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The aim of the research was to evaluate the influence of antagonists of specific beta-adrenergic receptor subtypes on bowel motility following abdominal surgery in rat model of postoperative ileus. Bowel motility was measured by the intestinal transit of Evans blue introduced via orogastric tube after surgical procedures of skin incision, laparotomy and laparotomy with gut manipulation. Male rats were given individual adrenergic receptor subtypes antagonists intraperitoneally, and the influence of administered agents on intestinal transit of Evans blue was then evaluated. No statistically significant differences in the length of intestine in tested rats were observed. Propranolol administered prior to surgical procedure has shown protective effect on Evans blue migration in rats undergoing laparotomy and gut manipulation. Intestinal dye transit for propranolol doses of 10, 30 and 45 mg/kg was 18.00 ± 1.88c m, 23.75 ± 1.71 cm and 22.5 ± 2.43 cm, respectively, and for last two doses, statistically significant increase of dye passage was noted, compared to Evans blue transit of 11.00 ± 2.43 cm in the control group. No acceleration of dye migration was seen following administration of beta1-, beta2- and beta3-selective adrenergic receptor antagonist metoprolol, ICI 118.551 and SR58894A, respectively. Our research confirmed that propranolol at high doses, as seen by other researchers, improved bowel motility in early phase of postoperative ileus. That slight acceleration of intestinal dye transit after surgery with gut manipulation is rather connected with membrane-stabilizing action, than the receptor blocking effect, as this effect was not observed after the application of selective antagonists of respective subtypes of beta-adrenergic receptor.

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Von Hippel-Lindau (VHL) syndrome is a rare inherited cancer disease where the lack of VHL protein triggers the development of multisystemic tumors such us retinal hemangioblastomas (HBs), CNS-HBs, and clear cell renal cell carcinoma (ccRCC). Since standard therapies in VHL have shown limited response, leaving surgery as the only possible treatment, targeting of the ß2-adrenergic receptor (ADRB2) has shown therapeutic antitumor benefits on VHL-retinal HBs (clinical trial), VHL-CNS HBs, and VHL-ccRCC (in vitro and in vivo). In the present study, we wanted to look deep into the effects of the ADRB2 blockers propranolol and ICI-118,551 on two main aspects of cancer progression: (i) the changes on the inflammatory response of ccRCC cells; and (ii) the modulation on the Warburg effect (glycolytic metabolism), concretely, on the expression of genes involved in the cell reactive oxygen species (ROS) balance and levels. Accordingly, in vitro studies with primary VHL-ccRCC and 786-O cells measuring ROS levels, ROS-expression of detoxifying enzymes, and the expression of p65/NF-κB targets by RT-PCR were carried out. Furthermore, histological analyses of ccRCC samples from heterotopic mouse xenografts were performed. The obtained results show that ADRB2 blockade in ccRCC cells reduces the level of oxidative stress and stabilizes the inflammatory response. Thus, these data further support the idea of targeting ADRB2 as a promising strategy for the treatment of VHL and other non-VHL tumors.

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Serotonin is an important monoamine in the human body, playing crucial roles, such as a neurotransmitter in the central nervous system. Previously, our group reported that ß-adrenergic drugs (ICI 118,551, isoprenaline, and propranolol) influence the proliferation of breast cancer cells (MCF-7 cells) and their inherent production of adrenaline. Thus, we aimed to investigate the production of serotonin in MCF-7 cells, clarifying if there is a relationship between this production and the viability of the cells. To address this question, briefly, we treated the MCF-7 cells with ICI 118,551, isoprenaline, and propranolol, and evaluated cellular viability and serotonin production by using MTT, Sulforhodamine B (SRB) and Neutral Red (NR) assays, and HPLC-ECD analysis, respectively. Our results demonstrate that isoprenaline promotes the most pronounced endogenous synthesis of serotonin, about 3.5-fold greater than control cells. Propranolol treatment also increased the synthesis of serotonin (when compared to control). On the other hand, treatment with the drug ICI 118,551 promoted a lower endogenous synthesis of serotonin, about 1.1-fold less than what was observed in the control. Together, these results reveal that MCF-7 cells can produce serotonin, and the drugs propranolol, isoprenaline and ICI 118,551 influence this endogenous production. For the first time, after modulation of the ß-adrenergic system, a pronounced cellular growth can be related to higher consumption of serotonin by the cells, resulting in decreased levels of serotonin in cell media, indicative of the importance of serotonin in the growth of MCF-7 cells.

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ß-blockers having specific affinities to ß-adrenergic receptors are routinely used to treat cardiovascular problems. Additionally, it has been demonstrated that these drugs can be effective in treating apoptosis-related diseases. The current study was conducted to investigate the cytotoxic and apoptotic effects of ß-1 selective esmolol, ß-2 selective ICI-118,551, and non-selective nadolol blockers on the cancerous and healthy lung cells. MTT test was used to evaluate cytotoxicity. Apoptotic actions were examined by using Annexin V-FITC/PI assay, JC-1 staining, ROS test, and the determination of the caspase-4 and -9, Bcl-2, Bax, Bax/Bcl-2, and JNK levels. Although the MRC-5 showed greater resistance than A549 cells, the ß-blockers at 150-250 µM exhibited different levels of cytotoxic effect on both lung cell lines. Esmolol was found to be the most ineffective blocker and the increases in Bcl-2 protein levels were appeared to be effective in resistance to this drug. The increases in reactive oxygen species (ROS) together with the increase in caspase-4 and Bax protein levels have been shown to play a role in ICI-118,551 induced lung cell death. Nadolol was the most effective blocker increasing the total apoptotic cell population in both lung cells, which was based on both mitochondrial and endoplasmic reticulum stress. When the selectivities of the ß-blockers are considered, it seems that ß-2 specific antagonism predominantly mediated the death of lung cells, and the overwhelming factors causing apoptosis mainly varied depending on the selectivity of the blockers.

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Fluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand-receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective ß2 -adrenoceptor (ß2 AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the ß2 AR (pKD >7.0) with good selectivity over the ß1 AR (pKD <6.0). The most potent and selective ligands being 8c (ICI 118,551-Gly-Ala-BODIPY-FL-X; ß2 AR pKD 7.48), 9c (ICI 118,551-ßAla-ßAla-BODIPY-FL-X; ß2 AR pKD 7.48), 12a (ICI 118,551-PEG-BODIPY-X-630/650; ß2 AR pKD 7.56), and 12b (ICI 118,551-PEG-BODIPY-FL; ß2 AR pKD 7.42). 9a (ICI 118,551-ßAla-ßAla-BODIPY-X-630/650) had the highest affinity at recombinant ß2 ARs (pKD 7.57), but also exhibited significant binding affinity to the ß1 AR (pKD 6.69). Nevertheless, among the red fluorescent ligands, 9a had the best imaging characteristics in recombinant HEK293 T cells and labeling was mostly confined to the cell surface. In contrast, 12a showed the highest propensity to label intracellular ß2 ARs in HEK293 T cell expressing exogenous ß2 ARs. This suggests that a combination of the polyethylene glycol (PEG) linker and the BODIPY-X-630/650 makes this ICI 118,551 derivative particularly susceptible to crossing the cell membrane to access the intracellular ß2 ARs. We have also used these ligands in combination with CRISPR/Cas9 genome-edited HEK293 T cells to undertake for the first time real-time ligand binding to native HEK293 T ß2 ARs at low native receptor expression levels. These studies provided quantitative data on ligand-binding characteristics but also allowed real-time visualization of the ligand-binding interactions in genome-edited cells using NanoBRET luminescence imaging.

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Von Hippel-Lindau (VHL), is a rare autosomal dominant inherited cancer in which the lack of VHL protein triggers the development of multisystemic tumors such us retinal hemangioblastomas (HB), CNS-HB, and clear cell renal cell carcinoma (ccRCC). ccRCC ranks third in terms of incidence and first in cause of death. Standard systemic therapies for VHL-ccRCC have shown limited response, with recurrent surgeries being the only effective treatment. Targeting of ß2-adrenergic receptor (ADRB) has shown therapeutic antitumor benefits on VHL-retinal HB (clinical trial) and VHL-CNS HB (in vitro). Therefore, the in vitro and in vivo antitumor benefits of propranolol (ADRB-1,2 antagonist) and ICI-118,551 (ADRB-2 antagonist) on VHL-/- ccRCC primary cultures and 786-O tumor cell lines have been addressed. Propranolol and ICI-118,551 activated apoptosis inhibited gene and protein expression of HIF-2α, CAIX, and VEGF, and impaired partially the nuclear internalization of HIF-2α and NFĸB/p65. Moreover, propranolol and ICI-118,551 reduced tumor growth on two in vivo xenografts. Finally, ccRCC patients receiving propranolol as off-label treatment have shown a positive therapeutic response for two years on average. In summary, propranolol and ICI-118,551 have shown antitumor benefits in VHL-derived ccRCC, and since ccRCCs comprise 63% of the total RCCs, targeting ADRB2 becomes a promising drug for VHL and other non-VHL tumors.

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Despite vast literature on catecholaminergic neuromodulation of auditory cortex functioning in general, knowledge about its role for long-term memory formation is scarce. Our previous pharmacological studies on cortex-dependent frequency-modulated tone-sweep discrimination learning of Mongolian gerbils showed that auditory-cortical D1/5 -dopamine receptor activity facilitates memory consolidation and anterograde memory formation. Considering overlapping functions of D1/5 -dopamine receptors and ß-adrenoceptors, we hypothesised a role of ß-adrenergic signalling in the auditory cortex for sweep discrimination learning and memory. Supporting this hypothesis, the ß1/2 -adrenoceptor antagonist propranolol bilaterally applied to the gerbil auditory cortex after task acquisition prevented the discrimination increment that was normally monitored 1 day later. The increment in the total number of hurdle crossings performed in response to the sweeps per se was normal. Propranolol infusion after the seventh training session suppressed the previously established sweep discrimination. The suppressive effect required antagonist injection in a narrow post-session time window. When applied to the auditory cortex 1 day before initial conditioning, ß1 -adrenoceptor-antagonising and ß1 -adrenoceptor-stimulating agents retarded and facilitated, respectively, sweep discrimination learning, whereas ß2 -selective drugs were ineffective. In contrast, single-sweep detection learning was normal after propranolol infusion. By immunohistochemistry, ß1 - and ß2 -adrenoceptors were identified on the neuropil and somata of pyramidal and non-pyramidal neurons of the gerbil auditory cortex. The present findings suggest that ß-adrenergic signalling in the auditory cortex has task-related importance for discrimination learning of complex sounds: as previously shown for D1/5 -dopamine receptor signalling, ß-adrenoceptor activity supports long-term memory consolidation and reconsolidation; additionally, tonic input through ß1 -adrenoceptors may control mechanisms permissive for memory acquisition.

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5-Hydroxytryptamine2A (5-HT2A) receptors are enriched in layers I and Va of the rat prefrontal cortex and neocortex and their activation increases the frequency of glutamatergic excitatory post-synaptic potentials/currents (EPSP/Cs) onto layer V pyramidal cells. A number of other G-protein coupled receptors (GPCRs) are also enriched in cortical layers I and Va and either induce (α1-adrenergic and orexin2) or suppress (metabotropic glutamate2 [mGlu2], adenosine A1, µ-opioid) both 5-HT-induced EPSCs and head twitches or head shakes induced by the phenethylamine hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI). Another neurotransmitter receptor also localized to apparent thalamocortical afferents to layers I and Va of the rat prefrontal cortex and neocortex is the ß2-adrenergic receptor. Therefore, we conducted preliminary electrophysiological experiments with rat brain slices examining the effects of epinephrine on electrically-evoked EPSPs following bath application of DOI (3 µM). Epinephrine (0.3-10 µM) suppressed the late EPSPs produced by electrical stimulation and DOI. The selective ß2-adrenergic receptor antagonist ICI-118,551 (300 nM) resulted in a rightward shift of the epinephrine concentration-response relationship. We also tested the selective ß2-adrenergic receptor agonist clenbuterol and the antagonist ICI-118,551 on DOI-induced head twitches. Clenbuterol (0.3-3 mg/kg, i.p.) suppressed DOI (1.25 mg/kg, i.p.)-induced head twitches. This clenbuterol effect appeared to be at least partially reversed by the selective ß2-adrenergic receptor antagonist ICI-118,553 (0.01-1 mg/kg, i.p.), with significant reversal at doses of 0.1 and 1 mg/kg. Thus, ß2-adrenergic receptor activation reverses the effects of phenethylamine hallucinogens in the rat prefrontal cortex. While Gi/Go-coupled GPCRs have previously been shown to suppress both the electrophysiological and behavioral effects of 5-HT2A receptor activation in the mPFC, the present work appears to extend this suppressant action to a Gs-coupled GPCR. Furthermore, the modulation of 5-HT2A receptor activation-induced glutamate release onto mPFC layer V pyramidal neurons apical dendrites by a range GPCRs in rat brain slices appears to results in behaviorally salient effects of relevance when screening for novel CNS therapeutic drugs.

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Altered sensitivity to the chronotropic effect of catecholamines and a reduction in the ß1/ß2-adrenoceptor ratio have previously been reported in right atria of stressed rats, human failing heart, and aging. In this report, we investigated whether left atrial inotropism was affected by foot-shock stress. Male rats were submitted to 3 foot-shock sessions and the left atrial inotropic response, adenylyl cyclase activity, and ß-adrenoceptor expression were investigated. Left atria of stressed rats were supersensitive to isoprenaline when compared with control rats and this effect was abolished by ICI118,551, a selective ß2-receptor antagonist. Schild plot slopes for the antagonism between CGP20712A (a selective ß1-receptor antagonist) and isoprenaline differed from unity in atria of stressed but not control rats. Atrial sensitivity to norepinephrine, as well as basal and forskolin- or isoprenaline-stimulated adenylyl cyclase activities were not altered by stress. The effect of isoprenaline on adenylyl cyclase stimulation was partially blocked by ICI118,551 in atrial membranes of stressed rats. These findings indicate that foot-shock stress equally affects inotropism and chronotropism and that ß2-adrenoceptor upregulation contributes to the enhanced inotropic response to isoprenaline.

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Drug seeking is maintained by encounters with drug-associated cues, and disrupting retrieval of these drug-cue associations would reduce the risk of relapse. Retrieval of cocaine-associated memories is dependent on ß-adrenergic receptor (ß-AR) activation, and blockade of these receptors induces a persistent retrieval deficit. Whether retrieval of cocaine-associated memory is mediated by a specific ß-AR subtype, however, remains unclear. Using a cocaine conditioned place preference (CPP) procedure, we examined whether retrieval of a cocaine CPP memory is mediated collectively by ß1- and ß2-ARs, or by one of these ß-AR subtypes alone. We show that co-blockade of ß1- and ß2-ARs abolished CPP expression on that and subsequent drug-free CPP tests, resulting in a long-lasting retrieval deficit that prevented subsequent cocaine-induced reinstatement. To dissociate the necessity of either ß1- or ß2-ARs alone, we administered subtype-specific antagonists prior to retrieval. Administration of a ß1-AR antagonist before the initial CPP trial dose-dependently reduced expression of a CPP on that and subsequent drug-free trials as compared to vehicle administration. In contrast, administration of a ß2-AR antagonist had no effect on initial CPP expression, although the highest dose reduced subsequent CPP expression. Importantly, either ß1- or ß2-AR blockade prior to an initial retrieval trial prevented subsequent cocaine-induced reinstatement. Our findings indicate that the ß1-AR subtype mediates retrieval of a cocaine CPP, and that acutely blocking either ß1- or ß2-ARs can prevent subsequent cocaine-induced reinstatement. Thus, ß-AR antagonists, particularly ß1-ARs antagonists, could serve as adjuncts for addiction therapies to prevent retrieval of drug-associated memories and provide protection against relapse.

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Studies on the relative contribution of ß 1- and ß 2-adrenoceptors (AR) generally employ selective ß 1- and ß 2-AR antagonists such as CGP 20712A and ICI 118,551, respectively, and assume that antagonism by one of these compounds indicates mediation by the respective AR subtype. Here, we evaluated the ß 2-AR-selectivity of ICI 118,551 in ventricular muscle strips of transgenic mice lacking ß 1-AR (ß 1-KO), ß 2-AR (ß 2-KO), or both (ß 1/ß 2-KO). Strips were electrically driven and force development was measured. In wild type (WT), ICI 118,551 (100 nmol/L) shifted the concentration-response curve (CRC) for adrenaline by about 0.5 log units to the right, corresponding to the known affinity of ICI 118,551 to ß 1-AR but not to ß 2-AR. Conversely, the phosphodiesterase inhibitor rolipram (10 µmol/L) shifted the CRC to the left, but did not enlarge the ICI 118,551 shift, indicating exclusive ß 1-AR mediation even when PDE4 is inactive. In line with this, rolipram and ICI 118,551 had similar effects in ß 2-KO than in WT. In contrast, ß 1-KO did not show any inotropic reaction to adrenaline (+/- rolipram). In WT, the ß 1-AR selective antagonist CGP 20712A (100 nmol/L) shifted the CRC for isoprenaline by 2.1 log units, corresponding to the affinity of CGP 20712A to ß 1-AR. Rolipram increased the sensitivity to adrenaline independently of the presence of CGP 20712A. We conclude that effects sensitive to the ß 2-AR antagonist ICI 118,551 are not necessarily ß 2-AR-mediated and CGP 20712A-resistant effects cannot be simply interpreted as ß 2-AR-mediated. Catecholamine effects in murine ventricles strictly depend on ß 1-AR, even if PDE 4 is blocked.

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Fenoterol, a ß2-adrenoceptor agonist, has anti-apoptotic action in cardiomyocytes and induces a specific pattern of downstream signaling. We have previously reported that exposure to fenoterol (5 µM) results in a delayed positive inotropic effect which is related to changes in both Ca2+ transient and NO. Here, the changes in reactive oxygen species (ROS) production in response to the fenoterol administration and the involvement of ROS in effect of this agonist on contractility were investigated in mouse isolated atria. Stimulation of ß2-adrenoceptor increases a level of extracellular ROS, while intracellular ROS level rises only after removal of fenoterol from the bath. NADPH-oxidase inhibitor (apocynin) prevents the increase in ROS production and the Nox2 isoform is immunofluorescently colocalized with ß2-adrenoceptor at the atrial myocytes. Treatments with antioxidants (N-acetyl-L-cysteine, NADPH inhibitors, exogenous catalases) significantly inhibit the fenoterol induced increase in the contraction amplitude, probably by attenuating Ca2+ transient and up-regulating NO production. ROS generated in a ß2-adrenoceptor-dependent manner can potentiate the activity of some Ca2+ channels. Indeed, inhibition of ryanodine receptors, TRPV-or L-type Ca2+- channels shows a similar efficacy in reduction of positive inotropic effect of both fenoterol and H2O2. In addition, detection of mitochondrial ROS indicates that fenoterol triggers a slow increase in ROS which is prevented by rotenone, but rotenone has no impact on the inotropic effect of fenoterol. We suggest that stimulation of ß2-adrenoceptor with fenoterol causes the activation of NADPH-oxidase and after the agonist removal extracellularly generated ROS penetrates into the cell, increasing the atrial contractions probably via Ca2+ channels.

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Norepinephrine (NE) responses are larger in renal and femoral veins compared to phenylephrine (PE). These differences may be due to the subtypes of adrenoceptor involved in these responses or to the involvement of local modulatory mechanisms. Therefore, the present study investigated in organ bath the adrenoceptor subtypes involved in the NE and PE responses in both renal and femoral veins as well as the influence of local mechanisms related to NO and to prostanoids upon these responses. The obtained data showed that the NE responses in these veins were not significantly modified by the selective inhibition of ß1 or ß2-adrenoceptors as well as AT1 or AT2 receptors. However, yohimbine reduced the NE Rmax in renal veins and, in parallel, right shifted the NE concentration-response curves in femoral veins. In both veins, prazosin reduced the NE Rmax and the clonidine induced a measurable contraction. The endothelium removal attenuated the NE responses in femoral veins, thereby abolishing the differences of NE and PE responses. Furthermore, the NE responses in renal and femoral veins were attenuated by indomethacin, which suppressed the statistical difference in relation to the PE response. In conclusion, a synergism between α1- and α2-adrenoceptors is essential to assure full NE contractile responses in both renal and femoral veins. Thus, by acting simultaneously in these adrenoceptors, NE induces more pronounced contractile responses, in comparison to PE, not only in renal but also in femoral veins. Moreover, this pronounced NE response in both renal and femoral veins appears to involve endothelium-derived vasoconstrictor prostanoids.

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ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora crispa has been used in folkloric medicine for the control of blood pressure. We previously found that an extract of Tinospora crispa and its constituents effect the heart rate and blood pressure in anesthetized rats. AIM OF THE STUDY: The aim was to investigate the effects and mechanisms of the Tinospora crispa extract and bioactive components on the rat isolated left atria. MATERIALS AND METHODS: Air-dried stems of Tinospora crispa were extracted with water, followed by partitioning with chloroform, ethyl acetate, and finally by n-butanol. The n-butanol soluble material was concentrated and dried under reduced pressure and lyophilized to obtain a crude powder (Tinospora crispa extract). The active components of Tinospora crispa extract were separated by column chromatography and preparative HPLC. The effects and mechanisms of the n-butanol extract and the bioactive purified components (adenine, uridine, adenosine, salsolinol, tyramine, higenamine, syringin, (-)-litcubinine, borapetoside A, borapetoside B, borapetoside D and borapetoside E) were studied in isolated left atria from normal and reserpinized rats. RESULTS: Tinospora crispa extract caused an increase in the force of contraction of the electrical field stimulated left atrium. This effect was inhibited by propranolol, atenolol, ICI-118,551, phentolamine and atropine. The positive inotropic effect on the reserpenized isolated left atrium of the Tinospora crispa extract was significantly inhibited by propranolol, atenolol and ICI-118,551. Phentolamine, on the other hand, caused potentiation and the effect was inhibited when propranolol was also added. Higenamine caused an increase in the force of contraction of the electrical field stimulated left atrium and this effect was significantly inhibited by ICI-118,551 and atenolol but not by phentolamine. Reserpine did not significantly shift the concentration-response curve (C-R curve) of the inotropic effect of the higenamine. ICI-118,551 and atenolol caused a parallel shift of the C-R curve to the right of about 8 and 33 fold, respectively. At low concentrations salsolinol caused a slight increase in the force of contraction of the left atrium, but at higher concentrations a decrease was observed. The negative inotropic effect of salsolinol was significantly inhibited by propranolol and atropine. In the reserpinized isolated left atrium, the negative inotropic effect of salsolinol was potentiated and again this effect was significantly inhibited by propranolol and atropine. Tyramine caused a positive inotropic effect, and this effect was inhibited by propranolol or by pretreatment of the rat with reserpine. Adenosine caused a negative inotropic effect, while uridine caused a slight positive inotropic effect on the left atrium. This effect was significantly inhibited by DPCPX. CONCLUSIONS: Crude extract of Tinospora crispa exert a positive inotropic effect on the electrical field stimulated isolated left atria that results from the concerted action of 5 bioactive compounds: higenamine, salsolinol, tyramine, adenosine and uridine. Higenamine, salsolinol (at low concentration) and tyramine acted via the adrenergic receptors to increase the force of the atrial contraction, whereas a high concentration of salsolinol acted indirectly by stimulating the release of acetylcholine. Adenosine and uridine acted via the purinergic pathways to cause negative inotropic effects on the isolated left atria.

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