The Intracellular Calcium Store

Removal of extracellular Ca does not significantly impair either the initial development of tension (as opposed to the maintained contraction) or the transient p eak in cytosohc Ca in Fura-2-loaded ASM which occur in response to agonist stimulation, but it does abolish the plateau level of raised Ca during the maintained phase (Fig. 9.2). 

The sr has been identified as the major intracellular source of activator Ca in both smooth and striated muscle (Somlyo etal., 1981 Bond etcd., 1984 Somlyo, 1985). The sr of smooth muscle is a system of membranous tubules which has components closely underlying the surface plasmalemmal membrane, as well as deeper portions contiguous with the double membrane of the 

Cytosolic Ca is transferred into the store by a 105 kDa Ca, Mg -ATPase situated in the sr membrane. This enzyme pump undergoes Ca -dependent phosphorylation (Sumida et /., 1984). It is also regulated through cAMP-mediated phosphorylation of phos-pholamban (Raeymaekers etal., 1986 Eggprmont etal., 1988 Twort and van Breemen, 1989), as a result of which elevation of cytosolic cAMP lowers cytosolic Ca by stimulating uptake into the sr. 

The Ca content of the sr is determined by a balance between the activity of the Ca -ATPase pump and mechanisms which release Ca from the store (Fig. 9.3). These mechanisms include the opening of ion channels in the sr membrane in response to IP3 and passive leak of Ca out of the sr. Both of these processes involve the movement of Ca from the high concentration within the store, estimated to be 5 mM Ca (Leijten and van Breemen, 1984), to the low concentration (100 nM) within the cytosol. Functional studies in vascular smooth muscle have demonstrated that the quantity of Ca stored in the sr is sufficient to activate maximal 

Mitochondria are not involved in storing activator Ca in smooth muscle (Twort and van Breemen, 1989). Despite the importance of Ca transport systems within the mitochondria for controlling cellular metabolism, the endogenous Ca content of mitochondria is low (Bond etal., 1984), and mitochondria in smooth muscle only accumulate Ca at pathological (10 fiM) rather than physiological (100 nM) cytosolic Ca concentrations (Yamamoto and van Breemen, 1986). 

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The calcium leak channel (described below) is also a SOC, since the single channel activity of the leak channel and manganese influx through the leak channel are greatly increased in cultured myotubes by depletion of the intracellular calcium stores (Hopf et al., 1996a). 

Twort, C.H.C. (1994). The intracellular calcium store in airway smooth muscle. In Airways Smooth Muscle Biochemical Control of Contraction and Relaxation (eds D. Raeburn and M.A. Giembycz), pp. 97-115. Virkhauser Verlag, Basel. 

Twort, C. and van Breemen, C. (1989). Human airway smooth muscle in cell culture control of the intracellular calcium store. Pulm. Pharmacol. 2, 45-53. 

The localization of the IP3 receptor and the intracellular calcium stores of Purkinje cells... 

It is now generally accepted that both of the products of phosphatidylinositol 4,5-bisphosphate hydrolysis can function as intracellular second messengers. 1,2-Diacylglycerol can affect a variety of intracellular processes by activation of protein kinase C [ 148, 229, 230]. Inositol 1,4,5-trisphosphate, on the other hand, has been shown to release calcium ions from non-mitochondrial stores in a number of peripheral tissues and may thus be the link between the receptor and the intracellular calcium store in many pharmacological responses [231-233]. Furthermore, it remains a possibility that inositol phospholipid hydrolysis may also have a r61e in calcium gating [221,234]. If inositol phospholipid metabolism is closely coupled to receptor-mediated calcium mobilization, then this response may be a more general consequence of H,-receptor stimulation than other H,-responses. 

NADP can be converted to nicotinic acid adenine dinucleotide phosphate (NAADP), which has distinct functions in the regulation of intracellular calcium stores. The studies of these new roles of NAD(P) in metabolism are in their early stages, but they might soon help to better understand and explain the symptoms of niacin deficiency ( pellagra) [1]. 

The ion channel receptors are relatively simple in functional terms because the primary response to receptor activation is generated by the ion channel which is an integral part of the protein. Therefore, no accessory proteins are needed to observe the response to nicotinic AChR activation and the full functioning of the receptor can be observed by isolating and purifying the protein biochemically and reconstituting the protein in an artificial lipid membrane. In contrast, the G-protein-coupled receptors require both G-proteins and those elements such as phospholipase-C illustrated in Fig. 3.1, in order to observe the response to receptor activation (in this case a rise in intracellular calcium concentration resulting from the action of IP3 on intracellular calcium stores). 

Figure 3.1 Schematic representation of a generic excitatory synapse in the brain. The presynaptic terminal releases the transmitter glutamate by fusion of transmitter vesicles with the nerve terminal membrane. Glutamate diffuses rapidly across the synaptic cleft to bind to and activate AMPA and NMDA receptors. In addition, glutamate may bind to metabotropic G-protein-coupled glutamate receptors located perisynaptically to cause initiation of intracellular signalling via the G-protein, Gq, to activate the enzyme phospholipase and hence produce inositol triphosphate (IP3) which can release Ca from intracellular calcium stores...

If some of the electrophysiological effects of oxidant stress occur secondary to an elevation in intracellular calcium, it is important to consider the possible factors that may underlie the initial elevation of calcium. In the simplest analysis, elevation of cytosolic calcium may be due to (1) redistribution of intracellular calcium stores (2) increased calcium influx or (3) decreased calcium efflux. 

Two general mechanisms have been considered by which a depleted intracellular Ca2+ pool might communicate with the plasma membrane [4]. There is evidence that the IP3 receptor is associated with the cytoskeleton, and this association may tether the IP3 receptor to the plasma membrane. Depletion of intracellular calcium stores might cause a conformational change in the IP3 receptor, which could be conveyed to the plasma membrane via the cytoskeleton or by a more direct protein-protein interaction. Alternatively, signaling could occur... 

The sER also functions as an intracellular calcium store, which normally keeps the Ca level in the cytoplasm low. This function is particularly marked in the sarcoplasmic reticulum, a specialized form of the sER in muscle cells (see p. 334). For release and uptake of Ca " ", the membranes of the sER contain signal-controlled Ca channels and energy-dependent Ca ATPases (see p. 220). In the lumen of the sER, the high Ca " " concentration is buffered by Ca -binding proteins. 

Calcium ion facilitates the toxic actions of cardiac glycosides by accelerating the overloading of intracellular calcium stores that appears to be responsible for digitalis-induced abnormal automaticity. Hypercalcemia therefore increases the risk of a digitalis-induced arrhythmia. The effects of magnesium ion appear to be opposite to those of calcium. These interactions mandate careful evaluation of serum electrolytes in patients with digitalis-induced arrhythmias. 

Terentyev, D., Viatchenko-Karpinski, S., Gyorke, I., Volpe, P., Williams, S. C., and Gyorke, S. (2003). Calsequestrin Determines the Functional Size and Stability of Cardiac Intracellular Calcium Stores Mechanism for Hereditary Arrhythmia. Proc Natl Acad Sci USA 100(20) 11759-64. 

SOCs, also known as ICRAC (calcium-release activated channels), have been observed in a wide range of cell types (Parekh and Penner, 1997). The defining property is that depletion of intracellular calcium stores results in increased calcium influx at the plasma membrane. The actual SOC that carries this calcium influx may vary between cells, and cloning studies have identified transient receptor potential channel (TRPC) (Parekh and Penner, 1997, Vandebrouck et al., 2002b) and CRACM1 (Peinelt et al., 2006) as candidate genes. Also, the exact mechanism by which SOCs are activated by store depletion has only been partly elucidated, with a role suggested for a calcium sensor on the endoplasmic reticulum (see Peinelt et al., 2006) and for IP3 (Kiselyov et al., 1998). 

Very little work has been reported on the role of oxidative stress in osteoblasts. However, osteoblasts can be induced to produce intracellular ROS (Cortizo et al., 2000 Liu et al., 1999), which can cause a decrease in alkalinephosphatase (ALP) activity that is partially inhibited by vitamin E and cause cell death (Cortizo et al., 2000 Liu et al., 1999). Treatment of rat osteosarcoma ROS 17/2.8 cells with tumor necrosis factor-alpha (TNF-a) suppressed bone sialoprotein (BSP) gene transcription through a tyrosine kinase-dependent pathway that generates ROS (Samoto et al., 2002). H202 modulated intracellular calcium (Ca2+) activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores (Nam et al., 2002). 

Ca2+ levels, with one of the intracellular calcium-chelating, fluorescent probes, quin-2, fura-2 or indo-1, demonstrates that, in both cases, there is a rapid rise in intracellular Ca2+ as this ion is released from intracellular stores. Analysis of stimulated B lymphocytes, using the probe fura-2, indicates that if Ca2+ in the external medium is removed the intracellular Ca2+ level returns to basal levels in 5 to 7 minutes, but if there is Ca2+ present in the external media a sustained increase in intracellular Ca2+ is detected [36]. Such analysis suggests the opening of a plasma membrane calcium channel but the nature of the channel or mechanism of its opening are not presently known. It is possible that the opening of this channel could be stimulated by one of the inositol phosphates. 

Figure 6.1. Overview of cellular calcium transport. Calcium enters the cell through voltage- or ligand-gated channels (left). It is extmded by ATP-driven pumps or by sodium antiport (right). Both the mitochondria and the endoplasmic reticulum serve as intracellular calcium stores. The cytosolic concentration is kept at -100 nM under resting conditions. CaM Calmodulin.

Both cADP-rihose and NAADP act to increase cytosolic calcium concentrations, releasing calcium from intracellular stores via a receptor distinct from that which responds to inositol trisphosphate (Section 14.4.1). The responses to cADP-rihose and NAADP are additive, and they seem to act on different intracellular calcium stores (Jacohson et al., 1995 Patel et al., 2001). 

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