Adenosine A1 and A2A receptor effects on G-protein cycling in beta-adrenergic stimulated ventricular membranes

In the heart beta1-adrenergic (beta1R) and adenosine A1 (A1R) and A2A (A2AR) receptors modulate contractile and metabolic function. The interaction between these receptors was investigated at the level of G-protein cycling by determining the effect of receptor agonists on the binding of GTP to G-proteins and displacement of G alpha-subunit-bound GDP by GTP. Crude membranes from rat heart or brain were stimulated by agonists for beta1R (isoproterenol; ISO), A1R (chlorocyclopentyladenosine, CCPA) and A2AR (CGS-21680; CGS). GTP binding to membranes was increased by ISO (17%), CCPA (6%) and CGS (12%). Binding values observed with incubation using ISO and CCPA together were significantly less than values obtained by the incubation of individual agents alone. With ISO, GTP binding to G alpha(s) subunits as determined by immunoprecipitation was increased 79% in heart and 87% in brain. These increases were attenuated by CCPA, an effect that was inhibited by CGS. GDP release by membranes was increased 6.9% and 4.6% by ISO and CCPA, respectively. After co-incubation of these agonists, release was increased less than determined by the addition of the individual agent responses. CGS inhibited the reduced release caused by of CCPA. Adenylyl cyclase activity stimulated by ISO was attenuated 33% by CCPA, an effect inhibited by CGS. Together, these results indicate that A1R exert an antiadrenergic action at the level of beta1R stimulated G(s)-protein cycling and that A2AR reduce this action.     

Effect of adenosine-5'-O-(beta, gamma-dichloromethane)triphosphate on ATP-activated currents in sensory neurons in rats

The action of beta,gamma-dihalogenmethane derivatives of ATP on ATP receptors has been studied in the sensory neurons of rats. beta,gamma-Dichloromethane-ATP and beta,gamma-dibromomethane-ATP are competitive blockers and beta,gamma-difluoromethane-ATP is a partial agonist of ATP receptors. At present, beta,gamma-dichloromethane-ATP is the most efficient of the known competitive blockers of these receptors (Ki = 2.10(-5) M). The data obtained confirm that beta- and gamma-phosphates of ATP play an important role in the activation of ATP receptors.     

Beta-adrenoceptor mediated inhibition of behavioral action of desipramine and of central noradrenergic activity in forced swimming rats

The immobility-reducing action of desipramine (DMI) in forced swimming rats was attenuated by intracerebroventricular (i.c.v.) injection of isoproterenol (ISO) and potentiated by i.c.v. atenolol (ATE), a beta 1-adrenoceptor antagonist. The effect of ISO was blocked by ATE. When administered i.c.v. in normal rats, ISO reduced the contents of 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (MHPG-SO4), a major metabolite of noradrenaline, in the septal area, thalamus and hypothalamus while ATE had no effect in most of the brain regions. However, in forced swimming rats treated with DMI, ISO reduced MHPG-SO4 in 6 out of 8 brain regions tested and conversely, ATE increased the levels in the amygdala, septal area and hypothalamus. Similar to the behavioral effect, the effect of ISO was antagonized by ATE. These results support the hypothesis that central beta 1-adrenergic mechanisms inhibit the immobility-reducing action of DMI by reducing the activity of noradrenergic neurons in the brain.     

Isoproterenol suppresses cytokine-induced RANTES secretion in human lung epithelial cells through the inhibition of c-jun N-terminal kinase pathway

It has been reported that beta2-agonists may potentially exert some anti-inflammatory action in addition to bronchodilation that may contribute to their beneficial effects on asthma control. Bronchial epithelial cells are well known to respond to a range of stimuli by producing various biologically active mediators that can influence airway inflammation. RANTES (regulated on activation, normal T cells expressed and secreted) plays an important role in the pathophysiology of airway inflammation of asthmatics through its chemotactic activity for eosinophils. In this study, the authors investigated whether cytokine-induced RANTES release from BEAS-2B human bronchial epithelial cells could be modulated by beta-agonist isoproterenol (ISO). The possible involvement of c-jun N-terminal kinase (JNK) pathway was also studied. Combination of tumor necrosis factor-alpha and interleukin-1beta (cytokine mix) increased RANTES release from BEAS-2B cells and stimulated JNK activity. Similar to JNK inhibitor SP600125, ISO inhibited not only the production of RANTES but also the activation of JNK pathway in cytokine mix-stimulated BEAS-2B cells. The effect of ISO was mediated by the beta2-adrenoceptor, since it was blocked by ICI 118,551, a selective beta2-receptor antagonist, but not by atenolol, a selective beta1-receptor antagonist. Adenylyl cyclase activator forskolin reproduced the effects of ISO. Isoproterenol was found to inhibit the release of RANTES from the human bronchial epithelial cells, at least in part, through the inhibition of JNK signaling pathway.     

Effects of chronic isoproterenol administration on beta 1-adrenoceptors and growth of pancreas of young and adult rats

[3H]Dihydroalprenolol (DHA) binding of membranes of adult pancreas differed from that of pancreas of young rats, and the DHA binding in the presence of atenolol or butoxamine also was different in the two age groups. The adult pancreas had 93% beta 2- and 7% beta 1-adrenoceptors and did not exhibit an increased incorporation of [3H]thymidine into deoxyribonucleic acid (DNA) following 2 days of DL-isoproterenol (ISO) administration; in contrast, pancreas of the 20-day-old rat had 71% beta 2-adrenoceptors and 27% beta 1-adrenoceptors and exhibited a 34-fold increase over that of adult, and a 6-fold increase over that of the control 20-day-old pancreas. Acinar cell differentiation was also accelerated by a 7-day regimen of ISO administration from 13 to 20 days of age. These growth responses to ISO appear to be beta 1 mediated. The lack of beta 1-adrenoceptors in the adult may account for the failure of the adult pancreas to exhibit a growth response to ISO.     

Beta-adrenergic and antiadrenergic modulation of cardiac adenylyl cyclase is influenced by phosphorylation

Adenosine protects the myocardium of the heart by exerting an antiadrenergic action via the adenosine A1 receptor (A1R). Because beta 1-adrenergic receptor (beta 1R) stimulation elicits myocardial protein phosphorylation, the present study investigated whether protein kinase A (PKA) catalyzed rat heart ventricular membrane phosphorylation affects the beta 1R adrenergic and A1R adenosinergic actions on adenylyl cyclase activity. Membranes were either phosphorylated with PKA in the absence/presence of a protein kinase inhibitor (PKI) or dephosphorylated with alkaline phosphatase (AP) and assayed for adenylyl cyclase activity (AC) in the presence of the beta 1R agonist isoproterenol (ISO) and/or the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA). 32P incorporation into the protein substrates of 140-120, 43, and 29 kDa with PKA increased both the ISO-elicited activation of AC by 51-54% and the A1R-mediated reduction of the ISO-induced increase in AC by 29-50%, thereby yielding a total antiadrenergic effect of approximately 78%. These effects of PKA were prevented by PKI. AP reduced the ISO-induced increase in AC and eliminated the antiadrenergic effect of CCPA. Immunoprecipitation of the solubilized membrane adenylyl cyclase with the use of a polyclonal adenylyl cyclase VI antibody indicated that the enzyme is phosphorylated by PKA. These results indicate that the cardioprotective effect of adenosine afforded by its antiadrenergic action is facilitated by cardiac membrane phosphorylation.     

Action of melanophore-stimulating hormone on melanophores of the cyprinid fish Zacco temmincki

1. Alpha MSH was extremely effective in inducing melanosome dispersion in both innervated and denervated melanophores in isolated scales of Zacco termmincki. 2. The sensitivity of the melanophores to MSH did not change after denervation. 3. The MSH action was blocked by mersalyl, a SH inhibitor, but not by any of alpha and beta adrenergic blockers. 4. Ca2+ was required for the MSH action, but not for melanosome dispersion itself, since the beta adrenergic response was normally produced in the absence of this ion. 5. Mg2+, Sr2+ and Ba2+ could not replace the Ca2+. 6. Mn2+ reversibly inhibited the MSH action.     

Effect of IL-1beta on CRE-dependent gene expression in human airway smooth muscle cells

IL-1beta inhibits isoproterenol (ISO)-induced relaxation of cultured human airway smooth muscle (HASM) cells. The purpose of this study was to determine whether IL-1beta can also suppress ISO-induced cAMP response element (CRE)-dependent gene expression. ISO (10 microM) caused a marked increase in CRE-binding protein (CREB) phosphorylation, which was attenuated by IL-1beta (2 ng/ml). This effect of IL-1beta was abolished by the cyclooxygenase (COX) inhibitor indomethacin. To examine CRE-driven gene expression, we transiently transfected HASM cells with a construct containing CRE upstream of a luciferase reporter gene. ISO (6 h) caused a sixfold increase in luciferase activity. IL-1beta (24 h) alone also increased luciferase activity, although to a lesser extent (2-fold). However, the ability of ISO to elicit luciferase expression was markedly reduced in cells treated with IL-1beta. Indomethacin, the MEK and p38 inhibitors U-0126 and SB-203580, the protein kinase A inhibitor H-89, and dexamethasone each completely abolished the ability of IL-1beta to induce CRE-driven gene expression but only slightly increased the ability of ISO to induce CRE-driven gene expression in IL-1beta-treated cells. IL-1beta also attenuated dibutyryl cAMP-induced CRE-driven gene expression, but not dibutyryl cAMP-induced CREB phosphorylation. Tumor necrosis factor-alpha (10 ng/ml) also attenuated ISO-induced CRE-driven gene expression, even though it was without effect on ISO-induced cAMP formation or ISO-induced CREB phosphorylation. The results suggest that IL-1beta and tumor necrosis factor-alpha may attenuate the ability of beta-agonists to induce expression of genes with CRE in their regulatory regions at least in part through events downstream of CREB phosphorylation.     

Alcohol-free red wine inhibits isoproterenol-induced cardiac remodeling in rats by the regulation of Akt1 and protein kinase C alpha/beta II

There is increasing evidence that moderate consumption of red wine containing high amount of polyphenols and anthocyanins is associated with decreased incidence of cardiovascular morbidity and mortality. Therefore, we hypothesized that cardiac hypertrophy and fibrosis as well as Akt (protein kinase B, PKB) and protein kinase C (PKC) cascades can be beneficially influenced by an alcohol-free red wine (AFRW) extract rich in 14 types of polyphenols and 4 types of anthocyanins during cardiac remodeling. To test this assumption, rats were treated with isoproterenol (ISO) to induce postinfarction remodeling and were given tap water or AFRW ad libitum for 8 weeks. Control rats received vehicle instead of ISO. Heart mass/body mass and ventricle mass/body mass ratios, diameter of cardiomyocytes, phosphorylation of PKC alpha/beta II and protein kinase B/Akt, and deposition of collagen type III were determined from the hearts of all four groups of rats. All measured gravimetric parameters, myocyte diameters and the amount of collagen type III decreased, and the phosphorylation of PKC alpha/beta II was reduced in the ISO+AFRW group compared to the ISO group. AFRW induced activation of Akt, one of the best characterized cytoprotective pathways even without ISO treatment, and this activation was further increased in the ISO+AFRW group. These data suggest that AFRW treatment has a protective effect on hearts undergoing postinfarction remodeling by repressing hypertrophy-associated increased phosphorylation of PKC alpha/beta II and by activating Akt, providing a molecular mechanism for the cardioprotective effect of red wine polyphenols.     

Effect of adrenergic beta-receptor blockers on the oxygen consumption or narcotized rats and the calorigenic action of adrenaline

The adrenergic beta-receptor blockers: Propranolol, Alprenolol, Oxprenolol, Pindolol, Practolol, and Talinolol, inhibit the calorigenic action of adrenaline in experiments on rats. These findings doe not permit any quantitative comparison, because the beta blockers show considerable differences in their inherent action on the basic metabolism. THe cardioselective beta-blockers, Practolol and Talinolol, are without effect, while Propanolol increases, and Alprenolol, Oxprenolol and Pindolol lower the basic metabolism. The intrinsic activity of these compounds increases with the decrease of their hydrophobicity.     

Determination of radical yields in solid-state drugs as one technique to identify drugs that will withstand radiosterilization: radioresistance of beta blockers

This article describes a simple preliminary test to determine whether a drug is sufficiently radioresistant to withstand radiosterilization. The test is based on the electron spin resonance (ESR) detection of radicals produced after irradiation of a solid-state drug, assuming that these radicals are the precursors of the final products detected after dissolution of the drug. A calibration curve has therefore been established by measuring ESR spectra of l-alanine irradiated at different doses. The response factor to quantify the radicals is the normalized double integration (DI) of the whole first-derivative ESR spectrum. The curve gives the relationship between the normalized DI and the number of radicals. Eight beta blockers have been chosen and their radical yield determined. This is the first time that several different drugs of the same pharmacological group have been studied and compared. The results obtained are similar for seven of the eight beta blockers; the mean G value (excepted for nadolol) is 3 x 10(-9) mol/J. This means that beta blockers are radioresistant. The two most radiosensitive drugs (nadolol and esmolol hydrochloride) were also studied by high-performance liquid chromatography (HPLC). No significant loss of the active compound was detected, which confirms this radioresistant property. Moreover, no change in color or smell was observed. Using ESR and HPLC, beta blockers were identified as potential candidates for radiosterilization.     

Nerve growth factor-induced increase in [3H]thymidine incorporation into pancreas of young rats and its partial blockade by propranolol or partial sialoadenectomy

Administration of nerve growth factor (NGF) twice daily for 2 days to rats 11 days at the time of the initial injection resulted in a 6.6-fold increase in [3H]thymidine levels of pancreas, when comparison was made to levels of untreated controls. Isoproterenol (ISO), a beta-adrenergic agonist known to produce marked increase in [3H]thymidine incorporation into DNA of salivary glands, caused increases in levels of [3H]thymidine in pancreas that were similar in magnitude to those induced by NGF. The combined administration of ISO and NGF did not cause any increase above those observed with either agent alone. Administration of propranolol (3 mg/kg body wt) prior to administration of ISO prevented the usual ISO-induced increase in DNA synthesis, but propranolol in either a 3- or 9-mg/kg body wt dose, caused only a 50% inhibition of NGF-induced thymidine incorporation. In the absence of the submandibular-sublingual glands, the ISO failed to induce the usual high levels of thymidine incorporation, whereas NGF induced the same high levels observed in rats with submandibular glands intact. NGF did not alter the distribution of beta 1- and beta 2-adrenoceptors in the pancreas but did increase norepinephrine when the initial administration was at age 5 days, but not when it was given at age 10 days. Since NGF increased DNA synthesis in the absence of submandibular-sublingual glands, whereas ISO did not, this suggests that ISO requires NGF to induce beta 1-activation and subsequent synthesis and that NGF is a direct activator.     

Carvedilol and its new analogs suppress arrhythmogenic store overload-induced Ca2+ release

Carvedilol is one of the most effective beta blockers for preventing ventricular tachyarrhythmias in heart failure, but the mechanisms underlying its favorable antiarrhythmic benefits remain unclear. Spontaneous Ca(2+) waves, also called store overload-induced Ca(2+) release (SOICR), evoke ventricular tachyarrhythmias in individuals with heart failure. Here we show that carvedilol is the only beta blocker tested that effectively suppresses SOICR by directly reducing the open duration of the cardiac ryanodine receptor (RyR2). This unique anti-SOICR activity of carvedilol, combined with its beta-blocking activity, probably contributes to its favorable antiarrhythmic effect. To enable optimal titration of carvedilol's actions as a beta blocker and as a suppressor of SOICR separately, we developed a new SOICR-inhibiting, minimally beta-blocking carvedilol analog, VK-II-86. VK-II-86 prevented stress-induced ventricular tachyarrhythmias in RyR2-mutant mice and did so more effectively when combined with either of the selective beta blockers metoprolol or bisoprolol. Combining SOICR inhibition with optimal beta blockade has the potential to provide antiarrhythmic therapy that can be tailored to individual patients.     

Pain perception in mice lacking the beta3 subunit of voltage-activated calcium channels

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N- and P/Q-type calcium channels as well as by gene targeting of the alpha1B subunit (N-type). The accessory beta3 subunits of calcium channels are preferentially associated with the alpha1B subunit in neurones. Here we show that deletion of the beta3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cord in vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of beta3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (alpha1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.     

Effects of the infusion of non-selective beta-, and selective beta1- or beta2-adrenergic agonists, on body fat mobilisation in underfed or overfed non-pregnant heifers.

The aim of this study was to analyse the effects of the intravenous infusion of a non-selective beta-agonist (beta-A) (isoproterenol, ISO), and selective beta1-A (dobutamine, D) or beta2-A (terbutaline, T) on body fat mobilisation in non-pregnant heifers, in a 2 x 2 crossover design with two treatments (underfeeding or overfeeding) and two periods. The effect of the duration of submission to each energy level on basal and ISO-induced fat mobilisation was also studied. ISO had a high and significant lipolytic effect whatever the energy level. Nevertheless, the response area of non-esterified fatty acids (NEFA) to ISO in underfed cows was 1.7 times greater than that in overfed cows. T had a slight but significant lipolytic effect on NEFA plasma response in the underfed group. D had no lipolytic effect. Basal and ISO-stimulated plasma NEFA levels were similar after 1 or 4 weeks of underfeeding.     

Isorhapontigenin, a new resveratrol analog, attenuates cardiac hypertrophy via blocking signaling transduction pathways

Cardiac hypertrophy is a major cause of morbidity and mortality worldwide. The hypertrophic process is mediated, in part, by oxidative stress-mediated signaling pathways. We hypothesized that isorhapontigenin (ISO), a new resveratrol analog, inhibits cardiac hypertrophy by blocking oxidative stress and oxidative stress-mediated signaling pathways. We treated cardiomyocytes with angiotensin II (Ang II) with or without ISO and found that ISO inhibited Ang II-induced cardiac hypertrophy. These effects were associated with a decrease in the levels of reactive oxygen species and H2O2 and the content of intracellular malonaldehyde and an increase in the activities of superoxide dismutase and glutathione peroxidase. Ang II induced the phosphorylation of PKC, Erk1/2, JNK, and p38 in cardiomyocytes and such phosphorylation was inhibited by ISO. ISO also blocked the PKC-dependent PI3K-Akt-GSK3beta/p70S6K pathway. These effects lead to direct or indirect inhibition of NF-kappaB and AP-1 activation. Our results revealed that pretreatment with ISO significantly inhibited Ang II-mediated NF-kappaB through affecting the degradation and phosphorylation of IkappaBalpha and the activity of IKKbeta and AP-1 activation by influencing the expression of c-Fos and c-Jun proteins. In addition, we also established the molecular link between activation of PKC and MAPKs and activation of NF-kappaB and AP-1 in cardiomyocytes. We also found that ISO treatment significantly attenuated heart weight/body weight ratio by approximately 25%, decreased posterior wall thickness and left ventricle diastolic and systolic diameters, and increased 10% fractional shortening in an aortic-banded rat model. Furthermore, treatment with ISO significantly decreased cardiac myocyte size and systolic blood pressure. These findings suggest that ISO prevents the development of cardiac hypertrophy through an antioxidant mechanism involving inhibition of different intracellular signaling transduction pathways.     

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.     

Release of beta-endorphin-immunoreactivity from rat pituitary and hypothalamus in vitro: effects of isoproterenol, dopamine, corticotropin-releasing factor and arginine8-vasopressin

The release of beta-endorphin-immunoreactivity (beta E-IR) from rat pituitary anterior lobe (AL) quarters, neurointermediate lobes (NILs), and hypothalamic fragments was investigated in vitro. The beta-adrenoceptor agonist isoproterenol (ISO) and the hypothalamic neurohormone corticotropin-releasing factor (CRF) concentration-dependently stimulated the release of beta E-IR from superfused AL quarters and NILs, but not from incubated hypothalamic fragments. Dopamine (DA) inhibited the release of beta E-IR from NILs and hypothalamic tissue in a concentration-dependent manner, whereas it did not affect the release from AL quarters. Arginine8-vasopressin (AVP) stimulated the release of beta E-IR from AL quarters and hypothalamic fragments, but did not affect the release from NILs. The data indicate that the release of beta E-IR from cells in the pituitary lobes and in the hypothalamus is differentially regulated, but that common principles are involved. In particular, the results provide first direct evidence for an action of vasopressin as a stimulator of the release of POMC-derived peptides in the hypothalamus.