John G. McCarron

Myogenic contraction by modulation of voltage‐dependent calcium currents in isolated rat cerebral arteries.

1. Tissue blood flow and blood pressure are regulated by the spontaneous, myogenic, contraction developed by resistance arteries. However, the cellular mechanisms underlying myogenic contraction are not understood. In this study, the mechanisms of myogenic contraction in cerebral resistance arteries were investigated. 2. The vasoconstriction observed in response to increased pressure in cerebral resistance arteries (myogenic reactivity) was dependent on Ca2+ entry through voltage‐dependent Ca2+ channels, since it was abolished by Ca2+ removal and by dihydropyridine antagonists of voltage‐dependent Ca2+ channels. 3. Myogenic reactivity persisted in a high‐K+ saline, with reduced Ca2+, where membrane potential is presumed to be clamped. Therefore, membrane depolarization alone does not fully account for the increased voltage‐dependent Ca2+ channel opening. 4. Voltage‐dependent Ca2+ currents in single smooth muscle cells isolated from the resistance artery were substantially increased by applying positive pressure to the patch electrode evoking membrane stretch. 5. Myogenic reactivity remained unaffected by ryanodine and therefore was independent of internal ryanodine‐sensitive Ca2+ stores. 6. The myofilament Ca2+ sensitivity was not increased by elevated pressure in alpha‐toxin‐permeabilized arteries. However, pharmacological activation of protein kinase C or G proteins did increase the myofilament Ca2+ sensitivity. 7. Myogenic contraction over the pressure range 30‐70 mmHg could be accounted for by an increase in [Ca2+]i from 100 to 200 nM. 8. It is concluded that modest increases in [Ca2+]i within the range 100‐200 nM can account for that myogenic contraction, and that stretch‐evoked modulation of Ca2+ currents may contribute to the myogenic response.

The mitochondrial membrane potential and Ca2+ oscillations in smooth muscle.

Ca2+ uptake by mitochondria might both modulate the cytosolic Ca2+ concentration ([Ca2+]c) and depolarize the mitochondrial membrane potential (ΔΨm) to limit ATP production. To investigate how physiological Ca2+ signaling might affect energy production, ΔΨm was examined during Ca2+ oscillations in smooth muscle cells. In single, voltage-clamped smooth muscle cells, inhibition of mitochondrial Ca2+ accumulation inhibited inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-evoked Ca2+ release and prolonged the time required for restoration of [Ca2+]c following activation of plasmalemmal Ca2+ currents (ICa). Ca2+ could be released from mitochondria immediately (within 15 seconds) after a [Ca2+]c rise evoked by Ins(1,4,5)P3 or ICa. Despite this evidence of mitochondrial Ca2+ accumulation, no change in ΔΨm was observed during single or repetitive [Ca2+]c oscillations evoked by these conditions. Occasionally, spontaneous, repetitive, persistent Ca2+ oscillations were observed. In these cases, mitochondria displayed stochastic ΔΨm depolarizations, which were independent both of events in neighboring mitochondria and of the timing of the [Ca2+]c oscillations themselves. Such ΔΨm depolarizations could be mimicked by increased exposure to either fluorescence excitation light or the ΔΨm-sensitive dye tetramethylrhodamine ethyl ester (TMRE) and were inhibited by antioxidants (ascorbic acid, catalase, Trolox and TEMPO) or the mitochondrial permeability transition pore (mPTP)-inhibitor cyclosporin A (CsA). Individual mitochondria within smooth muscle cells might depolarize during repetitive Ca2+ oscillations or during oxidative stress but not during the course of single [Ca2+]c transients evoked by Ca2+ influx or store release.

Depolarization‐evoked increases in cytosolic calcium concentration in isolated smooth muscle cells of rat portal vein.

1. Ca2+ current through voltage‐dependent Ca2+ channels (ICa) and intracellular free Ca2+ concentration ([Ca2+]i) were measured simultaneously in rat portal vein smooth muscle cells using conventional whole‐cell voltage clamp technique and high temporal resolution microfluorimetry. 2. The relationship between depolarization‐evoked ICa and rise in [Ca2+]i was examined. The extracellular Ca2+ concentration dependence and the voltage dependence of the depolarization‐evoked increases in ICa and [Ca2+]i were similar. Both ICa and increased [Ca2+]i were blocked to a similar extent by nimodipine and cadmium and augmented by Bay K 8644. Furthermore, the time course of the measured increase in [Ca2+]i, closely followed the increase in [Ca2+]i expected from the time‐integrated ICa. These observations suggest that the depolarization‐evoked rise in [Ca2+]i was tightly coupled to ICa. 3. The cytosolic Ca2+ buffering capacity, determined as the ratio of the expected increase in [Ca2+]i (from ICa) divided by the measured increase in [Ca2+]i, was over 100. Therefore, less than 1 out of 100 Ca2+ ions entering the cell appears as a free Ca2+. 4. Ryanodine (30 microM), a blocker of the Ca(2+)‐induced Ca2+ release mechanism, had little effect on buffering capacity measured over the first 200 ms of the depolarizing voltage clamp pulse. Ryanodine also had little effect on the buffering capacity during 800‐1000 ms of the depolarizing voltage clamp pulse. Therefore, it was concluded that there is little Ca(2+)‐induced Ca2+ release from the stores in rat portal vein smooth muscle cells during depolarization‐evoked Ca2+ entry. 5. During brief depolarizations, the largest [Ca2+]i increase and ICa occurred at 0 mV. However, during steady‐state depolarization, the largest increase in [Ca2+]i occurred around ‐30 mV, and we estimate the peak steady‐state ICa to be about 0.6 pA.

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.

Selective uncoupling of individual mitochondria within a cell using a mitochondria-targeted photoactivated protonophore

Depolarization of an individual mitochondrion or small clusters of mitochondria within cells has been achieved using a photoactivatable probe. The probe is targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and releases the protonophore 2,4-dinitrophenol locally in predetermined regions in response to directed irradiation with UV light via a local photolysis system. This also provides a proof of principle for the general temporally and spatially controlled release of bioactive molecules, pharmacophores, or toxins to mitochondria with tissue, cell, or mitochondrion specificity.

Regulation of the cytosolic Ca2+ concentration by Ca2+ stores in single smooth muscle cells from rat cerebral arteries

1 There is no general agreement on the presence or role of Ca2+‐induced Ca2+ release in smooth muscle. In this paper, Ca2+‐induced Ca2+ release has been investigated in rat resistance‐sized superior cerebral arteries to determine its role in regulating the cytosolic Ca2+ concentration ([Ca2+]i. 2 Pressurized superior cerebral arteries developed spontaneous oscillations in diameter. These oscillations were abolished by ryanodine (an inhibitor of Ca2+‐induced Ca2+ release) and removal of extracellular Ca2+. This suggests, indirectly, that Ca2+‐induced Ca2+ release may regulate [Ca2+]i in the resistance arteries. 3 To determine if Ca2+‐induced Ca2+ release could regulate [Ca2+]i, single smooth muscle cells were isolated from the superior cerebral artery, voltage clamped in the whole cell configuration and high temporal resolution [Ca2+]i measurements made. The relationship between the Ca2+ current (ICa) and rise in [Ca2+]i was examined. 4 Depolarization triggered ICa and increased [Ca2+]i. The time course of the measured increase in [Ca2+]i closely followed the increase in [Ca2+]i expected from the time‐integrated ICa, although about 140‐fold more Ca2+ entered the cytosol than appeared as free Ca2+. When the cells were dialysed with ryanodine (30 μm), the Ca2+ transient evoked by the ICa was substantially reduced indicating that Ca2+ influx triggered Ca2+ release from an internal store. 5 Voltage pulses to negative membrane potentials were more effective in triggering Ca2+ release than pulses to positive potentials suggesting that the Ca2+‐induced Ca2+ release was voltage dependent. However, the release of Ca2+ from the internal store triggered by caffeine was voltage independent. These results suggest that the voltage dependence of Ca2+ release is indirect and possibly related to the plasmalemma unitary Ca2+ current magnitude. 6 The results establish that Ca2+‐induced Ca2+ release contributes to depolarization‐evoked increases in [Ca2+]i in rat resistance‐sized superior cerebral arteries over the physiological [Ca2+]i range (100‐200 nm). Compared with more positive membrane potentials the efficacy of Ca2+ in triggering release is high at physiological membrane potentials.

The phospholipase C inhibitor U-73122 inhibits Ca2+ release from the intracellular sarcoplasmic reticulum Ca2+ store by inhibiting Ca2+ pumps in smooth muscle

Background and purpose:  The sarcoplasmic reticulum (SR) releases Ca2+ via inositol 1,4,5‐trisphosphate receptors (IP3R) in response to IP3‐generating agonists. Ca2+ release subsequently propagates as Ca2+ waves. To clarify the role of IP3 production in wave generation, the contribution of a key enzyme in the production of IP3 was examined using a phosphoinositide‐specific phospholipase C (PI‐PLC) inhibitor, U‐73122.