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  • agonists simulate norepinephrine NE in binding to

    2024-02-07

    α2 agonists simulate norepinephrine (NE) in binding to presynaptic surface autoreceptors, which in turn mediates feedback inhibition of NE release. Another major control mechanism for noradrenergic neurotransmission is termination of signaling by presynaptic NE transporter (NET)-mediated NE reuptake [1], [14], [23]. The pivotal role of NET in fine-tuning of noradrenergic neurotransmission provides an opportunity for pharmaceutical interventions for such disorders as depression, attention-deficit hyperactivity disorder, and emotional disturbances. NET function also underlies the basis for diagnostic imaging with radiolabeled metaiodobenzylguanidine (MIBG), widely used for evaluation of neuroendocrine tumors [12], [21]. Thus, a better understanding of mechanisms that modulate NET function is important for the development of newer psychoactive compounds as well as for optimizing clinical MIBG imaging. The equivalent actions of α2 agonists and NE on α2 adrenergic receptors raise the question whether NET may also be a target for α2 agonists. Interestingly, several injectable anesthetic agents have been observed to cause suppression of NE reuptake [8], [9], [15], [17], [24]. Furthermore, there have been a couple of previous observations suggesting that such α2 agonists as xyalzine and clonidine may suppress MIBG uptake in neuroblastoma cells. Babich et al. evaluated the effects of various adrenergic receptor ligands on MIBG uptake in SK-N-SH Dihydro-β-erythroidine hydrobromide synthesis and found xyalzine and clonidine to be included among agents with an inhibitory effect [2]. Our group also previously observed a reduction of MIBG uptake in SK-N-SH cells by xylazine while evaluating the effects of anesthetic agents on cellular transport and in vivo biodistribution of MIBG [11]. However, neither of these previous studies attempted to investigate the mechanism underlying this effect. In this study, we thus hypothesized that α2 agonists can target NET to influence NE reuptake, and further investigated the mechanisms that mediate the response.
    Materials and methods
    Results
    Discussion Our study reveals that α2 adrenergic agonists including xylazine and dexmedetomidine acutely inhibit MIBG uptake in a dose-dependent manner. Our findings further suggest their direct interaction with NET in a manner that interferes with substrate transport function as the likely mechanism for the observed effect. After we showed several different α2 agonists to inhibit MIBG uptake, we concentrated further experiments on a few representative compounds based on their clinical applicability. Clonidine is the prototypical α2 agonist that is used to treat hypertension and occasionally used to reduce anesthetic requirements [6], [10]. Xylazine is widely used in veterinary medicine and preclinical studies for sedation, analgesia, and anesthesia of animals including laboratory mice [13]. Dexmedetomidine is a novel superselective α2 agonist widely used for sedation of critically ill or injured patients in an intensive care unit setting or as an adjunct for sedation and anesthesia in certain operations and invasive medical procedures [3]. Our results show that clonidine and xylazine effectively suppressed MIBG transport, while an even more potent inhibitory effect was observed for dexmedetomidine. Although the half-inhibitory concentrations of MIBG transport observed for these drug in our study exceeds the plasma concentrations obtained by usual anesthetic doses of the drugs [16], [22], it is comparable to or even lower than the reported concentrations required to achieve neuroprotection. Indeed, studies investigating this effect have used doses up to 2 orders of magnitude above that for anesthetic purposes [7], [18], [19], [20]. Compounds that inhibit NE reuptake either act as a substrate competitor or interfere with NET function by directly binding [23]. We first checked the reversibility of the inhibitory effect of xylazine by removing the drug from culture media following cell exposure, and found a complete restoration of NET function. When we pretreated the cells with the selective α2 antagonist yohimbine, no blocking effect on α2 agonist-induced suppression of MIBG uptake was observed. Immunoblots revealed that xylazine caused no change in total cellular or plasma membrane NET content. Furthermore, saturation kinetics demonstrated xylazine and dexmedetomidine to substantially elevate Km values for MIBG transport without significantly affecting Vmax values. Collectively, these findings point to reduced substrate affinity by competitive inhibition without change in plasma membrane transporter number as the mechanism through which α2 agonists suppress NET function.