L. Raeymaekers

Many important aspects of our life are regulated by the free cytosolic Ca2+ concentration. The intracellular Ca2+ signal is regulated both in space, frequency and amplitude. Each cell chooses a unique set of Ca2+ signals to control its function. Ca2+ signal transduction is based on rises in free cytosolic Ca2+ concentration. Ca2+ can come from the extracellular space or be released from intracellular stores. Extracellular Ca2+ enters the cell through various types of plasma-membrane Ca2+ channels and leaves the cell using Ca2+ pumps and Na+/Ca(2+)-exchangers. Ca2+ is accumulated in intracellular stores by means of Ca2+ pumps and is released via inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors. Mutations or abnormalities in one of the above mentioned Ca(2+)-transporting proteins can lead to disease. Skeletal-muscle pathology can be caused by abnormal ryanodine receptors (malignant hyperthermia, porcine stress syndrome, central core disease), plasma-membrane Ca2+ channels (hypokalemic periodic paralysis, muscular dysgenesis mice, paraneoplastic Lambert-Eaton myasthenia syndrome) or Ca2+ pumps (Brody disease). Neurologic disorders can be related to altered function of plasma-membrane Ca2+ channels (episodic ataxia type 2, spinocerebellar ataxia type 6, familial hemiplegic migraine, glutamate excitotoxicity, tottering, leaner, lethargic and stargazer mice), IP3 receptors (Lowe's oculocerebrorenal syndrome, manic depression, Alzheimer's disease, opisthotonos mice) and Ca2+ pumps (deafwaddler mouse and wriggle mouse sagami). Two skin diseases are caused by Ca(2+)-pump mutations (Darier disease and Hailey-Hailey disease). Incomplete X-linked congenital stationary night blindness is caused by a mutation in the plasma-membrane Ca2+ channels in rods and cones.

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The Ca(2+)-pump ATPases of the plasma membrane and of the endoplasmic reticulum play an important role in controlling the intracellular Ca(2+)-concentration. In this perspective it is not unexpected that these enzymes are modulated by different factors. The activity of the plasmalemmal (Ca2+ +Mg2+)ATPase is modified by the amount of negatively charged phospholipids surrounding the enzyme. Some evidence is presented indicating that in stomach and myometrium smooth muscle agonists inhibit the extrusion of Ca2+ by reducing the negatively charged phospholipids surrounding the plasmalemmal Ca(2+)-pump, while c-GMP dependent protein kinase would activate this Ca(2+)-pump by increasing this amount. The regulation of the Ca(2+)-pump of the endoplasmic reticulum depends on the phosphorylation of phospholamban by cAMP- and cGMP-dependent protein kinase. In the second part of this review, the heterogeneity of the intracellular Ca2+ compartments and a possible connection between the intracellular compartment and the extracellular solution are discussed. In addition, some data on the regulation of Ca2+ inside the nucleus are presented.

The cytoplasmic Ca2+ concentration plays a central role in the contraction of smooth muscle cells. The concentration of cytoplasmic Ca2+ is determined for an important part by the operation of Ca(2+)-transport ATPases which extrude Ca2+ from the cell or accumulate the ion in the endoplasmic reticulum. The present work concerns the characterization of the Ca(2+)-transport ATPases of smooth muscle by biochemical, immunochemical and recombinant DNA techniques. This study also includes the investigation of the regulation of the Ca(2+)-transport ATPases and of the expression of associated Ca(2+)-binding proteins. Methods were developed for the purification of endoplasmic reticulum and plasma membranes from smooth-muscle cells. From the study of the phosphorylated transport intermediates and the proteolytic breakdown products, and by using polyclonal and monoclonal antibodies we could conclude that two different Ca(2+)-transport ATPases are expressed in smooth-muscle cells. Of each of these types of Ca(2+)-transport ATPases different isoforms exist. These isoforms were further characterized at the cDNA level and by generating isoform-specific antibodies. One isoform of the plasma-membrane Ca(2+)-pump and two different organellar-type Ca(2+)-pumps have been cloned and sequenced. In smooth-muscle cells, the primary RNA transcripts of the gene of the SERCA2 Ca(2+)-transport ATPase are alternatively processed in three different ways. In neural tissues even a fourth mode of splicing occurs. These different splice modes can be explained by the analysis of the exon/intron structure of the SERCA2 gene. The regulation of the alternative RNA splicing was studied on the stable muscle-cell line BC3H1 during induced myogenic differentiation. From this study we could conclude that the mechanisms responsible for active Ca(2+)-transport in smooth-muscle cells partially resemble those of non-muscle cells, and partially resemble the corresponding system in cardiac cells, but not those in skeletal muscle. A similar conclusion was reached concerning the regulation of the Ca(2+)-transport ATPase of the endoplasmic reticulum via the phosphorylation of phospholamban, and for the expression of the Ca(2+)-binding protein calsequestrin.(ABSTRACT TRUNCATED AT 400 WORDS)

Smooth muscle cells contain two distinct Ca2+-transport ATpases with a different subcellular localization. The plasmalemmal Ca2+ pump has a relative molecular weight (Mr) of 140k and its phospho-intermediate level is increased by La3+. Its resemblance to the erythrocyte Ca2+ pump is further confirmed by its calmodulin-binding capacity and its antigenic properties. A 100k Ca2+-transport ATPase is localized in the endoplasmic reticulum. Its phospho-intermediate level is decreased by La3+, and it is antigenically related to the cardiac sarcoplasmic reticulum Ca2+-transport ATPase. These two different Ca2+-transport ATPases are present in both visceral and vascular smooth muscle, but tissue- and species-dependent differences in their relative amount have been observed. The endoplasmic-reticulum Ca2+-transport ATPase is regulated via phospholamban. Phosphorylation of this regulatory protein by cAMP-dependent as well as by cGMP-dependent protein kinase stimulates the endoplasmic-reticulum Ca2+ pump. The activity of the plasmalemmal Ca2+-transport ATPase can be modulated by calmodulin, negatively charged phospholipids, and by receptor-binding agonists. cGMP-dependent protein kinase also exerts a stimulatory effect on the plasmalemmal Ca2+ pump, but this effect is not mediated via a direct phosphorylation of the Ca2+ pump.

Changes of the intracellular Ca concentrations play a predominant though not unique role in the regulation of the force development of smooth muscle cells. Contraction is initiated by an influx of Ca2+ through the cell membrane or by the release of Ca2+ from intracellular storage sites. Ca influx occurs via voltage operated channels and receptor operated channels. The intracellular Ca release induced by agonists probably originates from the endoplasmic reticulum. The removal of Ca2+ from the cytoplasm occurs by extrusion across the plasmalemma and by reaccumulation in the endoplasmic reticulum. These active Ca2+ transport systems are catalysed by (Ca2+ +Mg2+) ATPases. Na-Ca exchange across the sarcolemma of smooth muscle is probably of minor importance since the (Ca2+ +Mg2+)ATPase activity of plasma membranes is higher than the activity of the Na+K+ ATPase, the ultimate energy source for Na+-dependent Ca2+ extrusion. The (Ca2+ +Mg2+)ATPase of the plasmalemma has a Mr of 130.000 and it is stimulated by calmodulin. It resembles the Ca2+ transport ATPase of erythrocyte membranes, including immunological cross-reactivity. The Ca2+ transport enzyme of the endoplasmic reticulum has a Mr of 100.000, is insensitive to calmodulin and resembles the Ca2+ pump of sarcoplasmic reticulum of skeletal muscle. However, antibodies against the Ca2+ pump of skeletal muscle do not cross react with the enzyme of smooth muscle. Subcellular fractionation of pig stomach smooth muscle indicates that in this tissue the large fraction of the (Ca2+ +Mg2+)ATPase activity is present in the plasma membrane while less activity is found in the endoplasmic reticulum.