Discovery and Identification of Novel 5-Hydroxy-4H-benzo[1,4]oxazin-3-one Derivatives as Potent ß2-Adrenoceptor Agonists through Structure-Based Design, Synthesis, and Biological Evaluation.

Although ß2-agonists are crucial for treatment of chronic respiratory diseases, optimizing ß2-agonistic activity and selectivity remains essential for achieving favorable therapeutic outcomes. A structure-based molecular design workflow was employed to discover a novel class of ß2 agonists featuring a 5-hydroxy-4H-benzo[1,4]oxazin-3-one scaffold, which potently stimulated ß2 adrenoceptors (ß2-ARs). Screening for the ß2-agonistic activity and selectivity led to the identification of compound A19 (EC50 = 3.7 pM), which functioned as a partial ß2-agonist in HEK-293 cells containing endogenous ß2-ARs. Compound A19 exhibited significant relaxant effects, rapid onset time (Ot50 = 2.14 min), and long duration of action (>12 h) on isolated guinea pig tracheal strips, as well as advantageous pharmacokinetic characteristics in vivo, rendering A19 suitable for inhalation administration. Moreover, A19 suppressed the upregulation of inflammatory cytokines and leukocytes and improved lung function in a rat model of COPD, thereby indicating that A19 is a potential ß2 agonist candidate for further study.

"Photo-Adrenalines": Photoswitchable ß2 -Adrenergic Receptor Agonists as Molecular Probes for the Study of Spatiotemporal Adrenergic Signaling.

ß2 -adrenergic receptor (ß2 -AR) agonists are used for the treatment of asthma and chronic obstructive pulmonary disease, but also play a role in other complex disorders including cancer, diabetes and heart diseases. As the cellular and molecular mechanisms in various cells and tissues of the ß2 -AR remain vastly elusive, we developed tools for this investigation with high temporal and spatial resolution. Several photoswitchable ß2 -AR agonists with nanomolar activity were synthesized. The most potent agonist for ß2 -AR with reasonable switching is a one-digit nanomolar active, trans-on arylazopyrazole-based adrenaline derivative and comprises valuable photopharmacological properties for further biological studies with high structural accordance to the native ligand adrenaline.

Development of novel ß2-adrenergic receptor agonists for the stimulation of glucose uptake - The importance of chirality and ring size of cyclic amines.

ß2-Adrenergic receptor (ß2AR) agonists have been reported to stimulate glucose uptake (GU) by skeletal muscle cells and are therefore highly interesting as a possible treatment for type 2 diabetes (T2D). The chirality of compounds often has a great impact on the activity of ß2AR agonists, although this has thus far not been investigated for GU. Here we report the GU for a selection of synthesized acyclic and cyclic ß-hydroxy-3-fluorophenethylamines. For the N-butyl and the N-(2-pentyl) compounds, the (R) and (R,R) (3d and 7e) stereoisomers induced the highest GU. When the compounds contained a saturated nitrogen containing 4- to 7-membered heterocycle, the (R,R,R) enantiomer of the azetidine (8a) and the pyrrolidine (9a) had the highest activity. Altogether, these results provide pivotal information for designing novel ß2AR agonist for the treatment of T2D.

Molecular determinants of ligand efficacy and potency in GPCR signaling.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) bind to extracellular ligands and drugs and modulate intracellular responses through conformational changes. Despite their importance as drug targets, the molecular origins of pharmacological properties such as efficacy (maximum signaling response) and potency (the ligand concentration at half-maximal response) remain poorly understood for any ligand-receptor-signaling system. We used the prototypical adrenaline-ß2 adrenergic receptor-G protein system to reveal how specific receptor residues decode and translate the information encoded in a ligand to mediate a signaling response. We present a data science framework to integrate pharmacological and structural data to uncover structural changes and allosteric networks relevant for ligand pharmacology. These methods can be tailored to study any ligand-receptor-signaling system, and the principles open possibilities for designing orthosteric and allosteric compounds with defined signaling properties.

Advances in adrenergic receptors for the treatment of chronic obstructive pulmonary disease: 2023 update.

INTRODUCTION: Strong scientific evidence and large experience support the use of ß2-agonists for the symptomatic alleviation of COPD. Therefore, there is considerable effort in discovering highly potent and selective ß2-agonists. AREAS COVERED: Recent research on novel ß2-agonists for the treatment of COPD. A detailed literature search was performed in two major databases (PubMed/MEDLINE and Scopus) up to September 2023." EXPERT OPINION: Compounds that preferentially activate a Gs- or ß-arrestin-mediated signaling pathway via ß- adrenoceptors (ARs) are more innovative. Pepducins, which target the intracellular region of ß2-AR to modulate receptor signaling output, have the most interesting profile from a pharmacological point of view. They stabilize the conformation of the ß2-AR and influence its signaling by interacting with the intracellular receptor-G protein interface. New bifunctional drugs called muscarinic antagonist-ß2 agonist (MABA), which have both muscarinic receptor (mAChR) antagonism and ß2-agonist activity in the same molecule, are a new opportunity. However, all tested compounds have been shown to act predominantly as mAChR antagonists or ß2-agonists. An intriguing idea is to utilize allosteric modulators that bind to ß2-ARs at sites different than those bound by orthosteric ligands to augment or reduce the signaling transduced by the orthosteric ligand.

Predicting the duration of action of ß2-adrenergic receptor agonists: Ligand and structure-based approaches.

Agonists of the ß2 adrenergic receptor (ADRB2) are an important class of medications used for the treatment of respiratory diseases. They can be classified as short acting (SABA) or long acting (LABA), with each class playing a different role in patient management. In this work we explored both ligand-based and structure-based high-throughput approaches to classify ß2-agonists based on their duration of action. A completely in-silico prediction pipeline using an AlphaFold generated structure was used for structure-based modelling. Our analysis identified the ligands' 3D structure and lipophilicity as the most relevant features for the prediction of the duration of action. Interaction-based methods were also able to select ligands with the desired duration of action, incorporating the bias directly in the structure-based drug discovery pipeline without the need for further processing.

Might It Be Appropriate to Anticipate the Use of Long-Acting Muscarinic Antagonists in Asthma?

A growing number of clinical trials are documenting that adding a long-acting muscarinic antagonist (LAMA) to established asthma treatment with an inhaled corticosteroid (ICS) and a long-acting ß2-agonist (LABA) is a treatment option that improves the health of patients with uncontrolled severe asthma even when therapy is optimized. These favorable results are the reason why the leading guidelines recommend triple therapy with ICS + LABA + LAMA in patients with asthma uncontrolled by medium- to high-dose ICS-LABA. However, we suggest adding LAMAs to ICS-LABAs at an earlier clinical stage. Such action could positively influence airflow limitation, exacerbations, and eosinophilic inflammation, conditions that are associated with acetylcholine (ACh) activity. It could also interrupt the vicious cycle related to a continuous release of ACh leading to the progressive expansion of neuronal plasticity resulting in small airway dysfunction. The utility of an earlier use of triple therapy in asthma should, in any case, be confirmed by statistically powered trials.

Experimental drugs in clinical trials for COPD: artificial intelligence via machine learning approach to predict the successful advance from early-stage development to approval.

INTRODUCTION: Therapeutic advances in drug therapy of chronic obstructive pulmonary disease (COPD) really effective in suppressing the pathological processes underlying the disease deterioration are still needed. Artificial Intelligence (AI) via Machine Learning (ML) may represent an effective tool to predict clinical development of investigational agents. AREAL COVERED: Experimental drugs in Phase I and II development for COPD from early 2014 to late 2022 were identified in the ClinicalTrials.gov database. Different ML models, trained from prior knowledge on clinical trial success, were used to predict the probability that experimental drugs will successfully advance toward approval in COPD, according to Bayesian inference as follows: ≤25% low probability, >25% and ≤50% moderate probability, >50% and ≤75% high probability, and >75% very high probability. EXPERT OPINION: The Artificial Neural Network and Random Forest ML models indicated that, among the current experimental drugs in clinical trials for COPD, only the bifunctional muscarinic antagonist - ß2-adrenoceptor agonists (MABA) navafenterol and batefenterol, the inhaled corticosteroid (ICS)/MABA fluticasone furoate/batefenterol, and the bifunctional phosphodiesterase (PDE) 3/4 inhibitor ensifentrine resulted to have a moderate to very high probability of being approved in the next future, however not before 2025.

Pathological changes in GPCR signal organisation: Opportunities for targeted therapies for triple negative breast cancer.

Triple negative breast cancer (TNBC) has the poorest prognosis compared to other breast cancer subtypes, due to a historical lack of targeted therapies and high rates of relapse. Greater insight into the components of signalling pathways in TNBC tumour cells has led to the clinical evaluation, and in some cases approval, of targeted therapies. In the last decade, G protein-coupled receptors, such as the ß2-adrenoceptor, have emerged as potential new therapeutic targets. Here, we describe how the ß2-adrenoceptor accelerates TNBC progression in response to stress, and the unique signalling pathway activated by the ß2-adrenoceptor to drive the invasion of an aggressive TNBC tumour cell. We highlight evidence that supports an altered organisation of GPCRs in tumour cells, and suggests that activation of the same GPCR in a different cellular location can control unique cell responses. Finally, we speculate how the relocation of GPCRs to the "wrong" place in tumour cells presents opportunities to develop targeted anti-cancer GPCR drugs with greater efficacy and minimal adverse effects.

Discovery of Clinical Candidate CHF-6366: A Novel Super-soft Dual Pharmacology Muscarinic Antagonist and ß2 Agonist (MABA) for the Inhaled Treatment of Respiratory Diseases.

The development of molecules embedding two distinct pharmacophores acting as muscarinic antagonists and ß2 agonists (MABAs) promises to be an excellent opportunity to reduce formulation issues and boost efficacy through cross-talk and allosteric interactions. Herein, we report the results of our drug discovery campaign aimed at improving the therapeutic index of a previous MABA series by exploiting the super soft-drug concept. The incorporation of a metabolic liability, stable at the site of administration but undergoing rapid systemic metabolism, to generate poorly active and quickly eliminated fragments was pursued. Our SAR studies yielded MABA 29, which demonstrated a balanced in vivo profile up to 24 h, high instability in plasma and the liver, as well as sustained exposure in the lung. In vitro safety and non-GLP toxicity studies supported the nomination of 29 (CHF-6366) as a clinical candidate, attesting to the successful development of a novel super-soft MABA compound.

α and ß catalytic subunits of cAMP-dependent protein kinase regulate formoterol-induced inflammatory gene expression changes in human bronchial epithelial cells.

BACKGROUND AND PURPOSE: It has been proposed that genomic mechanisms contribute to adverse effects often experienced by asthmatic subjects who take regular, inhaled ß2 -adrenoceptor agonists as a monotherapy. Moreover, data from preclinical models of asthma suggest that these gene expression changes are mediated by ß-arrestin-2 rather than PKA. Herein, we tested this hypothesis by comparing the genomic effects of formoterol, a ß2 -adrenoceptor agonist, with forskolin in human primary bronchial epithelial cells (HBEC). EXPERIMENTAL APPROACH: Gene expression changes were determined by RNA-sequencing. Gene silencing and genome editing were employed to explore the roles of ß-arrestin-2 and PKA. KEY RESULTS: The formoterol-regulated transcriptome in HBEC treated concurrently with TNFα was defined by 1480 unique gene expression changes. TNFα-induced transcripts modulated by formoterol were annotated with enriched gene ontology terms related to inflammation and proliferation, notably "GO:0070374~positive regulation of ERK1 and ERK2 cascade," which is an apparent ß-arrestin-2 target. However, expression of the formoterol- and forskolin-regulated transcriptomes were highly rank-order correlated and the effects of formoterol on TNFα-induced inflammatory genes were abolished by an inhibitor of PKA. Furthermore, formoterol-induced gene expression changes in BEAS-2B bronchial epithelial cell clones deficient in ß-arrestin-2 were comparable with those expressed by their parental counterparts. Contrariwise, gene expression was partially inhibited in clones lacking the α-catalytic subunit (Cα) of PKA and abolished following the additional knockdown of the ß-catalytic subunit (Cß) paralogue. CONCLUSIONS: The effects of formoterol on inflammatory gene expression in airway epithelia are mediated by PKA and involve the cooperation of PKA-Cα and PKA-Cß.

Beta2 -agonist increases skeletal muscle interleukin 6 production and release in response to resistance exercise in men.

OBJECTIVE: Several tissues produce and release interleukin-6 (IL-6) in response to beta2 -adrenergic stimulation with selective agonists (beta2 -agonists). Moreover, exercise stimulates muscle IL-6 production, but whether beta2 -agonists regulate skeletal muscle production and release of IL-6 in humans in association with exercise remains to be clarified. Thus, we investigated leg IL-6 release in response to beta2 -agonist salbutamol in lean young men at rest and in recovery from resistance exercise. DESIGN: The study employed a randomized controlled crossover design, where 12 men ingested either salbutamol (16 mg) or placebo for 4 days, followed by the last dose (24 mg) administered 1½ h before exercise. Arterial and femoral venous plasma IL-6 as well as femoral artery blood flow was measured before and ½-5 h in recovery from quadriceps muscle resistance exercise. Furthermore, vastus lateralis muscle biopsies were collected ½ and 5 h after exercise for determination of mRNA levels of IL-6 and Tumor Necrosis Factor (TNF)-α. RESULTS: Average leg IL-6 release was 1.7-fold higher (p = 0.01) for salbutamol than placebo, being 138 ± 76 and 79 ± 66 pg min-1 (mean ± SD) for salbutamol and placebo, respectively, but IL-6 release was not significantly different between treatments within specific sampling points at rest and after exercise. Muscle IL-6 mRNA was 1.5- and 1.7-fold higher (p = 0.001) for salbutamol than placebo ½ and 5 h after exercise, respectively, whereas no significant treatment differences were observed for TNF-α mRNA. CONCLUSIONS: Beta2 -adrenergic stimulation with high doses of the selective beta2 -agonist salbutamol, preceeded by 4 consecutive daily doses, induces transcription of IL-6 in skeletal muscle in response to resistance exercise, and increases muscle IL-6 release in lean individuals.

The beta2 -adrenergic receptor - a re-emerging target to combat obesity and induce leanness?

Treatment of obesity with repurposed or novel drugs is an expanding research field. One approach is to target beta2 -adrenergic receptors because they regulate the metabolism and phenotype of adipose and skeletal muscle tissue. Several observations support a role for the beta2 -adrenergic receptor in obesity. Specific human beta2 -adrenergic receptor polymorphisms are associated with body composition and obesity, for which the Gln27Glu polymorphism is associated with obesity, while the Arg16Gly polymorphism is associated with lean mass in men and the development of obesity in specific populations. Individuals with obesity also have lower abundance of beta2 -adrenergic receptors in adipose tissue and are less sensitive to catecholamines. In addition, studies in livestock and rodents demonstrate that selective beta2 -agonists induce a so-called 'repartitioning effect' characterized by muscle accretion and reduced fat deposition. In humans, beta2 -agonists dose-dependently increase resting metabolic rate by 10-50%. And like that observed in other mammals, only a few weeks of treatment with beta2 -agonists increases muscle mass and reduces fat mass in young healthy individuals. Beta2 -agonists also exert beneficial effects on body composition when used concomitantly with training and act additively to increase muscle strength and mass during periods with resistance training. Thus, the beta2 -adrenergic receptor seems like an attractive target in the development of anti-obesity drugs. However, future studies need to verify the long-term efficacy and safety of beta2 -agonists in individuals with obesity, particularly in those with comorbidities.

Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

In most living cells, the second-messenger roles for adenosine 3',5'-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch-like actions of Gαs (stimulatory G protein)-activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface ß2AR (ß2-adrenoceptor), a Gs-coupled GPCR (G protein-coupled receptor), evokes cAMP egress to the extracellular space. Increased extracellular cAMP levels ([cAMP]e) are long-lived in culture and are induced by receptor-dependent and receptor-independent mechanisms in such a way as to define a universal response class of increased intracellular cAMP levels ([cAMP]i). We find that HASM cells express multiple ATP-binding cassette (ABC) membrane transporters, with ABCC1 (ABC subfamily member C 1) being the most highly enriched transcript mapped to MRPs (multidrug resistance-associated proteins). We show that pharmacological inhibition or downregulation of ABCC1 with siRNA markedly reduces ß2AR-evoked cAMP release from HASM cells. Furthermore, inhibition of ABCC1 activity or expression decreases basal tone and increases ß-agonist-induced HASM cellular relaxation. These findings identify a previously unrecognized role for ABCC1 in the homeostatic regulation of [cAMP]i in HASM that may be conserved traits of the Gs-GPCRs (Gs-coupled family of GPCRs). Hence, the general features of this activation mechanism may uncover new disease-modifying targets in the treatment of airflow obstruction in asthma. Surprisingly, we find that serum cAMP levels are elevated in a small cohort of patients with asthma as compared with control subjects, which warrants further investigation.

Evaluation of ß-adrenergic ligands for development of pharmacological heart failure and transparency models in zebrafish.

Cardiovascular toxicity represents one of the most common reasons for clinical trial failure. Consequently, early identification of novel cardioprotective strategies could prevent the later-stage drug-induced cardiac side effects. The use of zebrafish (Danio rerio) in preclinical studies has greatly increased. High-throughput and low-cost of assays make zebrafish model ideal for initial drug discovery. A common strategy to induce heart failure is a chronic ß-adrenergic (ßAR) stimulation. Herein, we set out to test a panel of ßAR agonists to develop a pharmacological heart failure model in zebrafish. We assessed ßAR agonists with respect to the elicited mortality, changes in heart rate, and morphological alterations in zebrafish larvae according to Fish Embryo Acute Toxicity Test. Among the tested ßAR agonists, epinephrine elicited the most potent onset of heart stimulation (EC50 = 0.05 mM), which corresponds with its physiological role as catecholamine. However, when used at ten-fold higher dose (0.5 mM), the same compound caused severe heart rate inhibition (-28.70 beats/min), which can be attributed to its cardiotoxicity. Further studies revealed that isoetharine abolished body pigmentation at the sublethal dose of 7.50 mM. Additionally, as a proof of concept that zebrafish can mimic human cardiac physiology, we tested ßAR antagonists (propranolol, carvedilol, metoprolol, and labetalol) and verified that they inhibited fish heart rate in a similar fashion as in humans. In conclusion, we proposed two novel pharmacological models in zebrafish; i.e., epinephrine-dependent heart failure and isoetharine-dependent transparent zebrafish. We provided strong evidence that the zebrafish model constitutes a valuable tool for cardiovascular research.

Validation of Fenoterol to Study ß2-Adrenoceptor Function in the Rat Urinary Bladder.

Fenoterol is a ß2-adrenoceptor (AR)-selective agonist that is commonly used to investigate relaxation responses mediated by ß2-AR in smooth muscle preparations. Some data have questioned this because fenoterol had low potency in the rat urinary bladder when a muscarinic agonist was used as a pre-contraction agent and because some investigators proposed that fenoterol may act in part via ß3-AR. We designed the present study to investigate whether fenoterol is a proper pharmacological tool to study ß2-AR-mediated relaxation responses in the rat urinary bladder. Firstly, we have compared the effect of pre-contraction agents on fenoterol potency and found that fenoterol potency was about 1.5 log units greater against KCl than carbachol (pEC50 7.19 ± 0.66 and 5.62 ± 1.09 of KCl and of carbachol, respectively). To test the selectivity of fenoterol, we have determined the effects of the ß2-AR antagonist ICI 118,551 and the ß3-AR antagonist L 748,337 on relaxation responses to fenoterol. While 300 nM L 748,337 had little effect on the potency of fenoterol (pEC50 6.56 ± 0.25 and 6.33 ± 0.61 in the absence and presence of L 748,337, respectively), the relaxation curve for fenoterol was right-shifted in the presence 300 nM ICI 118,551 (pEC50 5.03 ± 0.18). Thus, we conclude that fenoterol is a proper pharmacological tool to assess ß2-AR-mediated responses in the rat urinary bladder and most likely in other smooth-muscle preparations containing multiple subtypes of the ß-AR.

Identification and characterization of an atypical Gαs-biased ß2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis.

G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For ß2-adrenergic receptors (ß2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and ß-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable ß-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: ß2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from ß-arrestin, in contrast to albuterol and C5-S C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from ß-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of ß2AR actions favorable for treating obstructive lung disease.

Mechanical loading alleviated the inhibition of ß2-adrenergic receptor agonist terbutaline on bone regeneration.

The long-term use of adrenergic medication in treating various conditions, such as asthma, increases the chances of bone fracture. Dynamic mechanical loading at a specific time is a method for improving bone quality and promoting healing. Therefore, we hypothesized that precisely controlling the mechanical environment can contribute to the alleviation of the negative effects of chronic treatment with the common asthma drug terbutaline, which is a ß2-adrenergic receptor agonist that facilitates bone homeostasis and defect repair through its anabolic effect on osteogenic cells. Our in vitro results showed that terbutaline can directly inhibit osteogenesis by impairing osteogenic differentiation and mineralization. Chronic treatment in vivo was simulated by administering terbutaline to C57BL/6J mice for 4 weeks before bone defect surgery and mechanical loading. We utilized a stabilized tibial defect model, which allowed the application of anabolic mechanical loading. During homeostasis, chronic terbutaline treatment reduced the bone formation rate, the fracture toughness of long bones, and the concentrations of bone formation markers in the sera. During defect repair, terbutaline decreased the bone volume, type H vessel, and total blood vessel volume. Terbutaline treatment reduced the number of osteogenic cells. Periostin, which was secreted mainly by Prrx1+ osteoprogenitors and F4/80+ macrophages, was inhibited by treating the bone defect with terbutaline. Interestingly, controlled mechanical loading facilitated the recovery of bone volume and periostin expression and the number of osteogenic cells within the defect. In conclusion, mechanical loading can rescue negative effects on new bone accrual and repair induced by chronic terbutaline treatment.

ß2-adrenergic receptor regulates ER-mitochondria contacts.

Interactions between the endoplasmic reticulum (ER) and mitochondria (Mito) are crucial for many cellular functions, and their interaction levels change dynamically depending on the cellular environment. Little is known about how the interactions between these organelles are regulated within the cell. Here we screened a compound library to identify chemical modulators for ER-Mito contacts in HEK293T cells. Multiple agonists of G-protein coupled receptors (GPCRs), beta-adrenergic receptors (ß-ARs) in particular, scored in this screen. Analyses in multiple orthogonal assays validated that ß2-AR activation promotes physical and functional interactions between the two organelles. Furthermore, we have elucidated potential downstream effectors mediating ß2-AR-induced ER-Mito contacts. Together our study identifies ß2-AR signaling as an important regulatory pathway for ER-Mito coupling and highlights the role of these contacts in responding to physiological demands or stresses.

Potassium-induced potentiation of subtetanic force in rat skeletal muscles: influences of ß2-activation, lactic acid, and temperature.

Moderate elevations of extracellular K+ concentration ([K+]o) occur during exercise and have been shown to potentiate force during contractions elicited with subtetanic frequencies. Here, we investigated whether lactic acid (reduced chloride conductance), ß2-adrenoceptor activation, and increased temperature would influence the potentiating effect of potassium in slow- and fast-twitch muscles. Isometric contractions were elicited by electrical stimulation at various frequencies in isolated rat soleus and extensor digitorum longus (EDL) muscles incubated at normal (4 mM) or elevated K+, in combination with salbutamol (5 µM), lactic acid (18.1 mM), 9-anthracene-carboxylic acid (9-AC; 25 µM), or increased temperature (30-35°C). Elevating [K+]o from 4 mM to 7 mM (soleus) and 10 mM (EDL) potentiated isometric twitch and subtetanic force while slightly reducing tetanic force. In EDL, salbutamol further augmented twitch force (+27 ± 3%, P < 0.001) and subtetanic force (+22 ± 4%, P < 0.001). In contrast, salbutamol reduced subtetanic force (-28 ± 6%, P < 0.001) in soleus muscles. Lactic acid and 9-AC had no significant effects on isometric force of muscles already exposed to moderate elevations of [K+]o. The potentiating effect of elevated [K+]o was still well maintained at 35°C. Addition of salbutamol exerts a further force-potentiating effect in fast-twitch but not in slow-twitch muscles already potentiated by moderately elevated [K+]o, whereas lactic acid, 9-AC, or increased temperature does not exert any further augmentation. However, the potentiating effect of elevated [K+]o was still maintained in the presence of these, thus emphasizing the positive influence of moderately elevated [K+]o for contractile performance during exercise.