-induced increase in cyclic AMP and relaxation in rat aortic smooth muscle: role of phosphodiesterase 3. Br J Pharmacol. 1996 Oct;119(3):471-8.

[4]. Isoprenaline stimulates gene transcription of the inhibitory G protein alpha-subunit Gi alpha-2 in rat heart. Circ Res. 1993 Mar;72(3):696-700.

[5]. Modulation of delayed rectifier potassium current, iK, by isoprenaline in rabbit isolated pacemaker cells. Exp Physiol. 2000 Jan;85(1):27-35.

[6]. Metabolism of isoprenaline in dog and man. Br J Pharmacol . 1972 Nov;46(3):458-72.

Pharmacodynamics
Isoproterenol is a non-selective β-adrenergic receptor agonist used to treat various heart conditions and bronchospasm during anesthesia. Isoproterenol has a short duration of action due to its rapid clearance and wide therapeutic index. Patients should be informed of the risks associated with using isoproterenol in treating cardiogenic shock after myocardial infarction, exacerbation of atrioventricular block, or induction of Adams-Scholes attacks.

C11H17NO3

211.257583379745

211.12

C, 62.54; H, 8.11; N, 6.63; O, 22.72

7683-59-2

Isoprenaline hydrochloride;51-30-9;Isoprenaline hemisulfate;299-95-6

3779

Typically exists as solid at room temperature

1.2±0.1 g/cm3

417.5±40.0 °C at 760 mmHg

165 - 170ºC

179.7±17.9 °C

0.0±1.0 mmHg at 25°C

1.579

0.25

4

4

4

15

187

0

CC(NCC(C1C=CC(O)=C(O)C=1)O)C

JWZZKOKVBUJMES-UHFFFAOYSA-N

InChI=1S/C11H17NO3/c1-7(2)12-6-11(15)8-3-4-9(13)10(14)5-8/h3-5,7,11-15H,6H2,1-2H3

4-[1-hydroxy-2-(propan-2-ylamino)ethyl]benzene-1,2-diol

isoproterenol; Isoprenaline; 7683-59-2; Isopropydrin; Novodrin; Asiprenol; Assiprenol; Bellasthman;

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

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.)