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.

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 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.

The sarcoplasmic reticulum and sarcolemma together form a passive Ca2+ trap in colonic smooth muscle.

In smooth muscle, active Ca(2+) uptake into regions of sarcoplasmic reticulum (SR) which are closely apposed to the sarcolemma has been proposed to substantially limit the increase in the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) following Ca(2+) influx, i.e. the 'superficial buffer barrier hypothesis'.

Intraluminal pressure is a stimulus for NFATc3 nuclear accumulation: role of calcium, endothelium-derived nitric oxide, and cGMP-dependent protein kinase.

The transcription factor NFAT (nuclear factor of activated T-cells) is implicated in cardiac hypertrophy and vasculogenesis. NFAT activation, reflecting dephosphorylation by the calcium-dependent phosphatase, calcineurin, and subsequent nuclear localization, is generally thought to require a sustained increase in intracellular calcium. However, in smooth muscle we have found that elevation of calcium by membrane depolarization fails to induce an increase in nuclear localization of the NFATc3 isoform.

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.

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.

Effects of muscarinic toxins MT2 and MT7, from green mamba venom, on m1, m3 and m5 muscarinic receptors expressed in Chinese Hamster Ovary cells.

Several small proteins called muscarinic toxins (MTs) have been isolated from venom of green mamba (Dendroaspis angusticeps). They have previously been shown in radioligand binding studies to have high selectivity and affinity for individual muscarinic receptor subtypes, but less is known of their functional effects. This study has examined the actions of two of these MTs, MT2 and MT7, using changes in cytosolic Ca(2+) ([Ca(2+)](i)) measured using the fluorescent indicator fura-2 in Chinese Hamster Ovary (CHO) cells stably transfected with individual muscarinic receptor subtypes, m1, m3 and m5.

Effects of muscarinic toxins MT1 and MT2 from green mamba on different muscarinic cholinoceptors.

MT1 and MT2, polypeptides from green mamba venom, known to bind to muscarinic cholinoceptors, behave like muscarinic agonists in an inhibitory avoidance task in rats. We have further characterised their functional effects using different preparations. MT1 and MT2 behaved like relatively selective muscarinic M1 receptor agonists in rabbit vas deferens, but their effects were not reversed by washing or prevented by muscarinic antagonists, although allosteric modulators altered responses to MT1.

Ca2+ signalling and Ca2+-activated K+ channels in smooth muscle.

In smooth muscle, transient subsarcolemma increases in Ca2+ of approximately 200 nM from the sarcoplasmic reticulum activate Ca2+-activated K+ channels (KCa) in the sarcolemma giving rise to spontaneous transient outward currents (STOCs). In the present study we have examined whether (1) STOCs are spatially restricted membrane currents, (2) single KCa channel activity is regulated by changes in bulk average cytosolic Ca2+ concentrations ([Ca2+]c) without concomitant local subsarcolemma Ca2+ changes, and (3) a relationship exists between the voltage-dependent Ca2+ current (ICa) and KCa channel activity. Guinea-pig single colonic myocytes were voltage clamped in the whole cell configuration (to measure macroscopic currents) and bulk average [Ca2+]c measured simultaneously using the dye Fura-2.

Agonist-induced phasic and tonic responses in smooth muscle are mediated by InsP(3).

Many cellular functions are regulated by agonist-induced InsP(3)-evoked Ca2+ release from the internal store. In non-excitable cells, predominantly, the initial Ca2+ release from the store by InsP(3) is followed by a more sustained elevation in [Ca2+](i) via store-operated Ca2+ channels as a consequence of depletion of the store. Here, in smooth muscle, we report that the initial transient increase in Ca2+, from the internal store, is followed by a sustained response also as a consequence of depletion of the store (by InsP(3)), but, influx occurs via voltage-dependent Ca2+ channels.

Ca(2+) regulation in guinea-pig colonic smooth muscle: the role of the Na(+)-Ca(2+) exchanger and the sarcoplasmic reticulum.

To study the contribution of the Na(+)-Ca(2+) exchanger to Ca(2+) regulation and its interaction with the sarcoplasmic reticulum (SR), changes in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) were measured in single, voltage clamped, smooth muscle cells. Increases in [Ca(2+)](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 InsP(3) (InsP(3)). Depletion of the SR of Ca(2+) (verified by the absence of a response to caffeine and InsP(3)) by either ryanodine (50 microM), to open the ryanodine receptors (RyRs), or thapsigargin (500 nM) or cyclopiazonic acid (CPA, 10 microM), to inhibit the SR Ca(2+) pumps, reduced neither the magnitude of the Ca(2+) transient nor the relationship between the influx of and the rise in [Ca(2+)](c) evoked by depolarisation.