Bosche et al. - A differential impact of lithium on endothelium-dependent but not on...

Progress in Neuro-Psychopharmacology & Biological Psychiatry 67 (2016) 98–106

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A differential impact of lithium on endothelium-dependent but not onendothelium-independent vessel relaxation

Bert Bosche a,b,⁎, Marek Molcanyi c, Thomas Noll d, Soham Rej e,f, Birgit Zatschler d, Thorsten R. Doeppner b,Jürgen Hescheler c, Daniel J. Müller g, R. Loch Macdonald a, Frauke V. Härtel d

a Division of Neurosurgery, St Michael's Hospital, Keenan Research Centre for Biomedical Science and the Li Ka Shing Knowledge Institute of St. Michael's Hospital, Department of Surgery,University of Toronto, Toronto, Ontario, Canadab Department of Neurology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germanyc Institute of Neurophysiology, Center of Physiology and Pathophysiology, University of Cologne, Cologne, Germanyd Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University of Dresden, Dresden, Germanye Division of Geriatric Psychiatry, Department of Psychiatry, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario, Canadaf Department of Psychiatry, Geri-PARTy Research Group, Jewish General Hospital, McGill University, Montréal, Québec, Canadag Pharmacogenetics Research Clinic, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health & Department of Psychiatry, University of Toronto,Toronto, Ontario, Canada

Abbreviations: ACH, acetylcholine; BD, bipolar disordereticulum; GSK-3β, glycogen synthase kinase-3 beta; IMPIP3, inositol trisphosphate; L-NMMA, L-NG-monomethylLiAc, lithiumacetate;MCA,middle cerebral artery;NOS, nierror of the mean; SNP, sodium nitroprusside; XeD, Xesto⁎ Corresponding author at: St Michael's Hospital,

Biomedical Science and the Li Ka Shing Knowledge Instituthe University of Toronto, 209 Victoria St., Toronto, Ontar

E-mail addresses: [email protected], BoscheB@s

http://dx.doi.org/10.1016/j.pnpbp.2016.02.0040278-5846/© 2016 Elsevier Inc. All rights reserved.

a b s t r a c t

Article history:Received 6 January 2016Received in revised form 6 February 2016Accepted 9 February 2016Available online 10 February 2016

Lithium is drug for bipolar disorders with a narrow therapeutic window. Lithium was recently reported to pre-vent stroke and protect vascular endothelium but tends to accumulate particularly in the brain and kidney.Here, adverse effects are common; however mechanisms are still vaguely understood. If lithium could also neg-atively influence the endothelium is unclear.Wehypothesize that at higher lithium levels, the effects on endothe-lium reverses — that lithium also impairs endothelial-dependent relaxation of blood vessels. Vessel grafts fromde-nerved murine aortas and porcine middle cerebral arteries were preconditioned using media supplementedwith lithium chloride or acetate (0.4–100mmol/L). Native or following phenylephrine-induced vasoconstriction,the relaxation capacity of preconditioned vessels was assessed by isometric myography, using acetylcholine totest the endothelium-dependent or sodium nitroprusside to test the endothelium-independent vasorelaxation,respectively. At the 0.4 mmol/L lithium concentration, acetylcholine-induced endothelium-dependent vessel re-laxation was slightly increased, however, diminished in a concentration-dependent manner in vessel graftspreconditioned with lithium at higher therapeutic and supratherapeutic concentrations (0.8–100 mmol/L). Incontrast, endothelium-independent vasorelaxation remained unaltered in preconditioned vessel grafts at anylithium concentration tested. Lithium elicits opposing effects on endothelial functions representing a differentialimpact on the endothelium within the narrow therapeutic window. Lithium accumulation or overdose reducesendothelium-dependent but not endothelium-independent vasorelaxation. The differentiallymodified endothe-lium-dependent vascular response represents an additional mechanism contributing to therapeutic or adverseeffects of lithium.

© 2016 Elsevier Inc. All rights reserved.

Keywords:LithiumAdverse effects of lithiumBipolar disorderStrokeEndotheliumVascular relaxationVascular autoregulationCerebrovascular autoregulationEndothelial function

1. Introduction

Lithium is a highly effective treatment for bipolar disorders (BD) andan adjuvant treatment for major depression with a narrow therapeuticwindow (Calkin and Alda, 2012; Geddes and Miklowitz, 2013;

rs; ER, endothelial endoplasmicase, inositol monophosphatase;arginine; LiCl, lithium chloride;tric oxide synthase; SE, standardspongin D.Keenan Research Centre forte of St. Michael's Hospital andio M5C 1N8, Canada.mh.ca (B. Bosche).

Mohammad andOsser, 2014). Recent studies have also identified sever-al potential protective effects of lithium inmany other neuropsychiatricand somatic diseases, including cerebrovascular disease (Gold et al.,2011; Lan et al., 2015), dementia (Gerhard et al., 2015), suicidality(Saunders and Hawton, 2013), diabetes mellitus (Svendal et al., 2012),and endothelial dysfunction (Bosche et al., 2013). In neuronal cells, lith-ium appears to primarily mediates its action, directly and indirectly,through inositol monophosphatase (IMPase), intracellular calcium con-centration (Berridge, 1989, 1993;Wasserman et al., 2004; Li et al., 2012;Berridge, 2014) and the glycogen synthase kinase-3 beta (GSK-3β) en-zyme,which in turnmay control a variety of intracellular effectormech-anisms (Gould andManji, 2005; Trepiccione and Christensen, 2010; Rejet al., 2015b). Interestingly, emerging research suggests that lithiumelicits similar intracellular signalingmechanisms in vascular endothelial

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cells, too (Ryglewski et al., 2007; Munaron and Fiorio Pla, 2009). There-by, the endothelium-protective effects of lithium may be important incerebrovascular disease, traumatic brain injury with disturbed vascularregulation (Rajkowska, 2000; Bosche et al., 2003; Dohmen and Boscheet al., 2007; Gold et al., 2011; Halcomb et al., 2013; Leeds et al., 2014),and even bipolar disorder (Goldstein and Young, 2013). In endothelialcells, lithium prevents the discharge of calcium from endogenous storesby inhibition of the inositol trisphosphate (IP3)-sensitive calcium chan-nels of the endothelial endoplasmic reticulum (ER) (Schäfer et al.,2001), thus, counteracting cells stress-induced calcium overload and con-ferring lithium a cytoprotective potential (Bosche et al., 2013), possiblythrough inhibition of GSK-3β (Rej et al., 2015b) and/or IMPase (Chiuand Chuang, 2010; Dutta et al., 2014). Functionally, maintenance of intra-cellular calcium homeostasis together with other lithium effects manifestas modified endothelium-mediated vasodilation (Förstermann andMünzel, 2006), a potential marker of preserved or improved endothelialhealth (Yoo and Kim, 2009; Grove et al., 2015).

In long-term or at supratherapeutic levels, i.e. above the generallyrecommended concentrations in humans (Rej et al., 2015a), lithiumcan impair particularly kidney and brain function (Laliberté et al.,2015), where it tends to accumulate (Lichtinger et al., 2013;Johnson, 1998). Historically, lithium toxicity has been linked to in-hibition of the GSK-3β or inositol monophosphate pathway leadingto disturbed cellular metabolism (Rybakowski et al., 2013;Trepiccione and Christensen, 2010) On the other hand, lithium ac-cumulation or toxicity may also be related to vascular hemodynam-ic abnormalities (Schou et al., 1968; Laliberté et al., 2015). Onecould postulate that the effect of lithium could be harmful to endo-thelium at high doses and impairs vasodilation, which may contrib-ute to tissue specific toxicity, e.g. in the brain and kidney (Lichtingeret al., 2013; Johnson, 1998).

How can lithium be protective to blood vessel endothelium at lowtherapeutic doses, but be harmful to blood vessels at higher orsupratherapeutic doses? We hypothesize that at high levels, the effectof lithium on endothelium reverses — that lithium impairs endothelium-dependent relaxation of blood vessels. To validate this hypothesis, anestablished approach was applied to study vascular function in vesselgrafts (Ebner et al., 2011; Mulvany and Halpern, 1977; Kopaliani et al.,2014), which recently modified and translated also for human use(Wilbring et al., 2013).

Yet, to our knowledge, the data presented here uniquely suggestedthat supratherapeutic and higher therapeutic, but not lower therapeuticlithium levels impair particularly endothelium-dependent vascular re-laxation underlining the concept of opposite effects of lithium at differ-ent therapeutic concentrations.

2. Material and methods

2.1. Ethics of the animal model

This experimental study was approved by the University Commis-sion on Animal Experiments with respect to the animal welfare regula-tions of Germany, in accordance to the European Communities CouncilDirective and to the National Institutes of Health (NIH) Guidelines. Forperforming the study, written permission was obtained from localauthorities.

2.2. Materials and drugs

All materials, reagents and drugs are described when mentionedwithin their respective method sub-sections (see below).

2.3. Cold storage solutions

The used cold storage Tiprotec™ solution (Dr. F. Köhler GmbH,Bensheim, Germany) contains the following substance concentrations

(all are given inmmol/L):α-ketoglutarate 2, aspartate 5, N-acetyl-histi-dine 30, glycine 10, alanine 5, tryptophan 2, sucrose 20, glucose 10, Cl−:103.1, H2PO4: 1, Na+: 16, K+: 93, Mg2+: 8, Ca2+: 0.05, deferoxamine:0.082 and LK 614: 0.017. The pH (at 20 °C) was 7.0 and the osmolaritywas 305mosmol/L. This standard solution served as the control precon-ditioning or was supplemented with the different lithium chloride(LiCl) or lithium acetate (LiAc) concentrations.

2.4. Murine and porcine vessel preparation

The vessel grafts were isolated from murine aortas or from porcinemiddle cerebral arteries (MCA). Vessel preparation was performed ac-cording to the slightly modified method for rodents as described in de-tail elsewhere (Ebner et al., 2011; Wilbring et al., 2013; Kopaliani et al.,2014). In brief, male CD57mice 8 to 10weeks of age (Charles River Lab-oratories, Sulzfeld, Germany) were sacrificed by cutting off the uppercervical spinal cord under deep anesthesia. Male swine (Sus scrofadomesticus, 24 to 26 weeks of age) were stunned by electroshock andsacrificed by exsanguination. All mice and swine were dissected imme-diately. The murine aortas (pars thoracalis without aortic arch) or theproximal part (M1 segment) of porcine middle cerebral arteries wererecovered and directly placed into a storage solution containing either1) Tiprotec™ solution only (Dr F. Köhler GmbH, Bensheim, Germany)or 2) modified Tiprotec™ solution supplemented with 0.4 to100 mmol/L lithium (LiCl/LiAc, Sigma-Aldrich, Taufkirchen, Germany)and stored at 4 °C for ≥48 hours (h).

2.5. Post-mortem model of vascular tone control

The post-mortem model with de-nerved vessel grafts was per-formed to investigate the isolated vessel reaction in response to differ-ent lithium concentrations independently of the influence of lithiumon the central and thereby also on the vegetative nerve system includ-ing its remote control of the vessel tone.

2.6. Type of vessel

The thoracic aorta (pars descendens) was chosen as the used vesseltype, because a) it is an elastic type artery containing both the ordinaryvascular smooth muscle cells (SMC) and the myointimal SMC in a rela-tively high number; moreover, because b) aortic endothelial cells wereused in our previous vessel graft and cell culture studies regarding cyto-solic [Ca2+] measurements after long-term and immediate use of lithi-um and its influence on the specific type of endothelial cells takenfrom the aorta (Schäfer et al., 2001; Bosche et al., 2013). Vessel graftsof the MCA (M1 segment) from porcine brains were additionally usedto investigate the influence of lithium on cerebral endothelium-depen-dent vasorelaxation.

2.7. Isometric force measurement

After cold storage aortic or cerebral vessel grafts (pipes or long rings,2 mm in length, 500–600 μm or 1150–1400 μm internal width, respec-tively) were transferred to a 90%/10% (vol/vol) mixture of phosphate-buffered saline (PBS) solution and Hank's balanced salt solution(HBSS, Sigma-Aldrich, Taufkirchen, Germany) gently warmed to37 °C/98.6 °F over 30 min. Then, the vessel grafts were studied,stretchedwith a resting tension equivalent to that obtained by exposureto an intraluminal pressure of 20 mmHg for maximal responses. Vesselringswere equilibrated for 10min prior to vasomotor analysis.Maximalcontraction was induced by exposure of vessel rings to a potassium-enriched solution (123.7 mmol/L KCl) and/or to the application of10 μmol/L phenylephrine (see below Vasoactive Agents). Vessel relaxa-tion toward acetylcholine (ACH; 10−8.5 to 10−5.5 mol/L) or sodium ni-troprusside (SNP; 10−8.5 to 10−5.5 mol/L) was tested after a plateauconstriction induced by 10 μmol/L phenylephrine to assess

Fig. 1. Effect of lithium-preconditioning at supratherapeutic concentrations on endothelium-dependent and endothelium-independent vasorelaxation. Vessel graft ofmurine aortaswerepreconditioned with Tiprotec™ solution alone as control (A), or Tiprotec™ supplementedwith 20 mM LiCl (B) or 100 mM LiCl (C) for 48 h. Afterwards the vessels wereprecontracted by phenylephrine (10 μmol/L) and the capacity of endothelium-dependentand endothelium-independent vasorelaxation was tested by additions of equal amounts of10−8.5, 10−7 or 10−5.5 M acetylcholine (ACH, red record line) or sodium nitroprusside(SNP, blue record line), respectively. Representative recordings of isometric forcemeasurements of n = 7 per group are given. Horizontal bars indicate recording time of2 min, vertical bars force development of 1 mN, horizontal bars 2 min, respectively.

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endothelium-dependent and endothelium-independent relaxations inaortic vessel grafts, respectively. Since middle cerebral arteries do notconstrict in response to the α1-adrenoceptor agonist phenylephrinethe vessel grafts were studied under native conditions at resting tensionequivalent to an intraluminal pressure of 20mmHg. For the analyses ofthe influence of the different lithium preconditions, we focused on thefollowing ACH and/or SNP additions: a) none, b) 10−8.5 mol/L,c) 10−7 mol/L) causing approximately half and d) 10−5.5 mol/L causingthe most pronounced vessel relaxation found in pilot experiments.

We measured vessels' isometric forces to constrict and relax using acommercially available myograph (DMT-610M, Power Laboratory/400;AD-Instruments, Spechbach, Germany). The experiments were conduct-ed according to thewell-established and slightlymodifiedmethod usinga Halpern–Mulvany-myograph (Mulvany and Halpern, 1977; Ebneret al., 2011; Wilbring et al., 2013; Kopaliani et al., 2014). For data acqui-sition, the tissue bath system 700MO™ was used with a PowerLab DataAcquisition System™; and data recording was performed withLabChart™ software (AD-Instruments, Spechbach, Germany).

2.8. Vasoactive agents

Phenylephrine (Sigma-Aldrich) was used to induce smoothmuscle cell (SMC)-mediated vasoconstriction. Acetylcholine(Sigma-Aldrich) was used to stimulate the endothelial NO produc-tion and thereby provoke endothelium-dependent vasodilatation.Sodium nitroprusside (Sigma-Aldrich) was applied to induce endo-thelium-independent vasodilatation by directly decreasing the vas-cular SMC tone.

2.9. Inhibition of nitric oxide synthase (NOS)

In some experiments, L-NG-monomethyl arginine (L-NMMA;Sigma-Aldrich, Germany), a potent inhibitor of the endothelial nitricoxide synthase (NOS)was used to inhibit the NOS before adding the va-soactive drugs. In brief, we used L-NMMA in a concentration of300 μmol/L to block the endothelium-dependent relaxation of the ves-sel grafts, aiming to proof whether this specific type of vessel relaxationis NOS mediated or independent of endothelial NOS activity.

2.10. Vessel denudation (removal of endothelium)

In another bench of experiments, the endothelium was denudatedfrom the vessel grafts according to the method previous described(Wilbring et al., 2013; Kopaliani et al., 2014). In short, denudation ofthe endothelium is a method to carefully remove endothelium fromthe basal membrane of vessels. We somewhat modified the methodusing a suture string for removing the endothelium. Denudation of theendothelium aimed to totally block the endothelium-dependent partof the vessel relaxation, or to independently investigate the vessel be-havior without endothelium.

2.11. Inhibition of IP3-sensitive Ca2+-release channel of the endothelialendoplasmic reticulum

For blocking of the IP3 sensitive Ca2+ release channel of the ER(Schäfer et al., 2001) 3 μM Xestospongin D (XeD, Calbiochem —Merck Millipore, Darmstadt, Germany) was added to the vesselgrafts. Immediately and 30 min after XeD addition ACH and SNP in-duced vessel relaxation was myographically measured after maxi-mal contraction.

2.12. Statistical analyses

Normality and variance equality was assessed using Kolmogorov–Smirnov- and Levene-tests. Normal distribution was found in all vari-ables and groups. Results are expressed as mean ± SE. Intergroup

differences were analyzed using one-way analysis of variance withpost-hoc multiple comparisons, or independent-sample t-test. p b 0.05was chosen as level of significance. Data analyses were performedusing GraphPad Prism 6 and IBM SPSS (IBM, Chicago, IL, USA).

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3. Results

3.1. Influence of lithium on endothelium-dependent vs.endothelium-independent vessel relaxation

The influence of lithium on twomodes of vascular response, the en-dothelium-dependent and endothelium-independent relaxation, wasstudied in vessel grafts exposed to ACH or SNP at concentration givenin Fig. 1A–C. In vessel grafts preconditioned with 20 or 100 mmol/LLiCl for 48 h (Fig. 1B and C), ACH-induced vasorelaxation became re-duced,whereas the effect of SNP on the vascular tonewas left unaltered,indicating that lithium affects the endothelium-dependent but not theendothelium-independent vascular response. In line with that, ACH aswell as SNP led to a profound equivalent relaxation in non-preconditioned vessel grafts (Fig. 1A).

In vessel grafts preconditioned with 20 mmol/L LiCl the direct com-parison of ACH- vs. SNP-induced vasorelaxation (Fig. 2) revealed a sig-nificant difference between the endothelium-dependent and -independent vasorelaxation at all ACH and SNP concentrations tested.Preconditioning with LiCl concentrations N20 mmol/L enlarged the sig-nificant difference between both modes of vasorelaxation (data notshown) reaching its maximum at a concentration of 100 mmol/L LiCl(Fig. 3A). In contrast, preconditioning at that high LiCl concentrationdid not affect SNP-induced reduction of the vascular tone (Fig. 3B), indi-cating that the discrepancy between ACH- and SNP-induced vasorelax-ation in lithium preconditioned vessel grafts is due to a loss ofendothelium-mediated vascular function.

3.2. Effects of lithium on vascular function seem to involve IP3-sensitivecalcium discharge of the ER and NOS activity in endothelium

To prove the concept that LiCl affects vascular function by targetingthe endothelial cells of the intima but not cells of the media, vesselswere denudated from endothelial cells by a classical mechanical ma-neuver. After denudation, ACH failed to induce vasorelaxation in LiCl-preconditioned as well as in non-preconditioned vessel grafts(Fig. 3C). In contrast, denudation did not change the SNP-induced vesselrelaxation at all (Fig. 3D), suggesting that lithium precondition targetsendothelium-dependent but not endothelium-independent vascularfunction. In line with that, L-NG-monomethyl arginine (L-NMMA,300 μmol/L), a potent inhibitor of the endothelial nitric oxide synthase(NOS) and, therefore, of endothelium-dependent vasorelaxation,abolished the relaxation induced by 10−5.5 mol/L ACH of both lithium-

Fig. 2. Comparisons of endothelium-dependent and endothelium-independent relaxation of(20 mM). Vessels were precontracted by phenylephrine (10 μmol/L) and vasorelaxationequivalent concentrations of (A) 10−8.5, (B) 10−7 or (C) 10−5.5 mol/L, respectively. Data of n =

preconditioned andnon-preconditioned vessel grafts (Fig. 3E).WhereasL-NMMA did not affect endothelium-independent vessel relaxation in-duced by an equivalent concentration of SNP (10−5.5 mol/L) (107.9 ±7.2% vs. 109.3 ± 6.1%, n = 7 per group, not significantly different).Xestospongin D (XeD) is a reversible inhibitor of the IP3-sensitiveCa2+-release channel of the ER. Exposure to 3 μmol/L XeD for 30 mindid not affect the vascular tone of the phenylephrine precontracted ves-sel grafts. However, it totally blocked the ACH-induced vasorelaxation,but not the SNP-induced one (Fig. 3F). The similar responds to XeD orLiCl preconditioning suggested that the impact of lithium on endotheli-um-mediated vasorelaxation is also reliant on inhibition of the IP3-sen-sitive Ca2+-release of the ER.

3.3. Preconditioning with supratherapeutic lithium levels causes aconcentration-dependent reduction of endothelium-dependentvasorelaxation

In a set of experiments, vessel grafts were preconditioned withsupratherapeutic LiCl concentrations of 10, 30 and 50 mmol/L. Subse-quently, the vesselswere precontractedwith phenylephrine and the ca-pacity of vasorelaxation in response to ACHwas determined. As shownin Fig. 4A the most pronounced differences in vasorelaxation were ob-served at an ACH concentration of 10−7 mol/L. At that concentration,a trend to reduced endothelium-dependent vasorelaxation was alreadyfound in vessel grafts preconditioned at lower supratherapeutic LiClconcentrations (10 mmol/L). At higher LiCl concentrations (30 and50 mmol/L) ACH-induced vasorelaxation was reduced in concentra-tion-dependent manner (Fig. 4A). To rule out a chloride ion effect, lith-ium acetate was used for preconditioning. A similar concentration-dependent reduction of endothelium-dependent vasorelaxation wasfound in preconditioned vessel grafts with 20 and 100 mmol/L LiAc(Fig. 4B).

3.4. Opposing effects of lithium at low vs. high therapeutic levels onendothelium-dependent vessel relaxation

Preconditioning of vessel grafts with LiCl at a therapeutic concentra-tion of 0.4 mmol/L slightly enhanced endothelium-dependent vasore-laxation compared to non-preconditioned controls (Fig. 5). However,preconditioning of the vessel grafts with LiCl at higher, but still thera-peutic concentration (0.8 mM), reduced the endothelium-dependentvasorelaxation, which had already been observed at supratherapeuticlithium concentrations; though, the opposing effect of 0.4 mmol/L

murine vessel grafts preconditions at supratherapeutic lithium chloride concentrationcapacity was tested by acetylcholine (ACH) vs. sodium nitroprusside (SNP) added in7 independent vessel grafts per group are given, *p b 0.05.

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Fig. 4. Effect of lithium preconditioning at supratherapeutic concentrations onendothelium-dependent vasorelaxation. (A) Murine vessel grafts were preconditionedat 10, 30 and 50 mmol/L lithium chloride for 48 h. Afterwards the vessels wereprecontracted by phenylephrine (10 μmol/L) and vasorelaxation capacity was tested byacetylcholine (ACH). Data of n = 10–11 vessel grafts of independent preparations aregiven. (B) Vessel grafts were treated as described in (A), however, preconditioned with20 or 100 mmol/L lithium acetate. Data of n = 4 vessel grafts of independentpreparations are given, *p b 0.05 compared to control.

Fig. 5. Effect of lithium preconditioning at therapeutic concentrations on endothelium-dependent vasorelaxation. Murine vessel grafts were preconditions at 0.4 or 0.8 mmol/Llithium chloride (LiCl) for 48 h. Afterwards the vessels were precontracted byphenylephrine (10 μmol/L) and vasorelaxation capacity was tested by acetylcholine(ACH). Data of n = 3–4 vessel grafts of independent preparations are given, #p b 0.05,0.4 vs. 0.8 mmol/L LiCl.

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lithium was significantly higher compared to 0.8 mmol/L lithium chlo-ride. Repeating these experiments using 0.4 and 0.8 mmol/L LiAc con-firmed the results of LiCl (at 10−7 M ACH, n = 4, p b 0.05, 0.4 vs.

Fig. 3. Impact of supratherapeutic lithiumconcentrations on endothelial function. Comparison o100 mmol/L) or non-preconditioned (control) murine vessel grafts. (A) ACH (10−5.5 mol/L) orvasorelaxation were determined after 1 min. (C) and (D) comparisons under identical experimvessel grafts. Dashed lines represent the controls in (A) and (B) without denudation, respectiveNG-monomethyl arginine (L-NMMA, 300 μmol/L), a pan-specific inhibitor of nitric oxide syindependent vessel grafts per group are given, *p b 0.05 compared to control. Please note thatsignificantly different, respectively. (F) Comparison ACH- and SNP-induced (both 10−5.5 mol/D (3 μmol/L), representative experiment, vertical bar 1.5 mN, horizontal bar 2 min.

0.8 mmol/L LiAc). The effects were similar across all higher therapeuticand supratherapeutic concentrations regardless of lithium salt, indicat-ing that the opposing effects on vascular function solely relies on the ac-tual concentration of lithium ions.

To test whether this opposing effect of lithium also occurs in the ce-rebral vascular provinces, proximal M1 segments of middle cerebral ar-teries, preconditioned at 0 (control), 0.4 and 0.8 mmol/L LiAc, werechallenged by 10−7 M ACH. As shown in Fig. 6, preconditioning of cere-bral arteries with 0.4 mmol/L significantly enhanced the ACH-inducedvasorelaxation, whereas vessel grafts preconditioned with 0.8 mmol/LLiAc significantly diminished the ACH-induced vasorelaxation. Compar-ing 0.4 vs. 0.8 mmol/L LiAc preconditioning also revealed a significantlydifferent endothelium-dependent vasorelaxation. An increase of theLiAc concentration and prolonged preconditioning (N48 h) led to fur-ther impairment of the endothelium-dependent vasorelaxation; pre-conditioning with 50 mmol/L LiAc, entirely diminished the ACH-induced (10−7 M) vasorelaxation. Taken together, the results shownin Figs. 5 and 6 suggest that lithium preconditioning induces opposingeffects on endothelium-dependent vasorelaxation and that this differ-ential impact of lithium on endothelial function is a systemic phenome-non not restricted to single vascular provinces.

4. Discussion

The present study is the first to demonstrate that high therapeuticand supratherapeutic lithium levels impair endothelial function byinterfering with endothelium-dependent (but not endothelium-inde-pendent) relaxation of blood vessels. This lithium effectmoreover inter-fered with endothelial integrity (denudation), NOS function and IP3-sensitive calcium release of the ER, and was found in two different

f endothelium-dependent and independent vasorelaxaton in lithium-preconditioned (LiCl;(B) SNP (10−5.5 mol/L) were add to phenylephrine (10 μmol/L) precontracted vessels andental conditions as in (A) and (B), however, after denudation of the endothelium from thely. (E) Comparison under identical experimental conditions as in (A), but in presence of L-nthase. Dashed line represents the control without L-NMMA as in (A). Data of n = 7controls vs. LiCl in (C) and (D) and non-L-NMMA treaded control vs. LiCl in (E) were notL) vessel relaxation in phenylephrine precontracted vessels after 30 min of Xestospongin

Fig. 6. Effect of lithium preconditioning at therapeutic concentrations on cerebralendothelium-dependent vasorelaxation. Porcine brain vessel grafts (isolated from theM1 segment of middle cerebral arteries) were preconditions at zero (control), 0.4 or0.8 mmol/L lithium acetate (LiAc) for minimum 48 h. Afterwards vasorelaxationcapacity of native vessels (resting tension 20 mm Hg) was tested by acetylcholine (ACH,10−7 M). Data of n = 7–8 vessel grafts of independent preparations are given in percent(%) in relation to the ACH-induced vasorelaxation at control conditions (0 mM LiAc/10−7 M ACH, 100%); *p b 0.01, LiAc 0.4 or LiAc 0.8 mmol/L vs. control, respectively;#p b 0.001, 0.4 vs. 0.8 mmol/L LiAc.

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vascular provinces, moreover independent of the lithium salt. Weshowed significantly different vessel relaxation responses to ACH after0.4 and ≥0.8 mmol/L lithium preconditions. Our findings have majorclinical implications as well as for future research. Firstly, impaired en-dothelium-dependent blood vessel relaxation may be a mechanismcontributing to acute lithium toxicity. Impaired endothelial functionand reduced vessel relaxation can have significant deleterious effects,particularly in highly vascularized organs such as the brain and the kid-neys that both control blood flow by vascular autoregulation. Even briefexposure to lithium at high therapeutic or supratherapeutic levels aslow as N0.8–1.0 mmol/L for b12–24 h can have dramatic clinical effectson renal and neurological function (Kessing et al., 2008; Kirkham et al.,2014; Laliberté et al., 2015; Behl et al., 2015). One of the first pioneers inlithium research, Mogens Schou had hypothesized that hemodynamicabnormalities precipitated lithium-associated acute kidney injury(Schou et al., 1968), although this has yet to be definitively validated.In this respect, our current study may lead to better understand organspecific effects of lithium on endothelial cells like in kidneys or brain,as well as in specific vascular province, in which lithium may interferewith systemic control mechanisms, the action of local vascular media-tors or the (cerebro)vascular autoregulation of blood flow (Boscheet al., 2010; Dohmen and Bosche et al., 2007).

Perhapsmost importantly, our findings suggest that the intracellulareffects of lithium on the endothelium may be completely oppositeunder supratherapeutic vs. therapeutic lithium levels. Since the inhibi-tion of IP3 and GSK-3β molecular pathways have been implicated inthe usual functioning of lithium, one could hypothesize that lithiumcauses hyper-functioning in those pathways (Berridge, 1993, 2014; Liet al., 2012). This mechanism deserves more detailed investigations. Inaddition to supratherapeutic lithium levels, we found that XeD, a specif-ic inhibitor of the IP3-sensitive Ca2+ release channel of the ER,completely blocked endothelium-dependent relaxation. This suggeststhat supratherapeutic lithium levels likewise inhibit vessel relaxationby interfering with the IP3-sensitive Ca2+ release from the ER. Underphysiologic conditions, Ca2+ released via this mechanism activates en-zymes like nitric oxide synthases eliciting endothelium-mediated

vascular relaxation. Accordingly, blockage of this pathway immediatelyimpaired endothelium-dependent vascular relaxation similar to thetreatment with supratherapeutic lithium levels for 48 h. However, pre-vious studies using therapeutic lithium levels have found that inhibitionof GSK-3β or IP3 pathways have been associated with improved vesselrelaxation (Dehpour et al., 2000; Riadh et al., 2011), such as shownhere for 0.4 mmol/L lithium. These animal studies used therapeuticlithium levels in vivo over weeks or months, and had found somewhatdifferent results. However, most parts of the findings are consistentwith the protective effects we also observed when using two-day0.4 mmol/L lithium exposure. On the other hand, different methodsand species may explain the minor conflicting parts of the results(Dehpour et al., 2000; Riadh et al., 2011). It is possible that the knownendothelium protective effect of lithium is reversed at higher lithiumconcentrations. This is supported by our data showing significant differ-ences of vessel relaxation responses to ACHafter 0.4 and 0.8mmol/L LiCland LiAc precondition in two different species and vascular provinces. Itcannot be fully excluded, however, that other lithium-related mecha-nisms are also involved (Grünfeld and Rossier, 2009; Chiu andChuang, 2010, 2012). Regarding the vascular field, the results of thepresent study that lithium preconditioning can maintain or even im-prove vascular function may help to understand lithium effects ob-served in a variety of other clinical settings. Classic or remote ischemic(pre)conditioning is an established concept to protect the kidney, heartand brain with endothelium-dependent vascular function being one ofthe important targets (González Arbeláez et al., 2013; Mergenthaleret al., 2013; Liu and Gong, 2015;Wang et al., 2015). This concept is sup-ported by recent studies showing that lithium-associated precondition-ing reduces the risk of stroke in bipolar disorder patients (Lan et al.,2015) and may support functional recovery after cortical stroke(Mohammadianinejad et al., 2014). Interestingly, studies on the molec-ular mechanism revealed that ischemic and lithium-induced pharmaco-logical preconditioning share final common pathways involvingsignaling elements like GSK-3β inhibition (Chiu and Chuang, 2010;Talab et al., 2012; González Arbeláez et al., 2013).Whether bothmaneu-vers of preconditioning may have synergistic effects remains an openquestion, yet. Nevertheless, in the light of recent clinical and experimen-tal evidence, our novel results can help to better understand theunderlining mechanisms of those remarkable clinical findings by mov-ing the lithium-endothelium interactions into focus. Future investiga-tions of the effects of therapeutic (and supratherapeutic) lithiumlevels on endothelial function and vascular tonemay yield great insightsinto intracellular effects of lithium throughout the vasculature andthereby the entire body.

Limitations of our study should be considered. We analyzed non-perfused, cold storage, vessel grafts. Cold-storage andmissing perfusionmight cause tissue bradytrophia and limited the time window for lithi-um exposure and cellular up-take. Thus, relatively higher lithium con-centrations within the solutions could lead to somewhat lowercellular levels and less toxic effects compared to in vivo conditionsexplaining our use of relatively high lithium concentrations in some ex-periments. Similarmethods and approaches to ours have also been usedby others (Ebner et al., 2011; Wilbring et al., 2013; Dutta et al., 2014).Lithium sometimes takes two weeks to produce therapeutic effects inbipolar disorder (Geddes and Miklowitz, 2013; Calkin and Alda, 2012).According to the central nerve system and the vasculature, however,lithium effects do not share same time courses. Still, acute lithiumtoxicity often manifests in less than 24 h particularly at levelsN1.5 mmol/L both in humans and animals (Laliberté et al., 2015), sug-gesting that we can be confident of our findingswith higher therapeuticand supratherapeutic lithium levels also in potentially bradytrophicvessels. On the other hand, this argument could partly explain thenon-significant response at low therapeutic lithium levels such as0.4 mmol/L compared to control, found in aortic vessel grafts whichcan barely represent all vascular provinces. This experiment could alsobe somewhat underpowered. Future studies in animals and humans

105B. Bosche et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 67 (2016) 98–106

will need to assess whether longer—term therapeutic dosing of lithium(e.g. 0.4–1.0 mmol/L) may be protective to endothelium, further clarifythe underlying molecular mechanisms and explore the specific lithiumlevel indicating the turning point from supporting to impairing effectson endothelium-dependent vessel relaxation.

5. Conclusion

We conclude that in contrast to the already known endothelial func-tion stabilizing effect of lithium at low concentrations, higher therapeuticand supratherapeutic lithium levels impair endothelium-dependent butnot endothelium-independent blood vessel relaxation. Our findingscharacterize a differential effect of lithium on the endothelium. Thesefindings seem to be novel and have two major implications: First, im-paired endothelium-dependent blood vessel relaxation represents anadditional mechanism contributing to lithium toxicity (e.g. renal andneurological). And second, effects of lithium on the endothelium aremore complex than previously known and can be completely oppositeevenwithin the narrow therapeutic window of lithium, but also beyondit. However, future animal and clinical research is needed to further ex-amine the mechanisms underlying the differential effects of lithium onendothelium and the vasculature.

Contributors

B. Bosche, T. Noll S. Rej, T. Doeppner and F.V. Härtel, designed thestudy. B. Bosche, M. Molcanyi, F.V. Härtel and B. Zatschler performedthe experiments and acquired the data, which B. Bosche, B. Zatschler,D.J. Müller, S. Rej, J. Hescheler and T. Noll analyzed. B. Bosche, M.Molcanyi, S. Rej, T Doeppner, D.J. Müller, R.L. Macdonald, T. Noll andF.V. Härtel wrote the article, which all authors reviewed and approvedfor publication.

Conflict of interest

BB got a travel grant and a speaker honorary from CSL Behring,Germany. RLM is chief scientific officer of Edge Therapeutics, Inc. BB isa member of the scientific advisory board of Edge Therapeutics. Theother authors declare no conflict of interest.

Acknowledgments

This work was supported by grants of the DeutscheForschungsgemeinschaft (DFG) to Dr. B. Bosche (BO 4229/1-1, BO4229/2-1).

Furthermore, RLM receives grant support from the Physicians Ser-vices Inc. Foundation, Brain Aneurysm Foundation, Canadian Institutesfor Health Research (CIHR), and Heart and Stroke Foundation ofCanada, SR receives support from a CIHR fellowship award. BB, RLM,and TN got material support of CSL Behring, Germany and Canada. Wethank Paul J. Turgeon and Matthew S. Yan for their valuable editing onthe manuscript.

References

Behl, T., Kotwani, A., Kaur, I., Goel, H., 2015. Mechanisms of prolonged lithium therapy-induced nephrogenic diabetes insipidus. Eur. J. Pharmacol. 755, 27–33. http://dx.doi.org/10.1016/j.ejphar.2015.02.04.

Berridge, M.J., 1989. The Albert Lasker Medical Awards. Inositol trisphosphate, calcium,lithium, and cell signaling. JAMA 262, 1834–1841.

Berridge, M.J., 1993. Inositol trisphosphate and calcium signaling. Nature 361, 315–325.Berridge, M.J., 2014. Calcium signaling and psychiatric disease: bipolar disorder and

schizophrenia. Cell Tissue Res. 357, 477–492.Bosche, B., Dohmen, C., Graf, R., Neveling, M., Staub, F., Kracht, L., Sobesky, J., Lehnhardt,

F.G., Heiss, W.D., 2003. Extracellular concentrations of non-transmitter amino acidsin peri-infarct tissue of patients predict malignant middle cerebral artery infarction.Stroke 34, 2908–2913.

Bosche, B., Graf, R., Ernestus, R.I., Dohmen, C., Reithmeier, T., Brinker, G., Strong, A.J., Dreier,J.P., Woitzik, J., Members of the Cooperative Study of Brain Injury Depolarizations

(COSBID), 2010. Recurrent spreading depolarizations after subarachnoid hemorrhagedecreases oxygen availability in human cerebral cortex. Ann. Neurol. 67, 607–617.http://dx.doi.org/10.1002/ana.21943.

Bosche, B., Schäfer, M., Graf, R., Härtel, F.V., Schäfer, U., Noll, T., 2013. Lithium preventsearly cytosolic calcium increase and secondary injurious calcium overload inglycolytically inhibited endothelial cells. Biochem. Biophys. Res. Commun. 434,268–272. http://dx.doi.org/10.1016/j.bbrc.2013.03.047.

Calkin, C., Alda, M., 2012. Beyond the guidelines for bipolar disorder: practical issues inlong-term treatment with lithium. Can. J. Psychiatr. 57, 437–445.

Chiu, C.T., Chuang, D.M., 2010. Molecular actions and therapeutic potential of lithium inpreclinical and clinical studies of CNS disorders. Pharmacol. Ther. 128, 281–304.http://dx.doi.org/10.1016/j.pharmthera.2010.07.006.

Chiu, C.T., Chuang, D.M., 2012. Neuroprotective action of lithium in disorders of the centralnervous system Zhong Nan Da Xue Xue Bao Yi Xue Ban. Front. Mol. Neurosci. 36,461–476.

Dehpour, A.R., Aghadadashi, H., Ghafourifar, P., Roushanzamir, F., Ghahremani, M.H.,Meysamee, F., Rassaee, N., Koucharian, A., 2000. Effect of chronic lithium administrationon endothelium-dependent relaxation in rat aorta. Clin. Exp. Pharmacol. Physiol. 27,55–59.

Dohmen, C., Bosche, B., Graf, R., Reithmeier, T., Ernestus, R.I., Brinker, G., Sobesky, J., Heiss,W.D., 2007. Identification and clinical impact of impaired cerebrovascular autoregu-lation in patients with malignant middle cerebral artery infarction. Stroke 38, 56–61.

Dutta, A., Bhattacharyya, S., Dutta, D., Das, A.K., 2014 Dec. Structural elucidation of the bind-ing site and mode of inhibition of Li(+) and Mg(2+) in inositol monophosphatase.FEBS J. 281 (23), 5309–5324. http://dx.doi.org/10.1111/febs.13070.

Ebner, A., Zatschler, B., Deussen, A., 2011. Evaluation of cold storage conditions for vesselsobtained from donor rats after cardiac death. J. Vasc. Surg. 54, 1769–1777. http://dx.doi.org/10.1016/j.jvs.2011.06.080.

Förstermann, U., Münzel, T., 2006. Endothelial nitric oxide synthase in vascular disease:from marvel to menace. Circulation 113, 1708–1714.

Geddes, J.R., Miklowitz, D.J., 2013. Treatment of bipolar disorder. Lancet 381, 1672–1682.http://dx.doi.org/10.1016/S0140-6736(13)60857-0.

Gerhard, T., Devanand, D.P., Huang, C., Crystal, S., Olafsom, M., 2015. Lithium treatment andrisk for dementia in adults with bipolar disorder: population-based cohort study. Br.J. Psychiatry 207, 46–51. http://dx.doi.org/10.1192/bjp.bp.114.154047.

Gold, A.B., Herrmann, N., Lanctot, K.L., 2011. Lithium and its neuroprotective and neuro-trophic effects: potential treatment for post-ischemic stroke sequelae. Curr. Drug Tar-gets 12, 243–255.

Goldstein, B.I., Young, L.T., 2013. Toward clinically applicable biomarkers in bipolar disor-der: focus on BDNF, inflammatory markers, and endothelial function. Curr. PsychiatryRep. 15, 425.

González Arbeláez, L.F., Pérez Núñez, I.A., Mosca, S.M., 2013. Gsk-3β inhibitors mimic thecardioprotection mediated by ischemic pre- and postconditioning in hypertensiverats. Biomed Res. Int. 2013, 317456. http://dx.doi.org/10.1155/2013/317456.

Gould, T.D., Manji, H.K., 2005. Glycogen synthase kinase-3: a putative molecular target forlithiummimetic drugs. Neuropsychopharmacology 30, 1223–1237. http://dx.doi.org/10.1038/sj.npp.1300731.

Grove, T., Taylor, S., Dalack, G., Ellingrod, V., 2015. Endothelial function, folatepharmacogenomics, and neurocognition in psychotic disorders. Schizophr. Res. 164,115–121. http://dx.doi.org/10.1016/j.schres.2015.02.006.

Grünfeld, J.P., Rossier, B.C., 2009. Lithium nephrotoxicity revisited. Nat. Rev. Nephrol. 5,270–276. http://dx.doi.org/10.1038/nrneph.2009.43.

Halcomb, M.E., Gould, T.D., Grahame, N.J., 2013. Lithium, but not valproate, reduces im-pulsive choice in the delay-discounting task in mice. Neuropsychopharmacology 38,1937–1944. http://dx.doi.org/10.1038/npp.2013.89.

Johnson, G., 1998. Lithium—early development, toxicity, and renal function.Neuropsychopharmacology 19, 200–205. http://dx.doi.org/10.1016/S0893-133X(98)00019-0.

Kessing, L.V., Sondergard, L., Forman, J.L., Andersen, P.K., 2008. Lithium treatment and riskof dementia. Arch. Gen. Psychiatry 65, 1331–1335.

Kirkham, E., Skinner, J., Anderson, T., Bazire, S., Twigg, M.J., Desborough, J.A., 2014. Onelithium level N1.0mmol/L causes an acute decline in eGFR: findings from a retrospec-tive analysis of a monitoring database. BMJ, e006020 (Open 4).

Kopaliani, I., Martin, M., Zatschler, B., Bortlik, K., Müller, B., Deussen, A., 2014. Cell-specificand endothelium-dependent regulations of matrix metalloproteinase-2 in rat aorta.Basic Res. Cardiol. 109, 419. http://dx.doi.org/10.1007/s00395-014-0419-8.

Laliberté, V., Yu, C., Rej, S., 2015. Acute renal and neuro-toxicity in older lithium users:how can we manage and prevent these events in late-life mood disorders?J. Psychiatry Neurosci. 4, 29–30. http://dx.doi.org/10.1503/jpn.14035.

Lan, C.C., Liu, C.C., Lin, C.H., Lan, T.Y., McInnis, M.G., Chan, C.H., et al., 2015. A reduced riskof stroke with lithium exposure in bipolar disorder: a population-based retrospectivecohort study. Bipolar Disord. http://dx.doi.org/10.1111/bdi.12336 (Sep 23. [Epubahead of print]).

Leeds, P.R., Yu, F., Wang, Z., Chiu, C.T., Zhang, Y., Leng, Y., Linares, G.R., Chuang, D.M., 2014.A new avenue for lithium: intervention in traumatic brain injury. ACS Chem.Neurosci. 5, 422–433. http://dx.doi.org/10.1021/cn500040g.

Li, X., Frye, M.A., Shelton, R.C., 2012. Review of pharmacological treatment in mood disor-ders and future directions for drug development. Neuropsychopharmacol. Rev. 37,77–101. http://dx.doi.org/10.1038/npp.2011.198.

Lichtinger, J., Gernhäuser, R., Bauer, A., Bendel, M., Canella, L., Graw, M., Krücken, R.,Kudejova, P., Mützel, E., Ring, S., Seiler, D., Winkler, S., Zeitelhack, K., Schöpfer, J.,2013. Position sensitive measurement of lithium traces in brain tissue with neutrons.Med. Phys. 40, 023501. http://dx.doi.org/10.1118/1.4774053.

Liu, Z., Gong, R., 2015. Remote ischemic preconditioning for kidney protection: GSK3β-centric insights into the mechanism of action. Am. J. Kidney Dis. 66, 846–856.http://dx.doi.org/10.1053/j.ajkd.2015.06.026.

106 B. Bosche et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 67 (2016) 98–106

Mergenthaler, P., Muselmann, C., Sünwoldt, J., Isaev, N.K., Wieloch, T., Dirnagl, U., Meisel,A., Ruscher, K., 2013. A functional role of the cyclin-dependent kinase inhibitor 1(p21(WAF1/CIP1)) for neuronal preconditioning. J. Cereb. Blood Flow Metab. 33,351–355. http://dx.doi.org/10.1038/jcbfm.2012.213.

Mohammad, O., Osser, D.N., 2014. The psychopharmacology algorithm project at the Har-vard South Shore Program: an algorithm for acute mania. Harv. Rev. Psychiatry 22,274–294. http://dx.doi.org/10.1097/HRP.0000000000000018.

Mohammadianinejad, S.E., Majdinasab, N., Sajedi, S.A., Abdollahi, F., Moqaddam, M.M.,Sadr, F., 2014. The effect of lithium in post-stroke motor recovery: a double-blind,placebo-controlled, randomized clinical trial. Clin. Neuropharmacol. 37, 73–78.http://dx.doi.org/10.1097/WNF.0000000000000028.

Mulvany,M.J., Halpern,W., 1977. Contractile properties of small arterial resistance vesselsin spontaneously hypertensive and normotensive rats. Circ. Res. 41, 19–26.

Munaron, L., Fiorio Pla, A., 2009. Endothelial calciummachinery and angiogenesis: under-standing physiology to interfere with pathology. Curr. Med. Chem. 16, 4691–4703.

Rajkowska, G., 2000. Postmortem studies in mood disorders indicate altered numbers ofneurons and glial cells. Biol. Psychiatry 48, 766–777.

Rej, S., Elie, D., Musci, I., Looper, K.J., Segal, M., 2015a. Chronic kidney disease in lithium-treated older adults: a review of epidemiology, mechanisms, and implications forthe treatment of late-life mood disorders. Drugs Aging 32, 31–42.

Rej, S., Segal, M., Low, N.C., Musci, I., Holcroft, C., Shulman, K., Looper, K.J., 2015b. In thiscorrespondence, preliminary data in 100 lithium users found that urine osmolalitycorrelated with chronic kidney disease, diabetes mellitus, and hypothyroidism. Wehypothesize that GSK-3beta inhibition is a potential mechanism for lithium-associated medical illness. Med. Hypotheses 84, 602.

Riadh, N., Allagui, M.S., Bourogaa, E., Vincent, C., Croute, F., Elfeki, A., 2011. Neuroprotec-tive and neurotrophic effects of long term lithium treatment in mouse brain.Biometals 24, 747–757.

Rybakowski, J.K., Abramowicz, M., Szcepaniewicz, A., Michalak, M., Hauser, J., Czekalski, S.,2013. The association of glycogen synthase kinase-3beta (GSK-3beta) gene polymor-phism with kidney function in long-term lithium-treated bipolar patients. Int.J. Bipolar Disord. 1, 8.

Ryglewski, S., Pflueger, H.J., Duch, C., 2007. Expanding the neuron's calcium signaling rep-ertoire: intracellular calcium release via voltage-induced PLC and IP3R activation.PLoS Biol. 5, e66.

Saunders, K.E., Hawton, K., 2013. Clinical assessment and crisis intervention for the suicid-al bipolar disorder patient. Bipolar Disord. 15, 575–583.

Schäfer, M., Bahde, D., Bosche, B., Ladilov, Y., Schäfer, C., Piper, H.M., Noll, T., 2001. Modu-lation of early [Ca2+]i rise in metabolically inhibited endothelial cells byxestospongin C. Am. J. Physiol. Heart Circ. Physiol. 280, 1002–1010.

Schou, M., Amdisen, A., Trap-Jensen, J., 1968. Lithium poisoning. Am. J. Psychiatry 125,520–527.

Svendal, G., Fasmer, O.B., Engeland, A., Berk, M., Lund, A., 2012. Co-prescription of medi-cation for bipolar disorder and diabetes mellitus: a nationwide population-basedstudy with focus on gender differences. BMC Med. 10, 148.

Talab, S.S., Elmi, A., Emami, H., Nezami, B.G., Assa, S., Ghasemi, M., Tavangar, S.M.,Dehpour, A.R., 2012. Protective effects of acute lithium preconditioning againstrenal ischemia/reperfusion injury in rat: role of nitric oxide and cyclooxygenase sys-tems. Eur. J. Pharmacol. 681, 94–99. http://dx.doi.org/10.1016/j.ejphar.2012.01.042.

Trepiccione, F., Christensen, B.M., 2010. Lithium-induced nephrogenic diabetes insipidus:new clinical and experimental findings. J. Nephrol. 23 (Suppl. 16), 43–48.

Wang, Y., Reis, C., Applegate 2nd, R., Stier, G., Martin, R., Zhang, J.H., 2015. Ischemicconditioning-induced endogenous brain protection: applications pre-, per- or post-stroke. Exp. Neurol. 272, 26–40. http://dx.doi.org/10.1016/j.expneurol.2015.04.009.

Wasserman, M.J., Corson, T.W., Sibony, D., Cooke, R.G., Parikh, S.V., Pennefather, P.S., Li,P.P., Warsh, J.J., 2004. Chronic lithium treatment attenuates intracellular calciummo-bilization. Neuropsychopharmacology 29, 759–769.

Wilbring, M., Tugtekin, S.M., Zatschler, B., Ebner, A., Reichenspurner, H., Kappert, U.,Matschke, K., Deussen, A., 2013. Preservation of endothelial vascular function of sa-phenous vein grafts after long-time storage with a recently developed potassium-chloride and N-acetylhistidine enriched storage solution. Thorac. Cardiovasc. Surg.61, 656–662. http://dx.doi.org/10.1055/s-0032-1311549.

Yoo, S.Y., Kim, J.Y., 2009. Recent insights into the mechanisms of vasospastic angina. Ko-rean Circ. J. 39, 505–511. http://dx.doi.org/10.4070/kcj.2009.39.12.505.