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Oil and gas exploration in the Arctic can result in the release of polycyclic aromatic hydrocarbons (PAHs) into relatively pristine environments. Following the recent spill of approximately 17 500 tonnes of diesel fuel in Norilsk, Russia, May 2020, our study focussed on the effects of phenanthrene, a low molecular weight PAH found in diesel and crude oil, on the isolated atrial and ventricular myocytes from the heart of the polar teleost, the Navaga cod (Eleginus nawaga). Acute exposure to phenanthrene in navaga cardiomyocytes caused significant action potential (AP) prolongation, confirming the proarrhythmic effects of this pollutant. We show AP prolongation was due to potent inhibition of the main repolarising current, IKr, with an IC50 value of ~2 µM. We also show a potent inhibitory effect (~55%) of 1 µM phenanthrene on the transient IKr currents that protects the heart from early-after-depolarizations and arrhythmias. These data, along with more minor effects on inward sodium (INa) (~17% inhibition at 10 µM) and calcium (ICa) (~17% inhibition at 30 µM) currents, and no effects on inward rectifier (IK1 and IKAch) currents, demonstrate the cardiotoxic effects exerted by phenanthrene on the atrium and ventricle of navaga cod. Moreover, we report the first data that we are aware of on the impact of phenanthrene on atrial myocyte function in any fish species. © 2021 The Authors This work is supported by British Heart Foundation Grant PG/17/77/33125 to HAS and SNK and by the Interdisciplinary Scientific and Educational School of Moscow University «Molecular Technologies of the Living Systems and Synthetic Biology» as part of the Scientific Project of the State Order of the Government of Russian Federation to Lomonosov Moscow State University No.121032300071-8 to DVA. We thank Prof Jules Hancox, University of Bristol, UK, and Dr Fabien Brette, Université de Bordeaux for insightful comments. We are grateful to White Sea Biological Station director, Professor Alexander Tzetlin for supporting the study. We thank Valo and Valentina Sivonen for collecting the fish.

Neuronal nitric oxide synthase (nNOS) is considered a regulator of Cav1.2 L-type Ca2+ channels and downstream Ca2+ cycling in the heart. The commonest view is that nitric oxide (NO), generated by nNOS activity in cardiomyocytes, reduces the currents through Cav1.2 channels. This gives rise to a diminished Ca2+ release from the sarcoplasmic reticulum, and finally reduced contractility. Here, we report that nNOS inhibitor substances significantly increase intracellular Ca2+ transients in ventricular cardiomyocytes derived from adult mouse and rat hearts. This is consistent with an inhibitory effect of nNOS/NO activity on Ca2+ cycling and contractility. Whole cell currents through L-type Ca2+ channels in rodent myocytes, on the other hand, were not substantially affected by the application of various NOS inhibitors, or application of a NO donor substance. Moreover, the presence of NO donors had no effect on the single-channel open probability of purified human Cav1.2 channel protein reconstituted in artificial liposomes. These results indicate that nNOS/NO activity does not directly modify Cav1.2 channel function. We conclude that—against the currently prevailing view—basal Cav1.2 channel activity in ventricular cardiomyocytes is not substantially regulated by nNOS activity and NO. Hence, nNOS/NO inhibition of Ca2+ cycling and contractility occurs independently of direct regulation of Cav1.2 channels by NO. Electronic supplementary material The online version of this article (10.1007/s00424-019-02335-7) contains supplementary material, which is available to authorized users.

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

We have produced transgenic mice which overexpress cardiac Na(+)-Ca2+ exchange activity. Overexpression has been assessed by Western blot, Northern blot, and immunofluorescence. Functional overexpression was analyzed using membrane vesicles and isolated ventricular myocytes. In whole cell clamped myocytes dialyzed with 0.1-0.2 mM Fura-2, the magnitude of ICa and Ca2+i-transient triggered by ICa or caffeine were not significantly different in transgenic vs. control myocytes. In transgenic myocytes, activation of ICa, however, was followed by a large slowly inactivating transient inward current representing INa-Ca. This current depended on Ca2+ release as it was abolished when sarcoplasmic reticulum (SR) Ca2+ was depleted using thapsigargin. Cai-transients triggered by rapid application of 5 mM caffeine, even though equivalent in control and transgenic myocytes, activated larger INa-Ca (approximately 5 pA/pF at -90 mV) in transgenic vs. control myocytes (1.5 pA/pF). The decay rate of caffeine-induced Ca2+i-transient and INa-Ca was 2.5 times faster in transgenic than in control myocytes. 5 mM Ni2+ was equally effective in blocking INa-Ca in control or transgenic myocytes. In 9 out of 26 transgenic myocytes, but none of the controls, Ca2+ influx via the exchanger measured at +80 mV caused a slow rise in [Ca2+]i triggering rapid release of Ca2+ from the SR, SR Ca2+ release triggered by the exchanger at such potentials was accompanied by activation of transient current in the inward direction. In 2 mM Fura-2-dialyzed transgenic myocytes caffeine-triggered Cai-transients failed to activate INa-Ca even though the kinetics of inactivation of ICa slowed significantly in caffeine-treated myocytes. In 0.1 mM Fura-2-dialyzed transgenic myocytes 100 microM Cd2+ effectively blocked ICa and suppressed Cai-transients at -10 or +50 mV. Our data suggests that in myocytes overexpressing the exchanger, the content of intracellular Ca2+ pools and the signaling of its release by the Ca2+ channel vis-a-vis the Na(+)-Ca2+ exchanger were not significantly altered despite an up to ninefold increase in the exchanger activity. We conclude that the exchanger remains functionally excluded from the Ca2+ microdomains surrounding the DHP/ryanodine receptor complex.

Estrogenic and androgenic steroids can be synthesised in the brain and rapidly modulate synaptic transmission and plasticity through direct interaction with membrane receptors for estrogens (ERs) and androgens (ARs). We used whole cell patch clamp recordings in brainstem slices of male rats to explore the influence of ER and AR activation and local synthesis of 17β-estradiol (E2) and 5α-dihydrotestosterone (DHT) on the long-term synaptic changes induced in the neurons of the medial vestibular nucleus (MVN). Long-term depression (LTD) and long-term potentiation (LTP) caused by different patterns of high frequency stimulation (HFS) of the primary vestibular afferents were assayed under the blockade of ARs and ERs or in the presence of inhibitors for enzymes synthesizing DHT (5α-reductase) and E2 (P450-aromatase) from testosterone (T). We found that LTD is mediated by interaction of locally produced androgens with ARs and LTP by interaction of locally synthesized E2 with ERs. In fact, the AR block with flutamide prevented LTD while did not affect LTP, and the blockade of ERs with ICI 182,780 abolished LTP without influencing LTD. Moreover, the block of P450-aromatase with letrozole not only prevented the LTP induction, but inverted LTP into LTD. This LTD is likely due to the local activation of androgens, since it was abolished under blockade of ARs. Conversely, LTD was still induced in the presence of finasteride the inhibitor of 5α-reductase demonstrating that T is able to activate ARs and induce LTD even when DHT is not synthesized. This study demonstrates a key and opposite role of sex neurosteroids in the long-term synaptic changes of the MVN with a specific role of T-DHT for LTD and of E2 for LTP. Moreover, it suggests that different stimulation patterns can lead to LTD or LTP by specifically activating the enzymes involved in the synthesis of androgenic or estrogenic neurosteroids.