Resting cardiac sympathetic firing frequencies suppress terminal norepinephrine transporter uptake

https://doi.org/10.1016/j.autneu.2021.102794Get rights and content

Highlights

  • Cardiac single-terminal NET rate is rapidly suppressed by sympathetic nerve activity in a frequency-dependent manner

  • At low levels of resting sympathetic activity, NET is suppressed by prejunctional α2-adrenoceptors

  • Substrate competition at the transporter contributes to NET suppression at high levels of sympathetic activity

Abstract

The prejunctional norepinephrine transporter (NET) is responsible for the clearance of released norepinephrine (NE) back into the sympathetic nerve terminal. NET regulation must be tightly controlled as variations could have important implications for neurotransmission. Thus far, the effects of sympathetic neuronal activity on NET function have been unclear. Here, we optically monitor single-terminal cardiac NET activity ex vivo in response to a broad range of sympathetic postganglionic action potential (AP) firing frequencies.

Isolated murine left atrial appendages were loaded with a fluorescent NET substrate [Neurotransmitter Transporter Uptake Assay (NTUA)] and imaged with confocal microscopy. Sympathetic APs were induced with electrical field stimulation at 0.2–10 Hz (0.1–0.2 ms pulse width). Exogenous NE was applied during the NTUA uptake- and washout phases to investigate substrate competition and displacement, respectively, on transport.

Single-terminal NET reuptake rate was rapidly suppressed in a frequency-dependent manner with an inhibitory EF50 of 0.9 Hz. At 2 Hz, the effect was reversed by the α2-adrenoceptor antagonist yohimbine (1 μM) (p < 0.01) with no further effect imposed by the muscarinic receptor antagonist atropine (1 μM). Additionally, high exogenous NE concentrations abolished NET reuptake (1 μM NE; p < 0.0001) and displaced terminal specific NTUA during washout (1–100 μM NE; p < 0.0001). We have also identified α2-adrenoceptor-induced suppression of NET reuptake rate during resting stimulation frequencies, which could oppose the effect of autoinhibition-mediated suppression of exocytosis and thus amplify the effects of sympathetic drive on cardiac function.

Introduction

The Na+/Cl-dependent norepinephrine transporter (NET), located on the presynaptic membrane of sympathetic nerve terminals, is responsible for the high-affinity reuptake of released norepinephrine (NE) back into the nerve ending [termed uptake1; Iversen, 1963]. In the periphery, prejunctional NET is crucial for cardiovascular homeostasis as it represents up to 90% of neurotransmitter clearance in the heart (Goldstein et al., 1988; Esler et al., 1990) and is therefore an important determinant of noradrenergic volume transmission. Unsurprisingly, disruptions in NET function are often associated with sympathetic overactivity observed in cardiovascular pathologies such as hypertension, tachycardia, orthostatic intolerance (Schroeder and Jordan, 2012) and heart failure (Kaludercic et al., 2010; Liang et al., 1989).

Many factors influence NET function, including endogenous hormones (Apparsundaram et al., 1998; Mandela and Ordway, 2006b) and psychotropic drugs (Zhou, 2004; Weinshenker and Schroeder, 2007). Governed by kinases and phosphatases, they elicit changes in transporter trafficking, activity and/or expression either independently or in combination (Mandela and Ordway, 2006b). However, the effect of sympathetic nerve activity upon NET in intact tissue preparations remains unclear.

While the increase in NET function induced by membrane depolarisation is well established in in vitro systems (Savchenko et al., 2003; Mandela and Ordway, 2006a; Habecker et al., 2006; Sung et al., 2017), the findings ex/in vivo remains uncertain. Gillis (1963) demonstrated that, in perfused cat hearts, acute electrical stimulation of sympathetic nerve fibres increased neuronal NE retention in the atria. Corroborated findings were reported later by Chang and Chiueh (1969) in the rat submaxillary gland and Ungerer et al. (1996) in the rat heart. Contrastingly, depolarisation has been shown to reduce neuronal NE clearance in the cat spleen (Blakeley and Brown, 1964), guinea-pig vas deferens (Palaić and Panisset, 1969) and rat iris and salivary glands (Häggendal and Malmfors, 1969), with others reporting either no effect (Kirpekar and Wakade, 1968; Yamamoto and Kirpekar, 1972) or were left uncertain (Cervoni and Kirpekar, 1966). In those studies, NET function was assessed by measuring the total uptake of tritiated NE in homogenised tissue or metabolites in the perfusion effluent. However, some tissues also possess a non-neuronal mode of NE accumulation, termed uptake2, which is well-explored in the rat heart (Iversen, 1965) and present in other tissues and species (Almgren and Jonason, 1974; Major et al., 1978). As the studies of neurone depolarisation on NET generally did not utilise uptake2 inhibitors, observational differences could be explained by the additive effects of uptake1 and uptake2, as well as interspecies/inter-tissue differences in uptake2 kinetics in response to membrane depolarisation (Bönisch et al., 1985). For this reason, it is of particular importance to utilise a technique to study action potential (AP)-evoked NET regulation exclusively at the site of release.

So far, studies of NET function in real-time have been restricted to whole cell patch clamp recordings of transporter-induce currents (Sung et al., 2017), though it would be technically challenging to achieve from axon terminals in intact tissues ex/in vivo, particularly in the heart where there is a mixture of peripheral neuronal types as well as the repertoire of non-neuronal cell types (Shivkumar and Ardell, 2016). Recently, we developed a fluorescence-based optical technique that can discern individual noradrenergic terminals in cardiovascular tissue and dynamically monitor their reuptake function ex vivo in response to functional manipulations (Cao et al., 2020).

In the current study, we utilise a fluorescent technique to investigate the effects of a broad range of acute sympathetic firing frequencies on single-terminal cardiac NET reuptake rate. Furthermore, we also examined the influences of prejunctional α2-adrenergic autoreceptors and muscarinic heteroreceptors during nerve stimulation, as well as the contribution of NET substrate competition during uptake and displacement through the transporter.

Section snippets

Ethics approval

All animal experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and the ethics guidelines set out by the Animal Welfare and Ethical Review Body (AWERB) at the University of Birmingham. Procedures were performed under an approved UK Home Office license (PPL PFDAAF77F). Animals were housed in individually ventilated cages (n = 4 maximum per cage) in the Biomedical Services Unit at the University of Birmingham under standard conditions: 12:12 h light/dark

Sympathetic postganglionic APs suppress single-terminal NET reuptake rate

Exposure of the murine LAA to the fluorescent NET substrate, NTUA, revealed networks of fluorescent varicose noradrenergic nerve terminals (Fig. 1A). Relative changes in fluorescence during the fluorophore accumulation with time provides a measure of NET reuptake rate (Cao et al., 2020). NTUA was superfused concomitantly with EFS delivery for 6 min (Fig. 1B). In the absence of EFS [no stimulation (NS)], the accumulation of terminal-specific fluorescence increased in a linear fashion throughout

Main findings

We demonstrate here for the first time that acute sympathetic nerve activity at physiological firing rates can rapidly suppresses cardiac NET reuptake rate ex vivo at the spatiotemporal resolution of single noradrenergic nerve terminals. By directly measuring for changes in NET rate at the neuroeffector site in intact preparations, the optical technique utilised in this study provides new insight into dynamic mechanisms of transporter regulation and thus sympathetic volume transmission.

We also

Conclusion

In summary, this study utilised an optical fluorescence-based technique to detect for a frequency-dependent suppression of NET reuptake rate during physiological sympathetic firing activity. Using this technique, we overcome the methodological limitations associated with previous studies of membrane depolarisation and NET, which provide new insights into the regulation of transporter function at the site of release. We also uncover a role for the prejunctional α2AR to suppress NE clearance

Data availability statement

Data available on request from the authors.

CRediT authorship contribution statement

Experimental concepts and design were devised by LLC and KLB. Data was collected by LLC. LLC analysed the data. LLC, JMM, LF and KLB interpreted the data. Original drafting of the manuscript was performed by LLC and was edited by all authors.

Declaration of competing interest

None declared.

Acknowledgements

We would like thank Dr. Andrew Holmes (University of Birmingham) for his critical input and revision of the manuscript.

Funding received

this work was supported by the British Heart Foundation studentship [grant number FS/17/7/32651 to LLC, JMM, LF, KLB] and the British Heart Foundation Accelerator Award [AA/18/2/34218 to ICVS (LF)].

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