β adrenergic receptor

The whole-cell patch-clamp and intracellular perfusion techniques were used for studying the effects of a beta-2 adrenergic receptor activation on the L-type Ca current (ICa) in frog ventricular myocytes. The beta-2 adrenergic agonist Zinterol increased ICa in a concentration-dependent manner with an EC50 (i.e., the concentration of zinterol at which the response was 50% of the maximum) of 2.2 nM. The effect of zinterol was essentially independent of the membrane potential. The stimulatory effect of zinterol was competitively antagonized by ICI 118,551, a beta-2 adrenergic antagonist. The maximal stimulatory effect of zinterol was comparable in amplitude to the effect of a saturating concentration (1 or 10 microM) of isoprenaline, a nonselective beta adrenergic agonist. Moreover, 3-isobutyl-1-methylxanthine (100 microM), a nonselective phosphodiesterase inhibitor, or forskolin (10 microM), a direct activator of adenylyl cyclase, had no additive effects in the presence of 0.1 microM zinterol. Zinterol had a long lasting action on frog ICa because after washout of the drug, ICa returned to basal level with a time constant of 17 min. An application of acetylcholine (1 microM) during this recovery phase promptly reduced ICa back to its basal level suggesting a persistent activation of adenylyl cyclase due to a slow dissociation rate constant of zinterol from its receptor. Zinterol also increased ICa in rat ventricular and human atrial myocytes, and the maximal effect was obtained at 10 and 1 microM, respectively. In all three preparations, intracellular perfusion with 20 microM PKI(15-22), a highly selective peptide inhibitor of cAMP-dependent protein kinase, completely antagonized the stimulatory effect of zinterol on ICa. We conclude that beta-2 adrenergic receptor activation produces a strong increase in ICa in frog, rat and human cardiac myocytes which is due to stimulation of adenylyl cyclase and activation of cAMP-dependent phosphorylation. [2]

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In vitro arrhythmogenic effects of Zinterol [3]
To determine the underlying cellular mechanism of ventricular arrhythmias induced by zinterol, we measured contractions in isolated LV myocytes from control and HF rabbits. Field stimulation (0.5−4 Hz) of fluo-3 loaded myocytes (37°C) demonstrated that zinterol (1 μM) induced aftercontractions (AC's) in 7 of 7 HF myocytes, compared to 0 of 6 controls (p<0.01), an effect that was blocked by ICI-118,551 (100 nM, Fig.1, bottom). These ACs were associated with spontaneous SR Ca release or aftertransients. To verify that zinterol's effects were due to stimulation of β2-ARs, additional β2-AR stimulation studies were performed with 1 μM zinterol plus 300 nM of the β1-AR blocker CGP-20712A (CGP). Zinterol + CGP induced AC's (associated with aftertransients, Fig.2) in 7 of 8 HF myocytes, compared to 0 of 7 controls (p<0.01), an effect that was blocked by 100 nM ICI-118,551. Similar results were found in HF and control myocytes on glass slides without laminin, ruling out potential effects of laminin on β2-AR signaling.14 Lower concentrations of Zinterol (300nM) with CGP failed to induce aftertransients in 4 HF myocytes, consistent with a dose-dependent specific effect.

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Effects of β2-AR stimulation on cell shortening, Ca transients and SR Ca load [3]
Zinterol + CGP had no effects on cell shortening in control myocytes (at 1 or 3 Hz; Fig 3C). However, this β2-AR stimulation significantly increased cell shortening in HF myocytes (42% and 41% increase at 1 and 3 Hz, respectively, n=7, p<0.05; Fig 4C). In control rabbit myocytes, β2-AR stimulation (zinterol + CGP) did not increase Ca transient amplitude or SR Ca load (assessed by caffeine contractures) at either 1 or 3 Hz stimulation frequency (Fig 3A-B). However, in HF myocytes β2-AR stimulation significantly increased Ca transient amplitude and SR Ca load at 1 Hz (1.28 ± 0.05 vs 0.99 ± 0.06 and 1.82 ± 0.06 vs 1.42 ± 0.06 Δ F/Fo, n=12, p<0.05, Figs. 4A-B). Similar effects were seen at 3 Hz (Fig 4A-B). All these effects were blocked by the β2-AR blocker ICI-118,551 (Fig 4B). Thus, β2-AR stimulation enhances SR Ca content, Ca transients and contractions in HF, but not control myocytes, and this enhancement of SR Ca load and spontaneous SR Ca release may be arrhythmogenic in HF by enhancing aftercontractions and afterdepolarizations.5

To assess the effects of β2-AR stimulation on SERCA and NCX function, we measured the rate of [Ca]i decline during twitch and caffeine-induced Ca transients, respectively (Fig 5). Zinterol + CGP had no effect on tau of twitch [Ca]i decline in control myocytes, but significantly accelerated twitch [Ca]i decline in HF myocytes (156 ± 11 vs 199 ± 12 msec at 1 Hz, 104 ± 8 vs 125 ± 8 msec at 3 Hz, n=12,12, p<0.05). Zinterol + CGP had no effect on the tau of Ca decline of the caffeine transient either HF or control myocytes, suggesting unaltered NCX (Fig 5).

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β2-AR effects on Ca current in HF [3]
Figure 6A shows that in control myocytes, Zinterol (1 μM) increased ICa (e.g. by ∼40% for Em between −10 and +20 mV). This is much smaller than the 300% increase in ICa that we previously observed with 1 μM isoproterenol (combined β1- and β2-AR activation)5 in this same HF myocyte preparation. However, when we repeated the ICa measurements in control myocytes upon zinterol challenge in the presence of the β1-AR antagonist CGP, zinterol did not change ICa. This implies that the modest ICa stimulation by zinterol alone in control myocytes in Fig 6A is likely due to a minor crosstalk from the extremely potent β1-AR mediated effect on ICa. In HF myocytes, ICa was unaltered by zinterol (Fig. 6B). We conclude that β2-AR does not appreciably alter ICa in either control or HF rabbit myocytes.

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β2-AR effects on phospholamban phosphorylation [3]
We assessed the effects of β2-AR stimulation on PLB phosphorylation at Ser-16 (protein kinase A site) and Thr-17 (CaMKII site). At baseline, HF myocytes showed decreased Ser-16 and increased Thr-17 phosphorylation, as we have previously demonstrated.15 Control and HF rabbit myocytes were exposed to 1 μM Zinterol + 300 nM CGP 20712A in the presence or absence of 100 nM ICI118,551. HF (but not control) myocytes exhibited a 78% increase in PLB phosphorylation at the Ser-16 (PKA) site (p<0.05) but no significant change at the Thr-17 (CaMKII) site which was already elevated in HF (n= 5,4, Fig. 7A). The slight tendency for zinterol-induced increase in Thr-17 phosphorylation in HF (not significant) could be secondary to the zinterol-induced enhancement of Ca transients, which was seen only in HF myocytes.

n HF human myocytes, Zinterol (1 μM) + CGP (300 nM; 1 Hz, 37°C) significantly increased cell shortening (10.7 ± 3.0 vs 4.2 ± 0.9, n=6, p<0.05), and induced aftercontractions in 6 of 6 cells (e.g. see Fig 8A middle panel). In human HF myocytes loaded with fluo-3 (37°C; Fig 8), zinterol + CGP significantly increased both Ca transient amplitude (3.34 ± 0.72 vs 1.48 ± 0.18 ΔF/Fo, n=6, p<0.05, Fig 8B) and SR Ca load (assessed by caffeine Ca transient, 4.82 ± 0.60 vs 3.94 ± 0.44 ΔF/Fo, n=5, p<0.05, Fig 8B). Moreover, zinterol + CGP enhanced the rate of twitch [Ca]i decline (τ of 306 ± 26 vs 436 ± 85 msec, n=6, p<0.05, Fig 8C) consistent with enhanced SERCA activity, while the rate of [Ca]i decline of the caffeine contracture was unchanged (indicating unaltered NCX function after β2-AR). All of these effects of zinterol + CGP were reversed by the β2-AR antagonist ICI 118,551.

In rabbits with heart failure (HF), zictelol (2.5 μg/kg IV bolus over 5 seconds) induces ventricular arrhythmias, such as ventricular tachycardia (VT) and premature ventricular contractions (PVCs). In rabbits with heart failure, lower dosages of Zintrol (1 μg/kg iv, n=4) did not cause ventricular arrhythmias [3].

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1. The effects of (-)-isoprenaline and the new beta-adrenoceptor agonist, Zinterol/MJ-9184-1, on the lungs, on the cardiovascular system, and on slow contracting skeletal muscle have been compared in cats under chloralose anaesthesia.2. Both amines reduced the increases in airways resistance produced by 5-HT, depressed incomplete tetanic contractions of the soleus muscle, lowered the blood pressure and produced an increase in heart rate. In comparison with (-)-isoprenaline, MJ-9184-1 had a long duration of action.3. The effects of MJ-9184-1 and (-)-isoprenaline were antagonized by the beta-adrenoceptor antagonist, propranolol.4. MJ-9184-1 was approximately half as potent as (-)-isoprenaline in its effects on pulmonary resistance and soleus muscle contractility, and one seventh as potent in producing chronotropic effects in the heart.5. These results suggest that MJ-9184-1 possesses some specificity as a beta(2) receptor stimulant.[1]

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In vivo arrhythmogenic effects of β2-AR agonist Zinterol [3]
To determine whether stimulation of β2-ARs may contribute to the arrhythmogenic effects of isoproterenol, we performed in vivo drug infusion studies in 5 control and 6 HF rabbits. Zinterol (2.5 μg/kg i.v. bolus over 5 s) did not significantly alter heart rate or mean arterial blood pressure in either control or HF rabbits. Figure 1 shows that zinterol at this dose led to ventricular arrhythmias including premature ventricular complexes (PVCs) and runs of VT (up to 13 beats long) in 4 of 6 HF rabbits (vs 0 of 5 controls, p<0.01), an effect that was blocked by the β2-AR antagonist ICI 118,551 (ICI, 0.2 mg/kg). Zinterol at a lower dose (1μg/kg i.v, n=4) did not induce ventricular arrhythmias in HF rabbits. Intravenous zinterol (2.5 microg/kg) led to ventricular arrhythmias, including ventricular tachycardia up to 13 beats long in 4 of 6 HF rabbits (versus 0 of 5 controls, P<0.01), an effect blocked by beta(2)-AR antagonist ICI-118,551 (0.2 mg/kg).

β-Adrenergic receptor assays and competitive binding studies [3]
Saturation binding studies were carried out on LV homogenates by incubation with various concentrations (0−500 pM) of the β-AR antagonist [125I]-(−)Iodocyanopindolol as previously described.13 The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) were determined by Scatchard analysis using GraphPad Prism. For competitive binding studies, homogenates were incubated with 500 pM 125I-CYP plus increasing dilutions of 1 μM ICI-118,551, a selective β2-AR antagonist, or 100 nM CGP 20712A, a selective β1-AR antagonist. Results were adjusted to fmol/mg protein to calculate for the specific binding, and determine the percentage of β1 and β2 receptors.

Contraction, [Ca]i and patch clamp [3]
Ventricular myocytes were stored at 22°C and plated on laminin-pretreated glass-bottomed chambers. Cells were loaded with flou-3-acetoxymethyl ester and [Ca]i was measured as previously described.11 SR Ca-load was determined by rapid caffeine (10 mM) application after 20 stimulated pulses.5 Myocyte shortening was measured by video edge detection. The normal Tyrodes (NT) solution contained (in mmol/L): 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 5 HEPES, pH 7.4. Myocytes were field-stimulated (0.5−4 Hz, 20 beats, 37°C) followed by 10 seconds of observation for the presence of aftercontractions in the absence or presence of the β2-AR agonist Zinterol (300 nM or 1 μM) ± the β1-AR antagonist CGP 20712A (300 nM, Sigma) or ICI 118,551 (100 nM). In other studies, ruptured patch voltage clamp was done to measure L-type ICa ± similar doses of Zinterol and CGP with pipettes containing (mmol/L) CsOH 110, CsCl 20, EGTA 5, MgCl2 2, Aspartic acid 100, HEPES 5, Mg-ATP 2, Na3GTP 0.1, pH 7.2, 25°C. Membrane capacitance was measured from responses to 5-mV hyperpolarizing and depolarizing pulses.

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Phosphorylation of phospholamban [3]
Freshly-isolated control and HF rabbit myocytes were incubated for 10 min with Zinterol (1 μM), zinterol + CGP 20712A (300 nM) or zinterol + CGP + ICI 118,551 (100 nM). Cell pellets were then spun down and homogenized for protein quantification and Western blotting.

Animal/Disease Models: New Zealand white heart failure rabbits, regardless of gender [3]
Doses: 1.0 or 2.5 μg/kg
Route of Administration: intravenous (iv) (iv)bolus administration; over 5 seconds
Experimental Results: 2.5 μg/kg does not Dramatically change control rabbits or heart rate Heart rate or mean arterial blood pressure in failing rabbits. 2.5 μg/kg caused ventricular arrhythmias, including premature ventricular contractions (PVCs) and VT runs (up to 13 beats) in 4 of 6 HF rabbits (vs. 0 of 5 controls, p<0.01) . 1μg/kg does not cause ventricular arrhythmia in rabbits with heart failure.

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Rabbit heart failure model and myocyte isolation [3]
In New Zealand White rabbits of either sex, HF was induced by aortic insufficiency and 2−4 weeks later by abdominal aortic constriction (both during isoflurane anesthesia) as previously described.3 Progression of HF was assessed by two-dimensional echocardiography. HF rabbits were studied when LV end-systolic dimension exceeded 1.40 cm.3,5,10 At that stage, Intravenous bolus administration of Zinterol (1.0 or 2.5 μg/kg over 5 sec) ± the β2-AR blocker ICI-118,551 (0.2 mg/kg) was performed in conscious control and HF rabbits with monitoring of the surface ECG for at least 3 min. Protocols were approved by the University of Illinois at Chicago Animal Studies Committee. Rabbit left ventricular myocytes were isolated as described,5,10 with back flow across the incompetent aortic valve in HF rabbits blocked by a balloon-tipped catheter inflated in the LV outflow tract.

[1]. Pharmacological actions of a new -adrenoceptor agonist, MJ-9184-1, in anaesthetized cats. Br J Pharmacol. 1972 Nov;46(3):375-85.

[2]. Beta-2 adrenergic activation of L-type Ca2+ current in cardiac myocytes. J Pharmacol Exp Ther. 1997 Nov;283(2):452-61.

[3]. Arrhythmogenic effects of beta2-adrenergic stimulation in the failing heart are attributable to enhanced sarcoplasmic reticulum Ca load. Circ Res. 2008 Jun 6;102(11):1389-97.

Ventricular tachycardia in heart failure (HF) can initiate by nonreentrant mechanisms such as delayed afterdepolarizations. In an arrhythmogenic rabbit model of HF, we have shown that isoproterenol induces ventricular tachycardia in vivo and aftercontractions and transient inward currents in HF myocytes. To determine whether beta(2)-adrenergic receptor (beta(2)-AR) stimulation contributes, we performed in vivo drug infusion, in vitro myocyte and biochemical studies. Intravenous Zinterol (2.5 microg/kg) led to ventricular arrhythmias, including ventricular tachycardia up to 13 beats long in 4 of 6 HF rabbits (versus 0 of 5 controls, P<0.01), an effect blocked by beta(2)-AR antagonist ICI-118,551 (0.2 mg/kg). In field-stimulated myocytes (0.5 to 4 Hz, 37 degrees C), beta(2)-AR stimulation (1 micromol/L zinterol+300 nmol/L beta(1)-AR antagonist CGP-29712A) induced aftercontractions and Ca aftertransients in 88% of HF versus 0% of control myocytes (P<0.01). beta(2)-AR stimulation in HF (but not control) myocytes increased Ca transient amplitude (by 29%), sarcoplasmic reticulum (SR) Ca load (by 28%), the rate of [Ca](i) decline (by 28%; n=12, all P<0.05), and phospholamban phosphorylation at Ser16, but Ca current was unchanged. All of these effects in HF myocytes were blocked by ICI-118,551 (100 nmol/L). Although total beta-AR expression was reduced by 47% in HF rabbit left ventricle, beta(2)-AR number was unchanged, indicating more potent beta(2)-AR-dependent SR Ca uptake and arrhythmogenesis in HF. Human HF myocytes showed similar beta(2)-AR-induced aftercontractions, aftertransients, and enhanced Ca transient amplitude, SR Ca load and twitch [Ca](i) decline rate. Thus, beta(2)-AR stimulation is arrhythmogenic in HF, mediated by SR Ca overload-induced spontaneous SR Ca release and aftercontractions.[3]

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Zinterol has been used for many β2-AR studies, but at high doses (e.g. 10−5M) Zinterol may also stimulate β1-AR. We therefore performed most of these studies with zinterol in the presence of the β1-AR antagonist CGP 20712A (this was more difficult for in vivo studies where we found β1-AR blockade was not always well tolerated hemodynamically in rabbits with HF). In some in vitro studies, we also used zinterol alone and found results comparable to zinterol + CGP. This suggests that at the doses used, zinterol's effects were predominantly due to stimulation of β2-ARs, (with the exception that zinterol could slightly increase ICa via β1-AR). Overall, the ability to reverse zinterol's effects by the specific β2-AR blocker ICI-118,551 supports our conclusions about β2-AR-induced effects. Wang et al14 reported that myocytes on laminin-coated cover slips exhibited greater β2-AR responsiveness to zinterol. Our findings of enhanced arrhythmogenicity with β2-AR stimulation in vivo as well as in vitro (whether on laminin-coated cover slips or on glass cover slips without laminin) suggest that the laminin was not a confounding issue. Moreover, laminin may help simulate the extracellular protein environment in intact tissue that involves interactions with laminin and integrins.

C19H26N2O4S

378.48574

378.161

37000-20-7

Zinterol hydrochloride;38241-28-0

37990

White to off-white solid powder

1.282g/cm3

574.3ºC at 760mmHg

301.1ºC

1.615

3.952

4

6

8

26

526

0

CC(C)(CC1=CC=CC=C1)NCC(C2=CC(=C(C=C2)O)NS(=O)(=O)C)O

XJBCFFLVLOPYBV-UHFFFAOYSA-N

InChI=1S/C19H26N2O4S/c1-19(2,12-14-7-5-4-6-8-14)20-13-18(23)15-9-10-17(22)16(11-15)21-26(3,24)25/h4-11,18,20-23H,12-13H2,1-3H3

N-[2-hydroxy-5-[1-hydroxy-2-[(2-methyl-1-phenylpropan-2-yl)amino]ethyl]phenyl]methanesulfonamide

ZINTEROL; 37000-20-7; Zinterol [INN]; Zinterolum; Zinterolum [INN-Latin];

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)

DMSO : ~38 mg/mL (~100.40 mM)
H2O : ~1.89 mg/mL (~4.99 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.)