Regulation by FK506 and rapamycin of Ca²⁺ release from the sarcoplasmic reticulum in vascular smooth muscle: the role of FK506 binding proteins and mTOR

Abstract

Background and purpose:

The sarcoplasmic reticulum (SR), regulates the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto) in vascular smooth muscle. Release from the SR is controlled by two intracellular receptor/channel complexes, the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP₃R). These receptors may be regulated by the accessory FK506-binding protein (FKBP) either directly, by binding to the channel, or indirectly via FKBP modulation of two targets, the phosphatase, calcineurin or the kinase, mammalian target of rapamycin (mTOR).

Experimental approach:

Single portal vein myocytes were voltage-clamped in whole cell configuration and [Ca²⁺]_cyto measured using fluo-3. IP₃Rs were activated by photolysis of caged IP₃ and RyRs activated by hydrostatic application of caffeine.

Key results:

FK506 which displaces FKBP from each receptor (to inhibit calcineurin) increased the [Ca²⁺]_cyto rise evoked by activation of either RyR or IP₃R. Rapamycin which displaces FKBP (to inhibit mTOR) also increased the amplitude of the caffeine-evoked, but reduced the IP₃-evoked [Ca²⁺]_cyto rise. None of the phosphatase inhibitors, cypermethrin, okadaic acid or calcineurin inhibitory peptide, altered either caffeine- or IP₃-evoked [Ca²⁺]_cyto release; calcineurin did not contribute to FK506-mediated potentiation of RyR- or IP₃R-mediated Ca²⁺ release. The mTOR inhibitor LY294002, like rapamycin, decreased IP₃-evoked Ca²⁺ release.

Conclusions and implications:

Ca²⁺ release in portal vein myocytes, via RyR, was modulated directly by FKBP binding to the channel; neither calcineurin nor mTOR contributed to this regulation. However, IP₃R-mediated Ca²⁺ release, while also modulated directly by FKBP may be additionally regulated by mTOR. Rapamycin inhibition of IP₃-mediated Ca²⁺ release may be explained by mTOR inhibition.

Introduction

Regulation of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto) is vital to the control of vascular tone. The intracellular Ca²⁺ store, the sarcoplasmic reticulum (SR), critically regulates [Ca²⁺]_cyto, by controlling Ca²⁺ release (Bootman et al., 2001; McCarron et al., 2006). Release from the SR is mediated by two intracellular receptor complexes, the inositol 1,4,5-trisphosphate receptor (IP₃R) and the ryanodine receptor (RyR; nomenclature follows Alexander et al., 2008). IP₃Rs are activated primarily by IP₃ generated via G-protein- or tyrosine kinase-linked receptor activation (Bootman et al., 2001). In smooth muscle RyRs may also be activated pharmacologically (e.g. by caffeine) or when the Ca²⁺ content of the SR exceeds normal physiological levels (Burdyga and Wray, 2005; McCarron et al., 2006).

RyR and IP₃R are regulated by accessory proteins. Of particular importance are the FK506-binding proteins (FKBPs) (Cameron et al., 1995b; Dargan et al., 2002; Tang et al., 2002; Zheng et al., 2004) which were identified initially as the cytoplasmic receptor for the clinical immunosuppressant drugs FK506 and rapamycin (Harding et al., 1989). The association between FKBP and IP₃R or RyR is disrupted by FK506 and rapamycin, each of which binds to the accessory protein to form a drug-immunophilin protein complex and displaces FKBP from the channels (Cameron et al., 1995b; Bultynck et al., 2001a; Dargan et al., 2002).

The two major isoforms of the FKBP recognized as regulators of Ca²⁺ release channels are FKBP12 (the 12 kDa form of the protein) and FKBP12.6 (the 12.6 kDa form) (Tang et al., 2002; Wang et al., 2004; MacMillan et al., 2005b). FKBP12 and FKBP12.6 associate with IP₃R in various cell types (Cameron et al., 1995b; Cameron et al., 1995a; MacMillan et al., 2005b) suggesting that FKBP-dependent modulation of channel function may have an important role in Ca²⁺ signaling. Indeed, in some investigations removal of FKBP12 from the channel decreased IP₃R-mediated Ca²⁺ release (MacMillan et al., 2005b) and addition of exogenous FKBP12 increased IP₃R channel activity in bilayer studies (Dargan et al., 2002). Furthermore, rapamycin, which disrupts FKBP-IP₃R, decreased phenylephrine-induced contractions in rat vas deferens (Scaramello et al., 2009). These results suggest that FKBP12 potentiates IP₃R activity. In other studies FKBP12 may inhibit channel activity and removal of FKBP12 from the channel increased IP₃R-mediated Ca²⁺ release (Cameron et al., 1995b; Cameron et al., 1997).

FKBP12 also associates with RyR2 and RyR3 (Bultynck et al., 2001a; MacMillan et al., 2008) and with the skeletal muscle isoform, RyR1 (e.g. Carmody et al., 2001). An association between FKBP12.6 and RyR occurs in tracheal, pulmonary and coronary artery smooth muscle (e.g. Tang et al., 2002; Wang et al., 2004). RyR may be modulated by FKBPs to inhibit activity of the channel. Removal of FKBPs by either FK506 or rapamycin increased RyR channel open probability in lipid bilayers from coronary arterial smooth muscle (Tang et al., 2002) and cardiac muscle (Kaftan et al., 1996) or [Ca²⁺]_cyto in intestinal, colonic, bladder and pulmonary artery myocytes (Weidelt and Isenberg, 2000; Zheng et al., 2004; MacMillan et al., 2008). Conversely rebinding either FKBP12 or FKBP12.6, following their removal, decreases channel opening (Brillantes et al., 1994; Barg et al., 1997; Bultynck et al., 2001b).

However, evidence is not universally supportive of a role of FKBPs in regulating either RyR or IP₃R activity. In some studies no interaction between either RyR (Carmody et al., 2001; Wang et al., 2004; Zheng et al., 2004) or IP₃R (Bultynck et al., 2001a; Carmody et al., 2001; Zheng et al., 2004) and FKBP12 occurred. Other studies failed to demonstrate an association between FKBP12.6 and either RyR (types 1&3) or IP₃R (types 1–3) in pulmonary artery (Zheng et al., 2004). Functional studies also failed to detect any effect of FK506 or FKBP12 on IP₃-mediated Ca²⁺ release in various cell types, including smooth muscle (Bultynck et al., 2000; Bultynck et al., 2001a). Neither did removal nor addition of FKBP12 or FKBP12.6 alter RyR channel function (Timerman et al., 1996; Barg et al., 1997; Xiao et al., 2007). Nor did FK506 alter Ca²⁺ release via RyR in porcine coronary artery (Yasutsune et al., 1999) or cardiac myocytes (duBell et al., 1997) or induce contraction in renal, mesenteric, coronary or carotid arteries (Epstein et al., 1998).

In addition to binding to the channel, FKBPs may modulate signaling pathways by regulating kinase and phosphatase activity. Thus FKBPs may modulate Ca²⁺ release either directly by binding to the channel or indirectly via the kinase and phosphatase pathways modulated by FKBP. The different signaling pathways regulated by FKBPs, may account, at least in part, for the contradictory findings on FKBP modulation of IP₃R and RyR. For example, following the removal of FKBP from either IP₃R or RyR by FK506, the FK506-FKBP complex formed binds to and inhibits the Ca²⁺/calmodulin-dependent serine/threonine phosphatase calcineurin (Liu et al., 1991) Indeed, calcineurin may regulate RyR and IP₃R by forming part of the FKBP-channel complex (Cameron et al., 1995b; Cameron et al., 1995a; Cameron et al., 1997; Bandyopadhyay et al., 2000a; Shin et al., 2002). In support, calcineurin inhibitors increased caffeine- and ryanodine-induced Ca²⁺ release and the frequency and amplitude of Ca²⁺ oscillations in cardiac and skeletal muscle (Bandyopadhyay et al., 2000a; Shin et al., 2002). In COS-7 cells and cerebellar microsomes inhibition of calcineurin increased ATP-induced Ca²⁺ release and IP₃R activity respectively (Cameron et al., 1995a; Bandyopadhyay et al., 2000b).

Calcineurin may also regulate Ca²⁺ release via RyR (Bultynck et al., 2003; MacMillan et al., 2008) or IP₃R (Bultynck et al., 2003) independently of FKBPs or not at all (Kanoh et al., 1999). Indeed, the comparatively few studies in vascular smooth muscle failed to confirm a role for calcineurin in regulating [Ca²⁺]_cyto. Drugs which can inhibit calcineurin, cypermethrin and okadaic acid, for example, did not alter Ca²⁺ release via either RyR (MacMillan et al., 2008) or IP₃R (MacMillan and McCarron, unpublished data) in aortic myocytes. Neither did okadaic acid alter [Ca²⁺]_cyto in porcine coronary artery (Ashizawa et al., 1989; Hirano et al., 1989). Cyclosporin A, a calcineurin inhibitor which does not interact with FKBPs, did not alter [Ca²⁺]_cyto in pulmonary (Zheng et al., 2004) and coronary artery myocytes (Frapier et al., 2001) or SR [Ca²⁺] in aorta myocytes (Avdonin et al., 1999).

Rapamycin, a structural analogue of FK506, also binds to FKBP12 resulting in the formation of a rapamycin-FKBP12 complex. This complex does not inhibit calcineurin but binds to FKBP12-rapamycin-associated protein (Heitman et al., 1991; Peterson et al., 2000) also named mammalian target of rapamycin (mTOR) and inhibits its function (Brown et al., 1995). mTOR is a 289 kDa serine/threonine protein kinase and classified as a member of the phosphatidylinositol kinase-related kinase (PIKK) family. mTOR regulates a myriad of intracellular processes which include cell cycle progression and growth. Inhibition of mTOR by FKBP12-rapamycin may mediate the pharmacological actions of rapamycin (Brown et al., 1995; Sun et al., 2005) including the regulation of IP₃-mediated Ca²⁺ release in colonic smooth muscle (MacMillan et al., 2005b).

In view of the controversy which surrounds the effects of the drugs FK506 and rapamycin and the mechanism of action of FKBPs on intracellular Ca²⁺ release in vascular smooth muscle the present study was carried out in single myocytes from portal vein. Cells were voltage-clamped to avoid [Ca²⁺]_cyto changes which may have occurred via Ca²⁺ influx as a result of FK506- or rapamycin-evoked changes in membrane potential. The use of photolysed caged IP_3, to activate IP₃R, minimized the number of second messenger systems activated. This study has shown that pharmacological removal of FKBP from RyR with either FK506 or rapamycin, augmented caffeine-evoked Ca²⁺ release in portal vein myocytes. On the other hand, FK506 (FKBP-calcineurin inhibitor) augmented whereas rapamycin (mTOR inhibitor) reduced the IP₃-evoked [Ca²⁺]_cyto release. Neither cypermethrin nor okadaic acid (drugs which can inhibit calcineurin) altered caffeine- or IP₃-evoked [Ca²⁺]_cyto release. Thus, calcineurin is not required for the potentiation of RyR or IP₃R Ca²⁺ release to occur. The mTOR inhibitor LY294002, like rapamycin, decreased IP₃R Ca²⁺ release; mTOR inhibition may mediate the rapamycin-induced decrease in IP₃R Ca²⁺ release. Our results, in portal vein myocytes, suggest that RyR- and IP₃R-mediated Ca²⁺ release are modulated directly by FKBP. IP₃R-mediated Ca²⁺ release may be additionally modulated by mTOR.

Methods

Preparation of portal vein myocytes

All animal care and experimental procedures complied with the Animal (Scientific Procedures) Act UK 1986. Male guinea pigs (500–700 g) were humanely killed by cervical dislocation and immediate exsanguination, and the portal vein was removed quickly and transferred to an oxygenated (95% O₂–5% CO₂) physiological saline solution of the following composition (mM): NaCl 118.4, NaHCO₃ 25, KCl 4.7, NaH₂PO₄ 1.13, MgCl₂ 1.3, CaCl₂ 2.7 and glucose 11 (pH 7.4). From this tissue single vascular smooth muscle cells were isolated using a two-step enzymatic process. The vessel was initially treated (12 min at 34.5°C) with papain (1.7 mg·mL^–1), DTT (0.7 mg·mL^–1) and BSA (1.64 mg·mL^–1) in a low Ca²⁺ solution which contained (mM): sodium glutamate, 80; NaCl, 55; KCl, 6; MgCl₂, 1; CaCl₂, 0·1; Hepes, 10; glucose, 10; 0·2, EDTA (to remove heavy metals) (pH 7·3). During a second incubation, the tissue was further digested (14 min at 34.5°C) in the low Ca²⁺ solution containing BSA (1.64 mg·mL^–1), by collagenase (type F; 1.3–2 mg·mL^–1) and hyaluronidase (0.8–1 mg·mL^–1). The tissue was then rinsed several times with enzyme-free low Ca²⁺ solution containing BSA and then with the enzyme- and BSA-free low Ca²⁺ solution. Single smooth muscle cells were dispersed by trituration with a Pasteur pipette, stored at 4°C and used the same day. All experiments were conducted at room temperature (20–22°C).

Electrophysiological experiments

Cells were voltage clamped using conventional tight seal whole-cell recording (MacMillan et al., 2005a; 2008;). The composition of the extracellular solution was (mM): sodium glutamate 80, NaCl 40, tetraethylammonium chloride (TEA) 20, MgCl₂ 1.1, CaCl₂ 3, HEPES 10 and glucose 30 (pH 7.4 adjusted with NaOH 1M). The pipette solution contained (mM): Cs₂SO₄ 85, CsCl 20, MgCl₂ 1, HEPES 30, pyruvic acid 2.5, malic acid 2.5, KH₂PO₄ 1, MgATP 3, creatine phosphate 5, guanosine triphosphate 0.5, fluo-3 penta-ammonium salt 0.1 and caged Ins (1,4,5) P₃-trisodium salt 0.025 (pH 7.2 adjusted with CsOH 1M). Whole cell currents were amplified by an Axopatch 1D amplifier (Axon instruments, Union City, CA, USA), low pass filtered at 500 Hz (8-pole bessel filter; Frequency Devices, Haverhill, MA, USA), and digitally sampled at 1.5 kHz using a Digidata interface, pCLAMP software (version 6.0.1, Axon Instruments) and stored on a personal computer for analysis.

Assay of [Ca²⁺]_cyto

[Ca²⁺]_cyto was measured as fluorescence using the membrane-impermeable dye fluo-3 (penta-ammonium salt) introduced into the cell via the patch pipette (MacMillan et al., 2005a,b;). Fluorescence was quantified using a microfluorimeter which consisted of an inverted microscope (Nikon diaphot) and a photomultiplier tube with a bi-alkali photo cathode. Fluo-3 was excited at 488 nm (bandpass 9 nm) from a PTI Delta Scan (Photon Technology International Inc., London, UK) through the epi-illumination port of the microscope (using one arm of a bifurcated quartz fibre optic bundle). Excitation light was passed through a field stop diaphragm to reduce background fluorescence and reflected off a 505 nm long-pass dichroic mirror. Emitted light was guided through a 535 nm barrier filter (bandpass 35 nm) to a photomultiplier in photon counting mode. Interference filters and dichroic mirrors were obtained from Glen Spectra (London, UK). To photolyse caged IP₃ (25 µM) the output of a xenon flash lamp (Rapp Optoelektronik, Hamburg, Germany) was passed through a UG-5 filter to select UV light and merged into the excitation light path of the microfluorimeter using the second arm of the quartz bifurcated fibre optic bundle and applied to the caged compound. The nominal flash lamp energy was 57 mJ, measured at the output of the fibre optic bundle and the flash duration was approximately 1 ms. Fluorescence signals were expressed as ratios (F/F₀) of fluorescence counts (F) relative to baseline (control) values (taken as 1) before stimulation (F₀).

Statistical analysis

Results are expressed as means ± SEM. Student's t-tests were applied to test and control conditions, a value of P < 0.05 was considered significant.

Materials

Caged Ins (1,4,5) P₃-trisodium salt was purchased from Invitrogen (Paisley, UK). Fluo-3 penta-ammonium salt was purchased from TEF labs (Austin, Texas, USA). Rapamycin, cypermethrin and okadaic acid were each purchased from Calbiochem-Novabiochem (Beeston, Nottingham, UK) and papain was purchased from Worthington Biochemical Corporation (Lakewood, NJ, USA). All other reagents were purchased from Sigma (Poole, Dorset, UK). IP₃ was released from its caged compound by flash photolysis. Caffeine (10 mM) was applied by hydrostatic pressure ejection using a pneumatic pump (PicoPump PV 820, World Precision Instruments, Stevenage, Herts, UK). The concentration of caged, non-photolysed IP₃ refers to that in the pipette. Caffeine was dissolved in extracellular bathing solution, FK506 was dissolved in 100% ethanol (final bath concentration of the solvent, 0.05%, was by itself ineffective). Rapamycin, cypermethrin and okadaic acid were each dissolved in dimethyl sulphoxide (final bath concentration of the solvent, 0.01%, was by itself ineffective). Each drug (with the exception of caffeine) was perfused into the solution bathing the cells (∼5 mL per min). The calcineurin inhibitory peptide (CiP) based on the autoinhibitory fragment (ITSFEEAKGLDRINERMPPRRDAMP) was obtained from Sigma (Poole, UK) and introduced to the cell via the patch pipette filling solution.

Results

To determine the role of FKBPs in regulating Ca²⁺ release via RyRs and IP₃Rs, the effects of FK506 and rapamycin, each of which disrupt the FKBP-channel association, were examined on caffeine- and IP₃-induced Ca²⁺ release in voltage-clamped, single portal vein myocytes. Caffeine (10 mM), which activates RyR (Figure 1A), and photolysed caged IP₃ (25 µM, Figure 2A), which activates IP₃R, each reproducibly increased [Ca²⁺]_cyto. FK506 (10 µM; Figure 1A) and rapamycin (10 µM; Figure 1B) each significantly (P < 0.05) increased caffeine-induced Ca²⁺ release (ΔF/F₀) by 70 ± 11% and 59 ± 7% respectively [from 0.4 ± 0.02 (control) to 0.68 ± 0.08 (FK506, n= 3, Figure 1A) and 0.66 ± 0.11 (control) to 1.02 ± 0.16 (rapamycin, n= 7, Figure 1B)]. FK506 (10 µM; Figure 2A) also increased IP₃-induced Ca²⁺ release by 30 ± 1% (ΔF/F₀ from 0.93 ± 0.2 to 1.23 ± 0.2, n= 3). Rapamycin (10 µM; Figure 2B) on the other hand, significantly (P < 0.05) decreased the IP₃-evoked Ca²⁺ transient (ΔF/F₀) by 55 ± 8% from 1.78 ± 0.5 to 0.74 ± 0.14 (n= 3).

Figure 1.

FK506 or rapamycin increased the [Ca²⁺]_cyto rise evoked by caffeine in voltage clamped single portal vein myocytes. At −70 mV (iii) caffeine (CAF, 10 mM, ii) increased [Ca²⁺]_cyto (i) as indicated by F/F₀. FK506 (10 µM, n= 3, P < 0.05, A) and rapamycin (10 µM, n= 7, P < 0.05, B) each significantly increased the caffeine-evoked [Ca²⁺]_cyto transients (i). [Ca²⁺]_cyto, cytoplasmic Ca²⁺ concentration; F, fluorescence counts; F₀, baseline fluorescence counts.

Figure 2.

FK506 increased but rapamycin decreased IP₃-evoked Ca²⁺ increases in voltage clamped single portal vein myocytes. At −70 mV (ii) photolysed caged IP₃ (↑) increased [Ca²⁺]_cyto (i) as indicated by F/F₀. FK506 (10 µM, n= 3, P < 0.05, A) increased but rapamycin (10 µM, n= 3, P < 0.05, B) decreased the [Ca²⁺]_cyto transients (i). [Ca²⁺]_cyto, cytoplasmic Ca²⁺ concentration; F, fluorescence counts; F₀, baseline fluorescence counts; IP₃, inositol 1,4,5-trisphosphate.

FK506 and rapamycin each form a complex with FKBP to disrupt binding of FKBP to IP₃R and RyR. Disrupting FKBP binding to the channels is thus a feature which is common to the mechanism of action of both drugs. However, thereafter the action of the drug differs. The FK506-FKBP complex inhibits calcineurin whereas the rapamycin-FKBP complex inhibits mTOR. Therefore, the effects of FK506 and rapamycin on Ca²⁺ release may be either mediated by calcineurin or mTOR inhibition respectively, or by the removal of FKBP from the receptor.

Role of calcineurin in FK506-evoked potentiation of Ca²⁺ release

If the potentiation of RyR activity by FK506 was explained by inhibition of calcineurin, then the potentiation should be mimicked by calcineurin inhibitors. However, drugs which can inhibit calcineurin, cypermethrin (10 µM; Figure 3A) or okadaic acid (5 µM; Figure 3B), failed to alter caffeine-evoked Ca²⁺ transients [(ΔF/F₀) from 1.22 ± 0.25 (control) to 1.22 ± 0.26 (cypermethrin, n= 8, P > 0.05) and 0.65 ± 0.08 (control) to 0.65 ± 0.07 (okadaic acid, n= 5, P > 0.05) respectively]. Neither did the CiP prevent FK506 potentiation of caffeine-evoked Ca²⁺ release. CiP was administered into the cell via the pipette solution (because it is impermeant) and FK506 remained effective in increasing caffeine-evoked Ca²⁺ transients [(F/F₀) from 0.9 ± 0.09 (control) to 1.33 ± 0.09 (FK506, n= 7, P < 0.05); Figure 3C].

Figure 3.

Effect of calcineurin inhibition on caffeine-evoked Ca²⁺ increases in voltage-clamped single portal vein myocytes. Caffeine (CAF, 10 mM, ii) increased [Ca²⁺]_cyto (i) as indicated by F/F₀. Neither cypermethrin (10 µM, n= 8, A) nor okadaic acid (5 µM, n= 5, B) significantly (P > 0.05) altered the caffeine-evoked [Ca²⁺]_cyto transients (V_M−70 mV, iii). Following pretreatment with the calcineurin inhibitor CiP (100 µM, C) via the patch pipette filling solution, FK506 (10 µM) still significantly (P < 0.05) increased the caffeine-evoked [Ca²⁺]_cyto transients (V_M−70 mV, iii, n= 7). [Ca²⁺]_cyto, cytoplasmic Ca²⁺ concentration; CiP, calcineurin inhibitory peptide; F, fluorescence counts; F₀, baseline fluorescence counts.

If the potentiation of IP₃-mediated Ca²⁺ release by FK506 arose by inhibition of calcineurin, then inhibitors of the phosphatase should increase IP₃-mediated Ca²⁺ release. However, again, neither cypermethrin (10 µM; Figure 4A) nor okadaic acid (5 µM; Figure 4B) increased IP₃-mediated Ca²⁺ release [(ΔF/F₀) (from 2.02 ± 0.21 (control) to 2.03 ± 0.21 (cypermethrin, n= 5, P > 0.05) and 1.21 ± 0.19 (control) to 1.16 ± 0.19 (okadaic acid, n= 6, P > 0.05)]. Following calcineurin inhibition with CiP (Figure 4C), FK506 also remained effective in increasing IP₃-evoked Ca²⁺ transients [(F/F₀) from 1.39 ± 0.12 (control) to 2.00 ± 0.15 (FK506, n= 6, P < 0.05)].

Figure 4.

Effect of calcineurin inhibition on IP₃-evoked Ca²⁺ increases in voltage-clamped single portal vein myocytes. Photolysed caged IP₃ (↑) increased [Ca²⁺]_cyto (i) as indicated by F/F₀. Neither cypermethrin (10 µM, n= 5, A) nor okadaic acid (5 µM, n= 6, B) significantly (P > 0.05) altered the IP₃-evoked [Ca²⁺]_cyto transients (V_M−70 mV, ii). Following pretreatment with the calcineurin inhibitor CiP (100 µM, C), via the patch pipette filling solution, FK506 (10 µM) still significantly (P < 0.05) increased the IP₃-evoked [Ca²⁺]_cyto transients produced by photolysed caged IP₃ (V_M−70 mV, ii, n= 6). [Ca²⁺]_cyto, cytoplasmic Ca²⁺ concentration; CiP, calcineurin inhibitory peptide; F, fluorescence counts; F₀, baseline fluorescence counts; IP₃, inositol 1,4,5-trisphosphate.

Role of mTOR in rapamycin-evoked suppression of IP₃-mediated Ca²⁺ release

Interestingly, removal of FKBP by rapamycin decreased IP₃-mediated Ca²⁺ release. The decrease is unlikely to be explained by a reduction in the store's Ca²⁺ content by rapamycin's inhibition of the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA; Bultynck et al., 2000; Loughrey et al., 2007), because the Ca²⁺ transient in response to RyR activation with caffeine was significantly (P < 0.05) increased by the drug (Figure 1B). Had rapamycin inhibited SERCA the caffeine-evoked [Ca²⁺]_cyto rise would also have been reduced. Furthermore, inhibition of SERCA pump activity should increase steady-state [Ca²⁺]_cyto (Bultynck et al., 2000). No increase in steady-state [Ca²⁺]_cyto (measured as fluorescence) was observed either following FK506 [(F/F₀) from 1.06 ± 0.05 (control) to 1.09 ± 0.07 (FK506, n= 6, P > 0.05)] or rapamycin [(F/F₀) from 1.07 ± 0.04 (control) to 1.08 ± 0.04 (rapamycin, n= 10, P > 0.05)]. Moreover, neither FK506 nor rapamycin significantly altered the rate of Ca²⁺ removal from the cytoplasm following each of IP₃- or caffeine-evoked Ca²⁺ release. The 80–20% decay interval following IP₃- and caffeine-evoked Ca²⁺ release was 4.0 ± 1.0 s and 6.2 ± 1.4 s in controls and 4.7 ± 1.2 s and 6.3 ± 1.5 s in FK506, (each n= 3, P > 0.05) respectively. Similarly, the 80–20% decay interval following IP₃- and caffeine-evoked Ca²⁺ release was 5.8 ± 0.2 s and 6.2 ± 0.6 s in controls and 6.5 ± 0.9 s and 6.4 ± 0.5 s in rapamycin, (n= 3 and 7, P > 0.05) respectively.

Rapamycin-FKBP-mediated inhibition of mTOR may explain the inhibition of IP₃-mediated Ca²⁺ release. To test this, the effect of the mTOR inhibitor LY294002 (inhibits mTOR without first binding to FKBP) was examined on IP₃-evoked Ca²⁺ release. If the rapamycin-induced decrease in IP₃-mediated Ca²⁺ release arose by inhibition of mTOR, then an inhibitor of the kinase should also decrease IP₃-mediated Ca²⁺ release. LY294002 (20 µM; Figure 5) significantly (P < 0.05) decreased the IP₃-evoked Ca²⁺ transient (ΔF/F₀) by 52 ± 12% from 2.64 ± 0.4 to 1.24 ± 0.3 (n= 4).

Figure 5.

The mTOR inhibitor LY294002 decreased IP₃-evoked Ca²⁺ increases in voltage clamped single portal vein myocytes. Photolysed caged IP₃ (↑) increased [Ca²⁺]_cyto (i) as indicated by F/F₀. Addition of LY294002 (20 µM, n= 4, P < 0.05) decreased the IP₃-evoked [Ca²⁺]_cyto transients (i) (V_M−70 mV, ii). [Ca²⁺]_cyto, cytoplasmic Ca²⁺ concentration; F, fluorescence counts; F₀, baseline fluorescence counts; IP₃, inositol 1,4,5-trisphosphate; mTOR, mammalian target of rapamycin.

Discussion and conclusions

FK506 and rapamycin exert multiple effects on intracellular signaling via their effects on FKBP binding to RyRs and IP₃Rs, and by regulating the phosphatase calcineurin and the kinase mTOR. The multiple effects of the drugs explain the present results and account for apparently contradictory findings which exist in the literature on the role of FKBPs in Ca²⁺ signaling. The present study, in portal vein myocytes, suggests RyR is modulated directly by FKBP to decrease Ca²⁺ release via the channel. Neither calcineurin nor mTOR are required for FKBP modulation of RyR activity to occur. IP₃R activity, like RyR, is modulated directly by FKBP but, unlike RyR, also indirectly via the kinase mTOR. FKBP binding to IP₃R decreases Ca²⁺ release while inhibition of mTOR, by the FKBP-rapamycin complex, additionally decreases IP₃-mediated Ca²⁺ release.

An association between FKBPs and either RyR or IP₃R to regulate the activities of the channels has been reported in various cell types (e.g. Carmody et al., 2001; MacMillan et al., 2005b; 2008;) including vascular smooth muscle (Tang et al., 2002; Zheng et al., 2004). However, FKBPs may increase (Dargan et al., 2002; Su et al., 2003; MacMillan et al., 2005b), decrease (Cameron et al., 1995b; Zheng et al., 2004; MacMillan et al., 2008) or have no effect on channel activity (Barg et al., 1997; duBell et al., 1997; Kanoh et al., 1999; Yasutsune et al., 1999; Bultynck et al., 2000). The drugs FK506 and rapamycin each inhibit FKBP association with IP₃R and RyR but differ in that the FK506-FKBP complex inhibits calcineurin (Liu et al., 1991) whereas the rapamycin-FKBP complex inhibits mTOR (Brown et al., 1995). Therefore, the effects of FK506 and rapamycin may be either mediated by calcineurin or mTOR inhibition respectively, or by the removal of FKBP from the channels.

In the present study while FK506 increased, drugs which can inhibit calcineurin (cypermethrin or okadaic acid) failed to alter caffeine- or IP₃-evoked Ca²⁺ release. FK506 also remained effective in increasing the caffeine- and IP₃-evoked Ca²⁺ transients in the presence of CiP. These results suggest that calcineurin is unlikely to contribute to the FK506-induced potentiation of caffeine- or IP₃-evoked Ca²⁺ increases in portal vein. Had it done so the calcineurin inhibitors would have increased Ca²⁺ release and the effect of FK506 would have been inhibited. As FK506 and rapamycin each increased caffeine-induced Ca²⁺ release, a mechanism common to the action of both drugs, i.e. FKBP removal from the FKBP-RyR complex, may explain the increased Ca²⁺ release in the present study. This finding is consistent with our own previous results in colonic smooth muscle (MacMillan et al., 2008) and those of other investigators in various tissues which suggest that FK506 and rapamycin each increase the activity of RyR by disrupting FKBP-RyR association (Brillantes et al., 1994; Kaftan et al., 1996; Bultynck et al., 2000; Weidelt and Isenberg, 2000; Xiao et al. 2007).

On the other hand, while FK506 increased, rapamycin decreased IP₃-mediated Ca²⁺ release. The target for the rapamycin-FKBP12 complex has been identified as the kinase mTOR (Sabatini et al., 1994; Brown et al., 1995). The rapamycin-induced reduction in IP₃-mediated Ca²⁺ release in the present study may be mediated by inhibition of mTOR. In support, the inhibitor LY294002 which inhibits PI-3 kinase and mTOR (Brunn et al., 1996), but does not remove FKBP from the channel, reduced the IP₃-mediated Ca²⁺ release. As LY294002 mimicked the effects of rapamycin, inhibition of IP₃-mediated Ca²⁺ release by rapamycin may be mediated by mTOR. Thus, in addition to regulating vascular smooth muscle cell differentiation, proliferation and migration our study suggests mTOR also regulates Ca²⁺ release in vascular (portal vein) smooth muscle.

In addition to mTOR regulation of IP₃-mediated Ca²⁺ release, the present study has also shown that FKBP may modulate IP₃R directly in portal vein myocytes. This conclusion is derived from the finding that FK506 but not calcineurin inhibitors increased IP₃-mediated Ca²⁺ release. In our previous study in colonic smooth muscle, FKBP had little direct effect on IP₃-mediated Ca²⁺ release but indirectly modulated Ca²⁺ release through mTOR, to increase, and calcineurin, to inhibit, Ca²⁺ release (MacMillan et al., 2005b). Apart from the differences in the tissues used in the two studies, experimental conditions were otherwise identical. FKBPs, calcineurin and mTOR may each regulate RyR and IP₃R differently in various tissues and calcineurin appears to regulate neither IP₃R or RyR in portal vein. Similar findings were obtained in other vascular tissues. Calcineurin did not alter [Ca²⁺]_cyto in coronary (Ashizawa et al., 1989; Hirano et al., 1989) and pulmonary (Zheng et al., 2004) arteries nor SR [Ca²⁺] in aorta myocytes (Avdonin et al., 1999). Yet, the differences in IP₃R regulation by FKBP and calcineurin between colonic and portal vein myocytes, are not explained simply by peculiarities of vascular and gastrointestinal smooth muscle. We have investigated RyR and IP₃R regulation in two types of vascular smooth muscle and, again, interesting differences exist. While FKBP modulated RyR and IP₃R in portal vein, it neither modulated Ca²⁺ release or even associated with either RyR or IP₃R in aorta (MacMillan et al., 2005b; 2008;). Clearly, substantial tissue to tissue variation exists in the regulation of RyR and IP₃R by FKBPs and the effects of compounds such as FK506 and rapamycin.

In summary, the present study, in portal vein myocytes, suggest RyR-mediated Ca²⁺ release is modulated directly by FKBP as a result of the binding of the accessory protein to the channel. IP₃-mediated Ca²⁺ release may be modulated by both FKBP binding to IP₃R and also indirectly via the kinase mTOR. FK506 increased RyR and IP₃R channel activity whereas rapamycin decreased IP₃R activity. The opposing effects of FK506 and rapamycin on IP₃-mediated Ca²⁺ release suggest different roles for FKBPs and mTOR in IP₃R-mediated Ca²⁺ release in portal vein myocytes; FKBPs decrease RyR and IP₃R activity whereas mTOR increases IP₃R activity.

Glossary

Abbreviations:

[Ca²⁺]_cyto

cytoplasmic Ca²⁺ concentration

CiP

calcineurin inhibitory peptide

F

fluorescence counts

F₀

baseline fluorescence counts

FKBPs

FK506-binding proteins

IP₃

inositol 1,4,5-trisphosphate

IP₃R

inositol 1,4,5-trisphosphate receptor

mTOR

mammalian target of rapamycin

RyR

ryanodine receptor

SERCA

sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase

SR

sarcoplasmic reticulum

Conflicts of interest

None.