A large number of β-adrenergic receptor (β-AR) agonists and antagonists are widely used in the treatment of cardiovascular diseases and other diseases. Nonetheless, it remains unclear whether these commonly used β-AR drugs can activate downstream β- arrestin-biased signaling pathways. The objective of this study was to investigate β-arrestin2 recruitment effects of β-AR agonists and antagonists that were commonly used in clinical practice. We used TANGO (transcriptional activation following arrestin translocation) assay to detect the β-arrestin2 recruitment by β-AR ligands in HEK293 cell line (HTLA cells) stably transfected with tetracycline transactivator protein (tTA) dependent luciferase reporter and β-arrestin2-TEV fusion gene. Upon activation of β-AR by a β-AR ligand, β-arrestin2 was recruited to the C terminus of the receptor, followed by cleavage of the G protein-coupled receptors (GPCRs) fusion protein at the TEV protease-cleavage site. The cleavage resulted in the release of tTA, which, after being transported to the nucleus, activated transcription of the luciferase reporter gene. The results showed that β-AR non-selective agonists epinephrine, noradrenaline and isoprenaline all promoted β-arrestin2 recruitment at β1-AR and β2-AR. β1-AR selective agonists dobutamine and denopamine both promoted β-arrestin2 recruitment at β1-AR. β2-AR selective agonists procaterol and salbutamol promoted β-arrestin2 recruitment at β2-AR. β-AR non-selective antagonists alprenolol and pindolol promoted β-arrestin2 recruitment at β1-AR. β1-AR selective antagonists celiprolol and bevantolol showed β-arrestin2 recruitment at β1-AR. β2-AR selective antagonists butoxamine showed β-arrestin2 recruitment at β1-AR. These results provide some clues for the potential action of β-AR drugs, and lay a foundation for the screening of β-arrestin-biased β-AR ligands.
Humans ; beta-Arrestin 2/metabolism* ; HEK293 Cells ; Adrenergic beta-Agonists/pharmacology* ; Isoproterenol/pharmacology* ; Receptors, Adrenergic, beta-2/metabolism* ; Norepinephrine/pharmacology*

The cerebral vasospasm which often accompanies a subarachnoid hemorrhage from a ruptured intracranial aneurysm is one the chief reasons for morbidity and motarlity. Although the phenomenon still needs clarification, experimental evidence has indicated that alpha-blocking agents can modify this blood-induced spasm. A disappointing experience with these agents led to a clinical trial of the beta-adrenergic drug isoproterenol. In 1975 Sundt reported a good final result with the use of isoproterenol and lidocaine hydrochloride in the treatment of cerebral ischemia attributed to progressive vasospasm after a subarachnoid hemorrhage in human beings. We have reported our experience with the use of isoproterenol and lidocaine hydrochloride in 5 such cases. 3 were treated preoperatively and 2 postoperatively. Experience suggests that the drug regimen reported is useful when institute early after the onset of symptoms and is safe with proper monitoring techniques. The symptomatology of cerebral vasospasm, the reationable for this form of therapy, and the pharmacology of the drugs were discussed.
Brain Ischemia ; Humans ; Intracranial Aneurysm* ; Isoproterenol* ; Lidocaine* ; Pharmacology ; Spasm ; Subarachnoid Hemorrhage ; Vasospasm, Intracranial*

The present study investigated the effects of chikusetsu saponin Ⅳa(CHS Ⅳa) on isoproterenol(ISO)-induced myocardial hypertrophy in rats and explored the underlying molecular mechanism. ISO was applied to establish a rat model of myocardial hypertrophy, and CHS Ⅳa(5 and 15 mg·kg~(-1)·d~(-1)) was used for intervention. The tail artery blood pressure was measured. Cardiac ultrasound examination was performed. The ratio of heart weight to body weight(HW/BW) was calculated. Morphological changes in the myocardial tissue were observed by HE staining. Collagen deposition in the myocardial tissue was observed by Masson staining. The mRNA expression of myocardial hypertrophy indicators(ANP and BNP), autophagy-related genes(Atg5, P62 and beclin1), and miR199 a-5 p was detected by qRT-PCR. Atg5 protein expression was detected by Western blot. The results showed that the model group exhibited increased tail artery blood pressure and HW/BW ratio, thickened left ventricular myocardium, enlarged myocardial cells, disordered myocardial fibers with widened interstitium, and a large amount of collagen aggregating around the extracellular matrix and blood vessels. ANP and BNP were largely expressed. Moreover, P62 expression was up-regulated, while beclin1 expression was down-regulated. After intervention by CHS Ⅳa at different doses, myocardial hypertrophy was ameliorated and autophagy activity in the myocardial tissue was enhanced. Meanwhile, miR199 a-5 p expression declined and Atg5 expression increased. As predicted by bioinformatics, Atg5 was a target gene of miR199 a-5 p. CHS Ⅳa was capable of preventing myocardial hypertrophy by regulating autophagy of myocardial cells through the miR-199 a-5 p/Atg5 signaling pathway.
Animals ; Cardiomegaly/genetics* ; Isoproterenol ; Myocardium ; Myocytes, Cardiac ; Oleanolic Acid/analogs & derivatives* ; Rats ; Saponins/pharmacology*

OBJECTIVETo compare the bronchofilating and antiallergic effects with piclamilast with ciclamilast, the second-generation phosphodiesterase 4 (PDE4) selective inhibitors.

METHODSEffects of piclamilast and ciclamilast on airway smooth muscle (ASM) at resting tension, carbachol-induced contraction and the synergistic effect of two agents on isoproterenol-induced bronchorelaxation were evaluated in the isolated tracheal strips of guinea pig in a cumulative manner in vitro. Slow reaction substance of anaphylaxis (SRS-A) release from lung tissues of the sensitized guinea pigs after antigen challenge was examined by bioassay. Antiallergic effect of piclamilast, ciclamilast and rolipram on the isolated ASM of sensitized guinea pigs were evaluated with Schultz-Dale reaction.

RESULTSPiclamilast and ciclamilast showed bronchorelaxant effect in ASM at resting tension. EC50 values of piclamilast and ciclamilast were 1.00 x 10(-5) mol/L and 0.84 x 10(-5) mol/L. Piclamilast and ciclamilast could both enhance the bronchodilating effect of isoproterenol in the isolated ASM of guinea pig, reduce the amount of SRS-A released from lung tissues of the sensitized guinea pigs and also inhibit ovalbumin (OA)-induced bronchoconstruction (Schultz-Dale reaction).

CONCLUSIONThe results indicate the bronchodilating effect of ciclamilast is as potent as piclamilast, but the antiallergic effect of ciclamilast is significantly more potent than that of piclamilast.

Animals ; Anti-Allergic Agents ; pharmacology ; Benzamides ; pharmacology ; Bronchodilator Agents ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Guinea Pigs ; In Vitro Techniques ; Isoproterenol ; pharmacology ; Male ; Phosphodiesterase Inhibitors ; pharmacology ; Pyridines ; pharmacology

The present study aimed to observe the changes of contractile function and responsiveness to isoproterenol (ISO) in tail-suspended rat cardiomyocytes under simulated weightlessness condition. Tail-suspended rat model was used to simulate weightlessness on the ground. Twenty-four male Sprague-Dawley rats were randomly divided into the control and tail-suspended groups. After 4 weeks of suspension, the rats were injected with heparin (100 IU/100 g body weight, i.p.) and anesthetized with pentobarbital sodium (40 mg/kg body weight). The hearts were removed and the aortas were cannulated rapidly. The cannulated hearts were mounted on a Langendorff perfusion apparatus and perfused with constant flow. The perfusion pressure was monitored. The hearts were digested by 0.08% collagenase I at 37 degrees C. The ventricular tissues were chopped and the single myocytes were dispersed gently by a wide-tipped pipette. The contractile function was measured in the Edge Detector system within 6 h after isolation. The length and width of cardiomyocytes were measured without electric stimulation. Contractile curves of the single cardiomyocytes were recorded at stimulation frequency of 1.0, 2.0 and 4.0 Hz. To observe the responsiveness of cardiomyocytes to ISO, 1, 5 and 10 nmol/L ISO in Kreb's solution was perfused at a stimulation frequency of 2.0 Hz. The length and width of the left and right ventricular cardiomyocytes in tail-suspended group had little difference from that in the control group. The unloaded shortening amplitude increased as stimulation frequency elevated in both the control and tail-suspended groups. It was increased by (8.50±1.26)%, (9.00±1.38)%, (9.23±1.83)% in the left ventricular cardiomyocytes, and (9.80±2.48)%, (10.03±2.48)%, (10.28±2.27)% in the right ventricular cardiomyocytes in the control group at stimulation frequency of 1.0, 2.0 and 4.0 Hz. Compared with that in the control group, the unloaded shortening amplitude decreased by 12.2% and 10.9% in the left ventricular cardiomycytes (P<0.05), and 16.5% and 16.3% in the right ventricular cardiomyocytes (P<0.05) at stimulation frequency of 1.0 and 2.0 Hz in tail-suspended group. There was no significant difference in unloaded shortening amplitude at stimulation frequency of 4.0 Hz between the control and tail-suspended groups. Time to peak shortening (TPS) in tail-suspended group significantly reduced in both the left and right ventricular cardiomyocytes (P<0.05). Time from peak to 75% relaxation (TR(75)) in tail-suspended group significantly prolonged in both the left and right ventricular cardiomyocytes (P<0.05). No significant differences in shortening and relaxation rate (±dL/dt(max)) were observed between the control and tail-suspended groups. The unloaded shortening amplitude increased by (10.63±0.83)%, (35.06±5.22)% and (71.64±6.83)% in the control cardiomyocytes, but increased by (5.75±0.76)%, (23.97±4.50)% and (26.38±8.13)% in tail-suspended group during perfusion with 1, 5 and 10 nmol/L ISO (P<0.05, P<0.01). The unloaded shortening amplitude increased by (3.04±0.27)%, (9.81±2.66)% and (20.20±3.47)% in the control cardiomyocytes, but increased by (1.42±0.53)%, (3.83±1.71)% and (5.49±4.08)% in tail-suspended group during perfusion with 10, 50 and 100 nmol/L forskolin (P<0.05). The results obtained suggest that the unloaded shortening amplitude and responsiveness to ISO decrease in rat cardiomyocytes after 4-week tail-suspension.
Animals ; Electric Stimulation ; Hindlimb Suspension ; Isoproterenol ; pharmacology ; Male ; Myocytes, Cardiac ; drug effects ; Rats ; Rats, Sprague-Dawley ; Weightlessness Simulation

AIMTo observe the effect of LPC on the pacemaker current I(f) in ischemic myocardium and if the effect could be reversed by ISO.

METHODSBy using two microelectrode voltage clamp technique to measure and compare the amplitude of I(f) of ischemic myocardium in the presence of LPC and LPC add ISO.

RESULTSIschemia decreased the amplitude of I(f) at all membrane potential levels. Adding LPC 2 x 10(-5) mol/L to the ischemia-like solution, the amplitude of I(f) decreased further (n = 5, P < 0.05), it means that LPC aggravated the inhibitory effect of "ischemia" on the pacemaker activity. Adding LPC 2 x 10(-5) mol/L and ISO 1 x 10(-6) mol/L together to the ischemia-like solution, the amplitude of I(f) increased significantly at membrane potential -90 mV to - 120 mV (n = 8, P < 0.05) compared with ischemia condition, but still did not reach the levels before ischemia.

CONCLUSIONIn acute myocardial ischemia condition, toxic metabolite LPC accentuated its inhibitory effect on pacemaker current I(f), a local release and accumulation of catecholamine could not completely reverse their inhibitory effect.

Animals ; Isoproterenol ; metabolism ; Lysophosphatidylcholines ; pharmacology ; Membrane Potentials ; drug effects ; Microelectrodes ; Myocardial Ischemia ; metabolism ; physiopathology ; Myocardium ; Patch-Clamp Techniques ; Sheep

To study the effect and mechanism of Dendrobium candidum on isoproterenol-induced myocardial hypertrophy in rats, 60 healthy SD rats(30 males and 30 females) were randomly divided into 5 groups(12 in each group): normal group, model group, three D. candidum preventive administration groups(0.09, 0.18, 1.1 g·kg⁻¹). Except for the normal group, rats of other groups were injected back subcutaneously with ISO(5 mg·kg⁻¹) for 10 consecutive days. At the same time, preventive administration groups began to give different doses of the sample for 30 days and model group began to give normal saline. Left ventricular systolic pressure(LVSP) was measured in each group by common carotid artery cannulation, and the left ventricle(LW)/tibia length, heart weight index(HWI) and myocardial hydroxyproline(Hydro) content were calculated. Myocardial tissue HE staining and Masson staining were used to observe the myocardial structure and the degree of myocardial fibrosis respectively. Atrial natriuretic peptide(ANP), brain natriuretic peptide(BNP), and cardiac troponin I(cTN-I) concentration were measured by enzyme-linked immunosorbent assay(ELISA). The results showed that as compared with the normal group, the levels of ANP, BNP and cTN-I in plasma were significantly increased in ISO-induced hypertrophic rats; as compared with the model group, D. candidumcan inhibit ISO-induced ventricular pressure and ventricular hypertrophy, reduce myocardial collagen synthesis, improve myocardial fibrosis and ventricular remodeling, and significantly down-regulate ANP, BNP and cTN-I levels in plasma. This study shows that D. candidum has a protective effect on isoproterenol-induced cardiac hypertrophy.
Animals ; Cardiomegaly ; drug therapy ; Dendrobium ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Female ; Isoproterenol ; Male ; Myocardium ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo screen out the main components with no significant difference with Salvia miltiorrhiza diterpene quinones pharmacological action, in order to determine the compatible form of representative components that can describe the overall property of S. miltiorrhiza diterpene quinones.

METHODAccording to the results of the in vitro pharmacological experiment, the myocardial ischemia model of rats was induced through intraperitoneal injection of isoproterenol. The pharmacologic effects of S. miltiorrhiza diterpene quinones, combination with principal component A and combination with principal component B were compared in electrocardiogram (changes in J point), enzymology indicators (SOD, MDA, CK, LDH) and pathology (myocardial histological changes), so as to screen out the compatible form of representative components that can describe the overall property of S. miltiorrhiza diterpene quinones.

RESULTThe S. miltiorrhiza diterpenoid quinone high-dose group and the B high-dose group were similar in all pharmacological effects, with equal efficacy but no significant difference.

CONCLUSIONThe S. miltiorrhiza diterpenoid quinone high-dose group and the B high-dose group showed a certain therapeutic effect on ISO-induced myocardial ischemia. Therefore, the four components in the B high-dose group can be used as representative components of S. miltiorrhiza diterpene quinones.

Animals ; Diterpenes ; pharmacology ; Drug Evaluation, Preclinical ; Isoproterenol ; pharmacology ; Male ; Myocardial Ischemia ; drug therapy ; Quinones ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Salvia miltiorrhiza ; chemistry

OBJECTIVETo investigate the effects of a novel tripeptide analog of arginine on isoproterenol (ISO), a beta-adrenergic receptor agonist, induced the change in concentration transient cytosolic free calcium in cultured neonatal rat cardiac myocytes (CMs).

METHODSThe expression of inducible nitric oxide synthase (iNOS) was induced in cultured neonatal rat CMs by stimulation with lipopolysaccharide (LPS) and interleukin-6 (IL-6) for 24 h, and quantitatively measured using Western blotting. The CMs were incubated in the presence of the new arginine analog for 6 h and the changes of fluorescence signal of free calcium in response to isoproterenol (ISO) treatment were measured under laser scanning confocal microscope.

RESULTSIncubation of the CMs for 24 h in the presence of IL-6 and LPS resulted in significantly increased nitric oxide (NO) production, and also in increased iNOS protein accumulation in the cells. Specific inhibition of iNOS by the new arginine analog substantially inhibited NO production and increased the peak value of ISO-induced Ca2+ transient.

CONCLUSIONThe new arginine analog strongly inhibits IL-6 and LPS-induced NO production and increases beta-adrenergic responsiveness in cultured neonatal rat CMs.

Animals ; Animals, Newborn ; Arginine ; analogs & derivatives ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Isoproterenol ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Oligopeptides ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe the effect of Kanli Granule (KG) on myocardial mechanics in pressure overload induced diastolic heart failure (DHF) rats.

METHODSTotally 60 male Wistar rats were divided into the sham-operation group, the model group, the KG group, and the Valsartan group according to random digit table, 15 in each group. The pressure overload induced DHF model was established in all groups except the sham-operation group using abdominal aortic constriction surgery. Totally 7 rats died after modeling (with the mortality of 10. 67%) , and the rest 53 finished the following test. Rats in the KG group were administered with KG extract (calculated as 6. 75 g crude drug/kg) by gastrogavage. Rats in the Valsartan group were administered with Valsartan (7.2 µg/g) by gastrogavage. Equal volume of double distilled water was administered to rats in the model group and the sham-operation group by gastrogavage. All rats were intervened for 32 weeks. The response of isolated heart papillary muscle tonus to isoprenaline (ISO) and adenylate cyclase (Forskolin) was respectively observed. The enhancement phenomenon after resting development force (DF) of isolated heart papillary muscle tonus, and changes of DF in different Ca²⁺ concentrations were observed.

RESULTS(1) In the ISO response test: Compared with the sham-operation group, the amplifications of DF, ±df/dt, -df/dt were obviously elevated in the model group (P < 0.05). Compared with the model group, the amplifications of DF and ±df/dt were obviously lowered in the KG group (P < 0.01), and the amplification of ±df/dt was also reduced in the Valsartan group (P < 0.01). (2) In the Forskolin response test: Compared with the sham-operation group, the amplifications of DF and ±df/dt obviously increased in the model group (P < 0.05). Compared with the model group, the amplifications of DF and ±df/dt were obviously reduced in the KG group (P < 0.01), and the amplification of DF was also reduced in the Valsartan group (P < 0.05). (3) In post-resting DF enhancement test: Compared with the sham-operation group, the amplification of DF showed gradually decreasing tendency along with prolonged resting time in the model group, and they were obviously lowered at all time points (P < 0.05). Compared with the model group, the amplification of DF was gradually increasing along with prolonged resting time in the KG group. The amplification of DF at post-resting 240 s was obviously larger in the KG group than in the model group (P < 0.05). The amplification of post-resting DF still showed gradually decreasing tendency along with prolonged resting time in the Valsartan group, with increased amplifications of DF at post-resting 60 s and 120 s (P < 0. 05) (4) The amplifications of DF in different Ca²⁺ concentrations: Compared with the sham-operation group, the amplifications of DF were significantly elevated in different Ca²⁺ concentrations (1.75, 3.5, 7.0 mmol/L ) (P < 0.05, P < 0.01). Compared with the model group, there was no statistical difference in amplification of DF in different Ca²⁺ concentrations in the KG group (P > 0.05). The amplifications of DF in different Ca²⁺ concentrations were significantly reduced in the Valsartan group (P < 0.05).

CONCLUSIONSThe ISO response and the Forskolin response were enhanced in isolated heart papillary muscle tonus of pressure overload induced DHF rats; enhanced post-resting DF was reduced; DF in different supra-physiologic levels of Ca²⁺ was still enhanced. KG could significantly improve excessive enhancement of pressure overload induced DHF rats in ISO response and Forskolin response, and improve enhancement of post-resting myocardium.

Animals ; Colforsin ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Heart ; drug effects ; physiopathology ; Heart Failure, Diastolic ; drug therapy ; Isoproterenol ; pharmacology ; Male ; Random Allocation ; Rats ; Rats, Wistar