Altered vascular smooth muscle function in the ApoE knockout mouse during the progression of atherosclerosis.

OBJECTIVES: Relaxation of vascular smooth muscle (VSM) requires re-uptake of cytosolic Ca(2+) into the sarcoplasmic reticulum (SR) via the Sarco/Endoplasmic Reticulum Ca(2+) ATPase (SERCA), or extrusion via the Plasma Membrane Ca(2+) ATPase (PMCA) or sodium Ca(2+) exchanger (NCX). Peroxynitrite, a reactive species formed in vascular inflammatory diseases, upregulates SERCA activity to induce relaxation but, chronically, can contribute to atherogenesis and altered vascular function by escalating endoplasmic reticulum stress. Our objectives were to determine if peroxynitrite-induced relaxation and Ca(2+) handling processes within vascular smooth muscle cells were altered as atherosclerosis develops. METHODS: Aortae from control and ApoE(-/-) mice were studied histologically, functionally and for protein expression levels of SERCA and PMCA. Ca(2+) responses were assessed in dissociated aortic smooth muscle cells in the presence and absence of extracellular Ca(2+). RESULTS: Relaxation to peroxynitrite was concentration-dependent and endothelium-independent. The abilities of the SERCA blocker thapsigargin and the PMCA inhibitor carboxyeosin to block this relaxation were altered during fat feeding and plaque progression. SERCA levels were progressively reduced, while PMCA expression was upregulated. In ApoE(-/-) VSM cells, increases in cytosolic Ca(2+) [Ca(2+)]c in response to SERCA blockade were reduced, while SERCA-independent Ca(2+) clearance was faster compared to control. CONCLUSION: As atherosclerosis develops in the ApoE(-/-) mouse, expression and function of Ca(2+) handling proteins are altered. Up-regulation of Ca(2+) removal via PMCA may offer a potential compensatory mechanism to help normalise the dysfunctional relaxation observed during disease progression.

FK506 binding proteins: cellular regulators of intracellular Ca2+ signalling.

In many cell types the intracellular Ca(2+) store performs a central role in the regulation of the cytosolic Ca(2+) concentration ([Ca(2+)](c)), the elevation of which triggers diverse and fundamental activities from reproduction to apoptosis, as well as being the major trigger for contraction. Two distinct classes of Ca(2+) release channels, which mobilize Ca(2+) from the store, exist; the inositol 1,4,5-trisphosphate (IP(3)) receptor and the ryanodine receptor. Considerable attention has been directed towards the importance of modulatory proteins that interact with these channels including, FK506 binding proteins (FKBPs), FKBP12 and its isoform, FKBP12.6. Although FKBP12 was first identified as the principal intracellular target for the immunosuppressive drugs, FK506 and rapamycin, new insights into the role of FKBPs have since emerged. These regulatory proteins are reportedly important modulators of intracellular Ca(2+) release. FKBPs may regulate ryanodine and IP(3) receptors either directly, by binding to the cytoplasmic aspect of the channel, or indirectly via modulation of two targets, the phosphatase, calcineurin or the kinase, mammalian target of rapamycin (mTOR). Dissociation of FKBP12 or FKBP12.6 from either Ca(2+) release channel may increase, decrease or have no effect on ryanodine receptor- or IP(3) receptor-mediated Ca(2+) release. These important controversies may be attributed to FKBPs' ability to regulate the receptor indirectly via the kinase and phosphatase pathways modulated by the accessory proteins. This brief review discusses the regulation of intracellular ryanodine and IP(3) receptor Ca(2+) release channels by accessory FKBPs, with important implications for the role of FKBPs in the pathophysiology of a number of diseases.

Light-induced Ca(2+) transients observed in widefield epi-fluorescence microscopy of excitable cells.

We have investigated the possibility that variations in the level of intracellular Ca(2+) in excitable cells might be induced as an artifact of the incoherent illumination that is being used to monitor transient responses. In order to avoid the fluctuations in power of an arc lamp source, a microscope using a light emitting diode that was calibrated accurately at low power levels, was constructed to provide good control over the dose of light applied to the biological specimen. We report here that higher powers of illumination increased the probability of occurrence of Ca(2+) transients even in the sub-mW range normally used to measure such transients in epi-fluorescence work, suggesting that caution should be exercised when designing experiments and interpreting data.

Agonist-evoked Ca(2+) wave progression requires Ca(2+) and IP(3).

Smooth muscle responds to IP(3)-generating agonists by producing Ca(2+) waves. Here, the mechanism of wave progression has been investigated in voltage-clamped single smooth muscle cells using localized photolysis of caged IP(3) and the caged Ca(2+) buffer diazo-2. Waves, evoked by the IP(3)-generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) over a single though substantial length (approximately 30 microm) of the cell. During regenerative propagation, the wave-front was about 1/3 the length (approximately 9 microm) of the initiation site. The wave-front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP(3) may explain the amplitude changes. Ca(2+) was required for IP(3)-mediated wave progression to occur. Increasing the Ca(2+) buffer capacity in a small (2 microm) region immediately in front of a CCh-evoked Ca(2+) wave halted progression at the site. However, the wave front does not progress by Ca(2+)-dependent positive feedback alone. In support, colliding [Ca(2+)](c) increases from locally released IP(3) did not annihilate but approximately doubled in amplitude. This result suggests that local IP(3)-evoked [Ca(2+)](c) increases diffused passively. Failure of local increases in IP(3) to evoke waves appears to arise from the restricted nature of the IP(3) increase. When IP(3) was elevated throughout the cell, a localized increase in Ca(2+) now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP(3) and Ca(2+) are required for active propagation of the wave front to occur.

The sarcoplasmic reticulum Ca2+ store arrangement in vascular smooth muscle.

n vascular smooth muscle cells, Ca2+ release via IP(3) receptors (IP(3)R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) Ca2+ store contributes significantly to the regulation of cellular events such as gene regulation, growth and contraction. Ca2+ release from various regions of a structurally compartmentalized SR, it is proposed, may selectively activate different cellular functions. Multiple SR compartments with various receptor arrangements are proposed also to exist at different stages of smooth muscle development and in proliferative vascular diseases such as atherosclerosis. The conclusions on SR organization have been derived largely from the outcome of functional studies. This study addresses whether the SR Ca2+ store is a single continuous interconnected network or multiple separate Ca2+ pools in single vascular myocytes. To do this, the consequences of depletion of the SR in small restricted regions on the Ca2+ available throughout the store was examined using localized photolysis of caged-IP3 and focal application of ryanodine in guinea-pig voltage-clamped single portal vein myocytes. From one small site on the cell, the entire SR could be depleted via either RyR or IP(3)R. The entire SR could also be refilled from one small site on the cell. The results suggest a single luminally continuous SR exists. However, the opening of IP(3)R and RyR was regulated by the Ca2+ concentration within the SR (luminal [Ca2+]). As the luminal [Ca2+] declines, the opening of the receptors decline and stop, and there may appear to be stores with either only RyR or only IP(3)R. The SR Ca2+ store is a single luminally continuous entity which contains both IP(3)R and RyR and within which Ca2+ is accessed freely by each receptor. While the SR is a single continuous entity, regulation of IP3R and RyR by luminal [Ca2+] explains the appearance of multiple stores in some functional studies.

FK506-binding protein (FKBP12) regulates ryanodine receptor-evoked Ca2+ release in colonic but not aortic smooth muscle.

In smooth muscle, the ryanodine receptor (RyR) mediates Ca(2+) release from the sarcoplasmic reticulum (SR) Ca(2+) store. Release may be regulated by the RyR accessory FK506-binding protein (FKBP12) either directly, as a result of FKBP12 binding to RyR, or indirectly via modulation of the activity of the phosphatase calcineurin or kinase mTOR. Here we report that RyR-mediated Ca(2+) release is modulated by FKBP12 in colonic but not aortic myocytes. Neither calcineurin nor mTOR are required for FKBP12 modulation of Ca(2+) release in colonic myocytes to occur. In colonic myocytes, co-immunoprecipitation techniques established that FKBP12 and calcineurin each associated with the RyR2 receptor isoform (the main isoform in this tissue). Single colonic myocytes were voltage clamped in the whole cell configuration and cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) increases evoked by the RyR activator caffeine. Under these conditions FK506, which displaces FKBP12 (to inhibit calcineurin) and rapamycin, which displaces FKBP12 (to inhibit mTOR), each increased the [Ca(2+)](c) rise evoked by caffeine. Notwithstanding, neither mTOR nor calcineurin are required to potentiate caffeine-evoked Ca(2+) increases evoked by each drug. Thus, the mTOR and phosphatidylinositol 3-kinase inhibitor, LY294002, which directly inhibits mTOR without removing FKBP12 from RyR, did not alter caffeine-evoked [Ca(2+)](c) transients. Nor did inhibition of calcineurin by cypermethrin, okadaic acid or calcineurin inhibitory peptide block the FK506-induced increase in RyR-mediated Ca(2+) release. In aorta, although RyR3 (the main isoform), FKBP12 and calcineurin were each present, RyR-mediated Ca(2+) release was unaffected by either FK506, rapamycin or the calcineurin inhibitors cypermethrin and okadaic acid in single voltage clamped aortic myocytes. Presumably failure of FKBP12 to associate with RyR3 resulted in the immunosuppressant drugs (FK506 and rapamycin) being unable to alter the activity of RyR. The effects of these drugs are therefore, apparently dependent on an association of FKBP12 with RyR. Together, removal of FKBP12 from RyR augmented Ca(2+) release via the channel in colonic myocytes. Neither calcineurin nor mTOR are required for the FK506- or rapamycin-induced potentiation of RyR Ca(2+) release to occur. The results indicate that FKBP12 directly inhibits RyR channel activity in colonic myocytes but not in aorta.

Ion channels in smooth muscle: regulation by the sarcoplasmic reticulum and mitochondria.

In smooth muscle, Ca(2+) regulates cell division, growth and cell death as well as providing the main trigger for contraction. Ion channels provide the major access route to elevate the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) in smooth muscle by permitting Ca(2+) entry across the plasma membrane and release of the ion from intracellular Ca(2+) stores. The control of [Ca(2+)](c) relies on feedback modulation of the entry and release channels by Ca(2+) itself. Local rises in [Ca(2+)](c) may promote or inhibit channel activity directly or indirectly. The latter may arise from Ca(2+) regulation of ionic conductances in the plasma membrane to provide control of cell excitability and so [Ca(2+)](c) entry. Organelles such as mitochondria may also contribute significantly to the feedback regulation of ion channel activity by the control of Ca(2+) or redox status of the cell. This brief review describes the feedback regulation of Ca(2+) release from the internal Ca(2+) store and of plasma membrane excitability in smooth muscle.

Ins(1,4,5)P3 receptor regulation during 'quantal' Ca2+ release in smooth muscle.

Smooth muscle is activated by plasma-membrane-acting agonists that induce inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] to release Ca(2+) from the intracellular sarcoplasmic reticulum (SR) Ca(2+) store. Increased concentrations of agonist evoke a concentration-dependent graded release of Ca(2+) in a process called 'quantal' Ca(2+) release. Such a graded release seems to be incompatible with both the finite capacity of the SR store and the positive-feedback Ca(2+)-induced Ca(2+) release (CICR)-like process that is operative at Ins(1,4,5)P(3) receptors, which - once activated - might be expected to deplete the entire store. Proposed explanations of quantal release include the existence of multiple stores, each with different sensitivities to Ins(1,4,5)P(3), or Ins(1,4,5)P(3) receptor opening being controlled by the Ca(2+) concentration within the SR. Here, we suggest that the regulation of Ins(1,4,5)P(3) receptors by the Ca(2+) concentration within the SR explains the quantal Ca(2+)-release process and the apparent existence of multiple Ca(2+) stores in smooth muscle.

Ca2+ microdomains in smooth muscle.

In smooth muscle, Ca(2+) controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca(2+) to perform these multiple functions is the cell's ability to localize Ca(2+) signals to certain regions by creating high local concentrations of Ca(2+) (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca(2+) influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca(2+) store. A single Ca(2+) channel can create a microdomain of several micromolar near (approximately 200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca(2+)] and the rapid rates of decline target Ca(2+) signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca(2+) by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca(2+). In this review, the generation of microdomains arising from Ca(2+) influx across the plasma membrane and the release of the ion from the SR Ca(2+) store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.

In smooth muscle, FK506-binding protein modulates IP3 receptor-evoked Ca2+ release by mTOR and calcineurin.

Ca2+ release from the sarcoplasmic reticulum (SR) by the IP3 receptors (IP3Rs) crucially regulates diverse cell signalling processes from reproduction to apoptosis. Release from the IP3R may be modulated by endogenous proteins associated with the receptor, such as the 12 kDa FK506-binding protein (FKBP12), either directly or indirectly by inhibition of the phosphatase calcineurin. Here, we report that, in addition to calcineurin, FKPBs modulate release through the mammalian target of rapamycin (mTOR), a kinase that potentiates Ca2+ release from the IP3R in smooth muscle. The presence of FKBP12 was confirmed in colonic myocytes and co-immunoprecipitated with the IP3R. In aortic smooth muscle, however, although present, FKBP12 did not co-immunoprecipitate with IP3R. In voltage-clamped single colonic myocytes rapamycin, which together with FKBP12 inhibits mTOR (but not calcineurin), decreased the rise in cytosolic Ca2+ concentration ([Ca2+]c) evoked by IP3R activation (by photolysis of caged IP3), without decreasing the SR luminal Ca2+ concentration ([Ca2+]l) as did the mTOR inhibitors RAD001 and LY294002. However, FK506, which with FKBP12 inhibits calcineurin (but not mTOR), potentiated the IP3-evoked [Ca2+]c increase. This potentiation was due to the inhibition of calcineurin; it was mimicked by the phosphatase inhibitors cypermethrin and okadaic acid. The latter two inhibitors also prevented the FK506-evoked increase as did a calcineurin inhibitory peptide (CiP). In aortic smooth muscle, where FKBP12 was not associated with IP3R, the IP3-mediated Ca2+ release was unaffected by FK506 or rapamycin. Together, these results suggest that FKBP12 has little direct effect on IP3-mediated Ca2+ release, even though it is associated with IP3R in colonic myocytes. However, FKBP12 might indirectly modulate Ca2+ release through two effector proteins: (1) mTOR, which potentiates and (2) calcineurin, which inhibits Ca2+ release from IP3R in smooth muscle.

IP3-mediated Ca2+ increases do not involve the ryanodine receptor, but ryanodine receptor antagonists reduce IP3-mediated Ca2+ increases in guinea-pig colonic smooth muscle cells.

Smooth muscle responds to IP3-generating (sarcolemma acting) neurotransmitters and hormones by releasing Ca2+ from the sarcoplasmic reticulum (SR) via IP3 receptors (IP3Rs). This release may propagate as Ca2+ waves. The Ca2+ signal emanating from IP3 generation may be amplified by its activating further Ca2+ release from ryanodine receptors (RyRs) in the process of Ca2+-induced Ca2+ release (CICR). Evidence for this proposal has relied largely on the use of blocking drugs such as ryanodine, tetracaine and dantrolene, reportedly specific inhibitors of RyRs. Here we have examined whether or not Ca2+ released via IP3Rs subsequently activates RyRs. In addition, the specificity of the blocking agents has been assessed by determining the extent of their ability to block IP3-mediated Ca2+ release under conditions in which RyRs were not activated. IP3-evoked Ca2+ release and Ca2+ waves did not require or activate RyRs. However, the RyR blocking drugs inhibited IP3-mediated Ca2+ signals at concentrations thought to be selective for RyRs. In single colonic smooth muscle cells, voltage clamped in the whole cell configuration, carbachol (CCh) evoked propagating Ca2+ waves which were not inhibited by ryanodine when the sarcolemma potential was -70 mV. At -20 mV, at which potential the SR Ca2+ content was increased and RyRs activated, ryanodine inhibited the Ca2+ waves. Photolysed caged IP3 increased [Ca2+]c; ryanodine, by itself, did not reduce the IP3-evoked [Ca2+]c increase when the sarcolemma potential was maintained at -70 mV. However, after activation of RyRs by caffeine, in the continued presence of ryanodine, the IP3-evoked [Ca2+]c increase was inhibited. In other experiments, RyRs were activated (as evidenced by the occurrence of spontaneous transient outward currents) by depolarizing the sarcolemma to -20 mV and again ryanodine was effective in inhibiting IP3-evoked Ca2+ increase. Thus while ineffective by itself, ryanodine inhibited IP3-evoked Ca2+ increases, presumably by causing persistent opening of the channel and depleting the SR of Ca2+, after RyRs were activated. These experiments establish that IP3-evoked Ca2+ release and Ca2+ waves do not activate RyRs; had they done so ryanodine would have inhibited the Ca2+ increase. However, under conditions where ryanodine was ineffective against the IP3-evoked Ca2+ transient (i.e. when RyRs were not activated, e.g. at a membrane potential of -70 mV) tetracaine and dantrolene each blocked IP3-evoked Ca2+ increases. The results show that although IP3-mediated Ca2+ release does not activate RyRs, RyR blockers can inhibit IP3-mediated Ca2+ signals.

The sarcoplasmic reticulum, Ca2+ trapping, and wave mechanisms in smooth muscle.

The sarcoplasmic reticulum (SR) and apposed regions of the sarcolemma passively trap Ca2+ entering the cell to limit the rise in cytoplasmic Ca2+ concentration without SR pump involvement. When "leaky," the SR facilitates Ca2+ entry to the cytoplasm. SR Ca2+ release via inositol 1,4,5-trisphosphate receptors (IP(3)Rs) propagates as calcium waves; IP(3)Rs alone account for wave propagation.

Origin and mechanisms of Ca2+ waves in smooth muscle as revealed by localized photolysis of caged inositol 1,4,5-trisphosphate.

The cytosolic Ca(2+) concentration ([Ca(2+)](c)) controls diverse cellular events via various Ca(2+) signaling patterns; the latter are influenced by the method of cell activation. Here, in single-voltage clamped smooth muscle cells, sarcolemma depolarization generated uniform increases in [Ca(2+)](c) throughout the cell entirely by Ca(2+) influx. On the other hand, the Ca(2+) signal produced by InsP(3)-generating agonists was a propagated wave. Using localized uncaged InsP(3), the forward movement of the Ca(2+) wave arose from Ca(2+)-induced Ca(2+) release at the InsP(3) receptor (InsP(3)R) without ryanodine receptor involvement. The decline in [Ca(2+)](c) (the back of the wave) occurred from a functional compartmentalization of the store, which rendered the site of InsP(3)-mediated Ca(2+) release, and only this site, refractory to the phosphoinositide. The functional compartmentalization arose by a localized feedback deactivation of InsP(3) receptors produced by an increased [Ca(2+)](c) rather than a reduced luminal [Ca(2+)] or an increased cytoplasmic [InsP(3)]. The deactivation of the InsP(3) receptor was delayed in onset, compared with the time of the rise in [Ca(2+)](c), persisted (>30 s) even when [Ca(2+)](c) had regained resting levels, and was not prevented by kinase or phosphatase inhibitors. Thus different forms of cell activation generate distinct Ca(2+) signaling patterns in smooth muscle. Sarcolemma Ca(2+) entry increases [Ca(2+)](c) uniformly; agonists activate InsP(3)R and produce Ca(2+) waves. Waves progress by Ca(2+)-induced Ca(2+) release at InsP(3)R, and persistent Ca(2+)-dependent inhibition of InsP(3)R accounts for the decline in [Ca(2+)](c) at the back of the wave.

Cyclic ADP-ribose increases Ca2+ removal in smooth muscle.

Ca2+ release via ryanodine receptors (RyRs) is vital in cell signalling and regulates diverse activities such as gene expression and excitation-contraction coupling. Cyclic ADP ribose (cADPR), a proposed modulator of RyR activity, releases Ca2+ from the intracellular store in sea urchin eggs but its mechanism of action in other cell types is controversial. In this study, caged cADPR was used to examine the effect of cADPR on Ca2+ signalling in single voltage-clamped smooth muscle cells that have RyR but lack FKBP12.6, a proposed target for cADPR. Although cADPR released Ca2+ in sea urchin eggs (a positive control), it failed to alter global or subsarcolemma [Ca2+]c, to cause Ca2+-induced Ca2+ release or to enhance caffeine responses in colonic myocytes. By contrast, caffeine (an accepted modulator of RyR) was effective in these respects. The lack of cADPR activity on Ca2+ release was unaffected by the introduction of recombinant FKBP12.6 into the myocytes. Indeed in western blots, using brain membrane preparations as a source of FKBP12.6, cADPR did not bind to FKBPs, although FK506 was effective. However, cADPR increased and its antagonist 8-bromo-cADPR slowed the rate of Ca2+ removal from the cytoplasm. The evidence indicates that cADPR modulates [Ca2+]c but not via RyR; the mechanism may involve the sarcolemma Ca2+ pump.

Potassium channels underlying the resting potential of pulmonary artery smooth muscle cells.

1. The molecular identity of the K channels giving rise to the negative membrane potential of pulmonary artery smooth muscle cells has yet to be determined. 2. To date, most studies have focused on voltage-gated, delayed rectifier channels and their roles in mediating hypoxia-induced membrane depolarization. There is, however, strong evidence that an outwardly rectifying K+ conductance distinct from the classical delayed rectifier is involved. 3. Growing evidence that TASK-like channels can sense hypoxia and are present in pulmonary artery smooth muscle cells suggests that they may be responsible for the resting K+ conductance and resting potential. 4. The present review considers the evidence that particular K channels maintain the resting membrane potential of pulmonary artery smooth muscle cells and mediate the depolarizing response to hypoxia.