Thomas C. Muir

Mitochondrial regulation of the cytosolic Ca2+ concentration and the InsP3‐sensitive Ca2+ store in guinea‐pig colonic smooth muscle

1 Mitochondrial regulation of the cytosolic Ca2+ concentration ([Ca2+]c) in guinea‐pig single colonic myocytes has been examined, using whole‐cell recording, flash photolysis of caged InsP3 and microfluorimetry. 2 Depolarization increased [Ca2+]c and triggered contraction. Resting [Ca2+]c was virtually restored some 4 s after the end of depolarization, a time when the muscle had shortened to 50 % of its fully relaxed length. The muscle then slowly relaxed (t½= 17 s). 3 The decline in the Ca2+ transient was monophasic but often undershot or overshot resting levels, depending on resting [Ca2+]c. The extent of the overshoot or undershoot increased with increasing peak [Ca2+]c. 4 Carbonyl cyanide m‐chlorophenyl hydrazone (CCCP; 5 μM), which dissipates the mitochondrial proton electrochemical gradient and therefore prevents mitochondrial Ca2+ accumulation, slowed Ca2+ removal at high (> 300 nM) but not at lower [Ca2+]c and abolished [Ca2+]c overshoots. Oligomycin B (5 μM), which prevents mitchondrial ATP production, affected neither the rate of decline nor the magnitude of the overshoot. 5 During depolarization, the global rhod‐2 signal (which represents the mitochondrial matrix Ca2+ concentration, [Ca2+]m) rose slowly in a CCCP‐sensitive manner during and for about 3 s after depolarization had ended. [Ca2+]m then slowly decreased over tens of seconds. 6 Inhibition of sarcoplasmic reticulum Ca2+ uptake with thapsigargin (100 nM) reduced the undershoot and increased the overshoot. 7 Flash photolysis of caged InsP3 (20 μM) evoked reproducible increases in [Ca2+]c. CCCP (5 μM) reduced the magnitude of the [Ca2+]c transients evoked by flash photolysis of caged InsP3. Oligomycin B (5 μM) did not reduce the inhibition of the InsP3‐induced Ca2+ transient by CCCP thus minimizing the possibility that CCCP lowered ATP levels by reversing the mitochondrial ATP synthase and so reducing SR Ca2+ refilling. 8 While CCCP reduced the magnitude of the InsP3‐evoked Ca2+ signal, the internal Ca2+ store content, as assessed by the magnitude of ionomycin‐evoked Ca2+ release, did not decrease significantly. 9 [Ca2+]c decline in smooth muscle, following depolarization, may involve mitochondrial Ca2+ uptake. Following InsP3‐evoked Ca2+ release, mitochondrial uptake of Ca2+ may regulate the local [Ca2+]c near the InsP3 receptor so maintaining the sensitivity of the InsP3 receptor to release Ca2+ from the SR.

Origin and Mechanisms of Ca2+ Waves in Smooth Muscle as Revealed by Localized Photolysis of Caged Inositol 1,4,5-Trisphosphate

The cytosolic Ca2+ concentration ([Ca2+]c) controls diverse cellular events via various Ca2+ 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 [Ca2+]c throughout the cell entirely by Ca2+ influx. On the other hand, the Ca2+ signal produced by InsP3-generating agonists was a propagated wave. Using localized uncaged InsP3, the forward movement of the Ca2+ wave arose from Ca2+-induced Ca2+ release at the InsP3 receptor (InsP3R) without ryanodine receptor involvement. The decline in [Ca2+]c (the back of the wave) occurred from a functional compartmentalization of the store, which rendered the site of InsP3-mediated Ca2+ release, and only this site, refractory to the phosphoinositide. The functional compartmentalization arose by a localized feedback deactivation of InsP3 receptors produced by an increased [Ca2+]c rather than a reduced luminal [Ca2+] or an increased cytoplasmic [InsP3]. The deactivation of the InsP3 receptor was delayed in onset, compared with the time of the rise in [Ca2+]c, persisted (>30 s) even when [Ca2+]c had regained resting levels, and was not prevented by kinase or phosphatase inhibitors. Thus different forms of cell activation generate distinct Ca2+ signaling patterns in smooth muscle. Sarcolemma Ca2+ entry increases [Ca2+]c uniformly; agonists activate InsP3R and produce Ca2+ waves. Waves progress by Ca2+-induced Ca2+ release at InsP3R, and persistent Ca2+-dependent inhibition of InsP3R accounts for the decline in [Ca2+]c at the back of the wave.

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.

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.

Neuronal mediators of inhibitory junction potentials and relaxation in the guinea‐pig internal anal sphincter.

1. Inhibitory junction potentials (IJPs) and relaxations evoked in response to field stimulation (supramaximal voltage, 0.1 ms, single stimulus and 5 stimuli at 5‐40 Hz) of non‐adrenergic non‐cholinergic (NANC) nerves with atropine and phentolamine (each 1 microM) were measured in the guinea‐pig internal anal sphincter (gpIAS). The mean resting membrane potential was ‐44.2 +/‐ 0.2 mV (n = 1119 cells from 260 preparations). 2. NANC nerve stimulation evoked frequency‐dependent IJPs (19.7 +/‐ 1.1 mV, n = 165, 33 tissues to a single stimulus) and relaxations. IJPs consisted of two tetrodotoxin (1 microM)‐sensitive components: one was abolished by apamin (0.3 microM) and the P2‐purinoceptor antagonist suramin (100 microM); the other, smaller in amplitude, was sensitive to inhibitors of nitric oxide synthase (NOS, e.g. L‐NAME, 100 microM) and the nitric oxide (NO) scavenger oxyhaemoglobin (HbO, 10 microM). 3. ATP (1 mM), vasoactive intestinal polypeptide (VIP, 0.01‐0.25 microM) and pituitary adenylate cyclase‐activating peptide (PACAP(1‐27), 0.84 microM) each hyperpolarized and relaxed the gpIAS; only ATP responses resembled the evoked IJPs in time course. 4. The guanylyl cyclase inhibitor LY83583 (10 microM) abolished apamin‐insensitive IJPs and relaxations. The cGMP phosphodiesterase inhibitor M&B 22948 (30 microM) and 8‐Br‐cGMP (100 microM) each hyperpolarized the gpIAS. 5. Two components comprise the IJP and relaxation evoked in response to NANC nerve stimulation in the gpIAS. One, sensitive to apamin, resembles the response to ATP and is modulated by purinoceptor antagonists; the other, apamin and suramin insensitive, is inhibited by NO antagonists.

Ca2+ regulation in guinea‐pig colonic smooth muscle: the role of the Na+‐Ca2+ exchanger and the sarcoplasmic reticulum

To study the contribution of the Na+‐Ca2+ exchanger to Ca2+ regulation and its interaction with the sarcoplasmic reticulum (SR), changes in cytoplasmic Ca2+ concentration ([Ca2+]c) were measured in single, voltage clamped, smooth muscle cells. Increases in [Ca2+]c were evoked by either depolarisation (−70 mV to 0 mV) or by release from the SR by caffeine (10 mm) or flash photolysis of caged InsP3 (InsP3). Depletion of the SR of Ca2+ (verified by the absence of a response to caffeine and InsP3) by either ryanodine (50 μm), to open the ryanodine receptors (RyRs), or thapsigargin (500 nm) or cyclopiazonic acid (CPA, 10 μm), to inhibit the SR Ca2+ pumps, reduced neither the magnitude of the Ca2+ transient nor the relationship between the influx of and the rise in [Ca2+]c evoked by depolarisation. This suggested that Ca2+‐induced Ca2+ release (CICR) from the SR did not contribute significantly to the depolarisation‐evoked rise in [Ca2+]c. However, although Ca2+ was not released from it, the SR accumulated the ion following depolarisation since ryanodine and thapsigargin each slowed the rate of decline of the depolarisation‐evoked Ca2+ transient. Indeed, the SR Ca2+ content increased following depolarisation as assessed by the increased magnitude of the [Ca2+]c levels evoked each by InsP3 and caffeine, relative to controls. The increased SR Ca2+ content following depolarisation returned to control values in approximately 12 min via Na+‐Ca2+ exchanger activity. Thus inhibition of the Na+‐Ca2+ exchanger by removal of external Na+ (by either lithium or choline substitution) prevented the increased SR Ca2+ content from returning to control levels. On the other hand, the Na+‐Ca2+ exchanger did not appear to regulate bulk average Ca2+ directly since the rates of decline in [Ca2+]c, following either depolarisation or the release of Ca2+ from the SR (by either InsP3 or caffeine), were neither voltage nor Na+ dependent. Thus, no evidence for short term (seconds) control of [Ca2+]c by the Na+‐Ca2+ exchanger was found. Together, the results suggest that despite the lack of CICR, the SR removes Ca2+ from the cytosol after its elevation by depolarisation. This Ca2+ is then removed from the SR to outside the cell by the Na+‐Ca2+ exchanger. However, the exchanger does not contribute significantly to the decline in bulk average [Ca2+]c following transient elevations in the ion produced either by depolarisation or by release from the store.

Two Ca2+ entry pathways mediate InsP3‐sensitive store refilling in guinea‐pig colonic smooth muscle

1 Sarcolemma Ca2+ influx, necessary for store refilling, was well maintained, over a wide range (‐70 to + 40 mV) of membrane voltages, in guinea‐pig single circular colonic smooth muscle cells, as indicated by the magnitude of InsP3‐evoked Ca2+ transients. 2 This apparent voltage independence of store refilling was achieved by the activity of sarcolemma Ca2+ channels some of which were voltage gated while others were not. At negative membrane potentials (e.g. ‐70 mV), Ca2+ influx through channels which lacked voltage gating provided for store refilling while at positive membrane potentials (e.g. +40 mV) voltage‐gated Ca2+ channels were largely responsible. 3 Sarcolemma voltage‐gated Ca2+ currents were not activated following store depletion. 4 Removal of external Ca2+ or the addition of the Ca2+ channel blocker nimodipine (1 μM) inhibited store refilling, as assessed by the magnitude of InsP3‐evoked Ca2+ transients, with little or no change in bulk average cytoplasmic Ca2+ concentration. One hypothesis for these results is that the store may refill from a high subsarcolemma Ca2+ gradient. 5 Influx via channels, some of which are voltage gated and others which lack voltage gating, may permit the establishment of a subsarcolemma Ca2+ gradient. Store access to the gradient allows InsP3‐evoked Ca2+ signalling to be maintained over a wide voltage range in colonic smooth muscle.

Effects of calcium channel antagonists on action potential conduction and transmitter release in the guinea-pig vas deferens.

1 The effects of the Ca2+ channel antagonists amlodipine, cobalt, diltiazem, nifedipine and verapamil and the local anaesthetic lignocaine were investigated on action potential conduction in and on evoked transmitter release from sympathetic nerves in the guinea‐pig isolated vas deferens. Transmitter release was investigated by measurement of (a) evoked (trains of pulses at 1 and 2 Hz, 0.1‐0.5 ms supramaximal voltage) excitatory junction potentials (e.j.ps) using microelectrodes; tension was recorded simultaneously; (b) tritium (3H) overflow from vasa preincubated (37°C, 30 min) in Krebs solution containing either [3H]‐noradrenaline (NA, 25 μCi ml−1, 2 × 10−6 M NA) or [3H]‐adenosine (50 μCi ml−1, 1 × 10−6 M adenosine). 2 Amlodipine (0.5–2 × 10−4 M), verapamil (0.5‐2 × 10−4 M), diltiazem (1–8 × 10−4 M), lignocaine (0.1–2 × 10−3 M) and cobalt (2–6 × 10−2 M) in descending order of potency, but not nifedipine (1–5 × 10−3 M), increased the latency and inhibited, then abolished, the amplitude and number of action potentials in a concentration‐dependent manner. 3 Amlodipine (0.5–1 × 10−4 M), verapamil (1–2 × 10−4 M), diltiazem (1–5 × 10−4 M) and cobalt (1 × 10−3 M), in descending order of potency, but not nifedipine (5 × 10−4 M), inhibited then abolished evoked e.j.ps in a concentration‐dependent manner. Cobalt inhibited e.j.ps at a lower concentration than that (2–6 × 10−2 M) required to block action potential conduction. 4 In unstimulated tissues, the resting 3H overflow following preincubation with [3H]‐NA consisted largely of 4‐hydroxy 3‐methoxymandelic acid (VMA), 4‐hydroxy 3‐methoxy phenylglycol (MOPEG), 3,4 dihydroxyphenylglycol (DOPEG) and NA; stimulated tissues (300 pulses at 20 Hz, 0.5 ms supramaximal voltage) released mainly NA. Verapamil (0.1–1 × 10−4 M), amlodipine (0.05–1 × 10−4 M) and nifedipine (1–5 × 10−4 M), but not cobalt (2 × 10−3 M), increased, significantly, the resting overflow of 3H comprising mainly DOPEG. Cobalt (2 × 10−3 M) inhibited, significantly, the stimulation‐evoked overflow of 3H. 5 Verapamil (1 × 10−4 M) had little effect on the resting overflow of 3H following preincubation with [3H]‐adenosine. Diltiazem (5 × 10−4 M) and cobalt (2 × 10−3 M) both inhibited evoked 3H overflow. Nifedipine (5 × 10−4 M) was ineffective. 6 The effectiveness of Ca2+ channel antagonists at pre‐ and postjunctional sites differ; the results are discussed in terms of the selectivity of these drugs for each site and their differential effects on transmitter release.

Neuroeffector transmission in the guinea-pig internal anal sphincter: an electrical and mechanical study.

ATP (10(-7)-10(-4) M), ADP (10(-7)-10(-4) M), AMP (10(-7)-10(-4) M) and adenosine (10(-6)-10(-4) M) each hyperpolarized the membrane, inhibited spontaneous spike discharge and relaxed the guinea-pig internal anal sphincter. All experiments were carried out using intracellular microelectrode and simultaneous tension recording techniques in the presence of phentolamine (10(-6) M) and atropine (10(-6) M). ATP was the most effective and produced a concentration-dependent membrane potential change comparable in amplitude to that produced by field stimulation of non-adrenergic non-cholinergic (NANC) nerves. Inhibitory junction potentials, the accompanying relaxations and the responses to ATP (5 X 10(-6)-5 X 10(-5) M) were additive and were increased in K+-deficient and decreased in K+-rich solutions and inhibited by apamin (10(-7) M). A proteolytic enzyme, alpha-chymotrypsin (0.5 U/ml) preferentially antagonized the ability of vasoactive intestinal polypeptide (10(-7) M) to hyperpolarize the membrane and relax the sphincter. The electrical and mechanical responses to ATP (10(-5) M) and inhibitory nerve stimulation were only slightly reduced. The results are consistent with the view that ATP or a related adenine nucleotide may have a transmitter role in the guinea-pig internal anal sphincter.

Mechanisms underlying the electrical and mechanical responses of the guinea-pig internal anal sphincter to field stimulation and to drugs

1 The electrical membrane characteristics and the response of the circular muscle of the guinea‐pig internal anal sphincter (i.a.s.) to field stimulation were studied in vitro using intracellular microelectrodes and conventional mechanical recording techniques. 2 The i.a.s. developed its own tone (3–4 g), following initial stretch (1 g) and spontaneous spike potentials were evident. In the absence of spike potentials, tone declined and disappeared. Tone was not significantly reduced by phentolamine (1 × 10−6 M). The resting membrane potential, measured between spontaneous spike potentials, was − 45 ± 3.0 mV (n = 224); the space constant (Λ) was 1.13 ± 0.1 mm (n = 13). Spikes usually overshot by approximately 15 mV. 3 The frequency of spike potential discharge (1–3 Hz) varied with the degree of membrane depolarization, being increased in K+‐rich and decreased in K+‐deficient solutions or by the presence of Mn2+. It was not significantly affected by Cl−‐withdrawal but was increased in Na+‐deficient solutions with or without tetrodotoxin (TTX; 1 × 10−6 M). 4 Field stimulation (1–20 Hz, 0.5 ms, supramaximal voltage) produced inhibitory junction potentials (i.j.ps) and relaxed tone; at high frequencies (50 Hz or greater), contractions were observed but excitatory junction potentials (e.j.ps) were not. I.j.ps and relaxations were inhibited by apamin (1 × 10−6M), TTX (1 × 10−6M) but not by atropine (1 × 10−6M), phentolamine (1 × 10−6M) or hexamethonium (1 × 10−6M). 5 I.j.ps were reduced by hyperpolarization and enhanced by depolarization of the membrane by current pulses (15 s). The mean equilibrium potential for the i.j.p. was − 94 mV (correlation coefficient, γ = 0.71, n = 5, p < 0.001). I.j.ps were enhanced in K+‐deficient solutions and reduced in K+‐rich solutions. Together these results suggest that the i.j.p. is mediated by an increased GK. The absence of [Ca2+]o or the presence of Mn2+ (2 mM) abolished the i.j.p.; in contrast Na+‐deficient or Cl−‐free solutions were ineffective in this respect. 6 Tetraethylammonium (5–50 mM) abolished the i.j.p.; the accompanying relaxation was reduced by about 80%. The major aspect of the relaxation to nerve stimulation is mediated by membrane hyperpolarization.