Signal transduction in smooth muscle as studied by the subcellular membrane approach.

Many of the contractile regulatory events in smooth muscle reside in various cellular membrane components as functional membrane constituents that interact in a variably complex manner. The physiological handling of ionized calcium (Ca2+), which serves multiple roles as an extracellular signal, a second messenger, and an activator interacting directly with myofilaments to effectuate contractile responses, referred to as Ca2+ signalling processes, represents an integral part of a more complicated membrane transduction mechanism. The subcellular membrane approach toward the understanding of Ca2+ signalling as well as the transduction mechanisms involving membrane receptors, GTP binding proteins, ion channels, membrane-bound enzymes, and the production of intracellular second messengers has made a significant contribution in smooth muscle research for the past decade. This review summarizes the current state of knowledge about the multiplicity of interactions between Ca2+ and various membrane constituents in the surface membranes and sarcoplasmic reticulum, such as Ca2+ binding, Ca2+ ATPase pumps, Ca2+ channels, and Ca2+Na+ or related ion exchangers. A number of recent novel findings from this laboratory have also been discussed. First of all, the technical refinement of membrane separation and characterization, which permits better identification of neuronal membranes in highly innervated smooth muscle tissues, led to the distinction of prejunctional and postjunctional membrane receptors. Secondly, unlike the Ca(2+)-release channels labelled with [3H]inositol 1,4,5-trisphosphate, the other type of internal membrane Ca(2+)-release channels labelled by [3H]ryanodine has been identified only recently in smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and properties of plasma membrane from smooth muscle

A generalized approach to obtain relatively pure fractions of plasma membrane from smooth muscle tissues for studying calcium transport is described. The use of various markers for cellular membranes to establish the purity of various fractions is critically considered. Plasma membranes from rat myometrium have been isolated in a purity estimated to be 95-99%. Plasma membrane purifications to 70-80% have been achieved from rat mesenteric arteries and veins, canine tracheal smooth muscle, rabbit intestinal muscle, rat vas deferens, rat fundus, and dog gastric corpus. The ATP-dependent transport of Ca is correlated with the distribution of plasma membrane markers. Ca gradient of greater than 1000-fold have been achieved. ATP-dependent active Ca transport by plasma membranes could sometimes be stimulated by oxalate or phosphate. Anion activation of Ca active transport is not a marker for endoplasmic reticulum. In some smooth muscles (e.g., rat vas deferens) ATP-dependent Ca uptake did not correlate exclusively with the distribution of plasma membrane markers. Instead, the correlation seemed to be with NADPH-cytochrome reductase EC 1.6.2.5 activity (putative endoplasmic reticulum marker) as well as with plasma membrane markers. In all smooth muscles, active Ca transport appears to be a property of the plasma membrane; in some it may also be a property of the endoplasmic reticulum. Mitochondria actively transport Ca, but in most systems studied to date, the Km for Ca2+ for this transport is higher than that for plasma membrane. Thus the plasma membrane may be the major physiological mechanism of active transport for Ca out of cytoplasm of smooth muscle cells. In two plasma membrane fractions (from rat myometrium and mesenteric arteries) it has been possible to demonstrate the existence of an Na-Ca exchange system. Its contribution to lowering cytoplasmic Ca is unknown.     

Mg2+- or Ca2+-activated ATPase activities of plasma membranes isolated from vascular smooth muscle

Studies of ATP hydrolysis by various subcellular fractions isolated from rat mesenteric arteries and veins indicate that an apparent ATPase activity, which can be activated by Mg2+ or Ca2+, is primarily associated with the plasma membranes. Although both Mg2+-activated and Ca2+-activated ATPase activities under the optimal condition are substantially lower in venous than in arterial plasma membrane fraction, their dependence on the concentration of Mg2+ and Ca2+ are quite similar in arterial as well as venous plasma membrane fractions. No synergistic effect on ATP hydrolysis was observed in the presence of both Mg2+ and Ca2+. In addition, Mg2+-activated and Ca2+-activated ATPase activities show similar pH dependence, inhibition by deoxycholate, stability toward heat inactivation and substrate specificity. Furthermore, Mg2+-activated and Ca2+-activated ATPase activities were similarly reduced in vascular smooth muscles of spontaneously hypertensive rats. These results suggest that the activation of ATP hydrolysis by Mg2+ or Ca2+ may represent a single enzyme moiety in the plasma membrane of vascular smooth muscle. The possible involvement of such ATPase in the Ca2+ transport function of vascular smooth muscle is discussed.     

A role of calcium in altered sodium ion transport of hypertensives?

Research on the etiology of essential hypertension has led to many reports of altered ion transport in cells from hypertensive patients and animal models. Abnormalities in sodium and calcium ion gradients and transport in vascular smooth muscle, neuronal tissue, cardiac muscle as well as erythrocytes have been extensively investigated. It is not clear whether these abnormalities are of primary or secondary nature. The current knowledge of sodium and calcium ion transport in essential hypertension is briefly reviewed here. Furthermore, evidence is presented which suggests a role of calcium in the regulation of sodium transport activity.     

In vitro inhibition of calcium binding by alloxan and of calcium transport by isolated vascular smooth muscle microsomes

The primary purpose of this study was to investigate the possible direct toxic effect of alloxan on the Ca2+ handling by microsomal membranes isolated from rat mesenteric arteries. It was found that preincubation of the vascular muscle microsomal membranes with alloxan had a suppressive effect on both binding of Ca2+ (in the absence of ATP) and ATP-supported Ca2+ transport. Such an inhibition was time, dose, pH, and temperature dependent. ATP-supported Ca2+ transport was more susceptible to the inhibitory action of alloxan than Ca2+ binding. Unlike alloxan, another commonly used diabetogenic drug, streptozotocin, was not effective in causing such an in vitro inhibition of Ca2+ handling.     

Vascular Na+/Ca2+ exchanger: implications for the pathogenesis and therapy of salt-dependent hypertension

The Na+/Ca2+ exchanger is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on membrane potential and transmembrane ion gradients. In arterial smooth muscle cells, the Na+/Ca2+ exchanger is thought to participate in the maintenance of vascular tone by regulating cytosolic Ca2+ concentration. Recent pharmacological and genetic engineering studies have revealed that the Ca2+ influx mode of vascular Na+/Ca2+ exchanger type-1 (NCX1) is involved in the pathogenesis of salt-dependent hypertension. SEA0400, a specific Na+/Ca2+ exchange inhibitor that preferentially blocks the Ca2+ influx mode, lowers arterial blood pressure in salt-dependent hypertensive models, but not in normotensive rats or other types of hypertensive rats. Furthermore, heterozygous mice with reduced expression of NCX1 are resistant to development of salt-dependent hypertension, whereas transgenic mice with vascular smooth muscle-specific overexpression of NCX1 readily develop hypertension after high-salt loading. SEA0400 reverses the cytosolic Ca2+ elevation and vasoconstriction induced by nanomolar ouabain, as well as humoral factors in salt-loaded animals. One possibility is that circulating endogenous cardiotonic steroids may be necessary for NCX1-mediated hypertension. These findings help to explain how arterial smooth muscle cells in blood vessels contribute to salt-elicited blood pressure elevation and suggest that NCX1 inhibitors might be therapeutically useful for salt-dependent hypertension.     

Aggregation of smooth muscle membranes and its use in the preparation of plasma membrane enriched fraction from gastric fundus smooth muscle

Microsomal membranes isolated from rat gastric fundus smooth muscle by differential centrifugation aggregate substantially in the presence of the divalent metal ion Mg2+ or Ca2+. The magnitude of cation-induced membrane aggregation is higher for Ca2+ than for Mg2+, but the ion concentration required for half-maximum membrane aggregation (K0.5 value) is similar for Mg2+ and Ca2+. Cation-induced membrane aggregation is suppressed by high ionic strength and low pH of the medium. Cation-induced membrane aggregation of mitochondrial membrane and plasma membrane enriched fractions differ in the rate of aggregate formation, metal ion concentration dependence, and pH dependence. Such different properties of membrane aggregation were used to prepare a plasma membrane enriched fraction by conventional differential centrifugation. Subfractionation of the heterogeneous microsomal membranes by free-flow electrophoresis indicated that smooth muscle plasma membranes showed a higher electrophoretic mobility than the intracellular membranes. These results suggest that ionic interactions on the cell membrane surfaces differ from those on the intracellular membrane surfaces and that induction of membrane aggregation by Ca2+ or Mg2+ is a useful procedure for an effective and rapid preparation of plasma membrane enriched fraction from smooth muscle.     

An analytical approach to the preparation and characterization of subcellular membranes from canine mesenteric arteries

Subcellular membrane fractions were isolated from dog mesenteric arteries by differential and isopynic sucrose density gradient centrifugations. Isolated membrane fractions were characterized by marker enzyme activities, morphological features and sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns. Our results show that the microsomal fraction isolated by conventional differential centrifugation was highly heterogenous and contained substantial amount of plasma membranes which could be further enriched as a light density membrane fraction on a discontinuous sucrose density gradient. The microsomal fraction and its subfractions were vesicular in appearance under electron microscope and were capable of binding and actively transporting Ca2+. The binding of Ca2+ and ATP-supported Ca2+-transport in the presence or absence of oxalate paralleled the distribution of plasma membrane marker enzyme activities suggesting that plasma membranes in vascular smooth muscle may play a major role in handling Ca2+ and thus the control of contractile function.     

Hypoxia-induced migration in pulmonary arterial smooth muscle cells requires calcium-dependent upregulation of aquaporin 1

Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca(2+)](i)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca(2+)](i), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca(2+)](i), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca(2+)](i) that results in increased AQP1 protein levels and cell migration.     

Ion channels in smooth muscle: regulation by the sarcoplasmic reticulum and mitochondria

In smooth muscle, Ca(2+) regulates cell division, growth and cell death as well as providing the main trigger for contraction. Ion channels provide the major access route to elevate the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) in smooth muscle by permitting Ca(2+) entry across the plasma membrane and release of the ion from intracellular Ca(2+) stores. The control of [Ca(2+)](c) relies on feedback modulation of the entry and release channels by Ca(2+) itself. Local rises in [Ca(2+)](c) may promote or inhibit channel activity directly or indirectly. The latter may arise from Ca(2+) regulation of ionic conductances in the plasma membrane to provide control of cell excitability and so [Ca(2+)](c) entry. Organelles such as mitochondria may also contribute significantly to the feedback regulation of ion channel activity by the control of Ca(2+) or redox status of the cell. This brief review describes the feedback regulation of Ca(2+) release from the internal Ca(2+) store and of plasma membrane excitability in smooth muscle.     

Calcium and abnormal reactivity of vascular smooth muscle in hypertension

It has been well documented that vascular smooth muscle (VSM) reactivity, as well as calcium sensitivity in response to neurotransmitters is increased in a number of blood vessels in established hypertension. Regulation of VSM reactivity involves the interaction of neurotransmitters and blood-borne hormones with specific receptors on target cell membranes. This results in phospholipase-C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and the generation of two second messengers: inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG) both of which act synergistically to produce muscle contraction. We will summarize recent findings in this review which suggest that in essentially hypertensive patients and spontaneously hypertensive rats (SHR), the activation of phospholipase C in response to hormones is increased. Further, we will discuss how increases in phospholipase C activation via GTP-binding proteins may explain the observed increases in Ca2+ influx through potential- and receptor-operated Ca2+ channels, increased activation of protein kinase-C and increased [Ca2+]i in hormone-stimulated blood platelets and VSM cells in the hypertensive state. In addition to these defects, a decrease in the plasma membrane Ca2+ pump and Ca2+-binding proteins has been demonstrated in hypertension. Thus, it appears that the defect in Ca2+ metabolism in the hypertensive vessels is multifocal. All these defects in Ca2+ metabolism together may lead to an increase in peripheral vascular resistance with a concomitant increase in blood pressure.     

Endothelin-1 increases intracellular calcium mobilization but not calcium uptake in rabbit vascular smooth muscle cells

Conflicting evidence has been reported regarding the role of endothelin-1, a potent vasconstrictor peptide, in stimulating extracellular calcium influx in rabbit vascular smooth muscle. The objective of this study was to elucidate the effects of endothelin-1 on transmembrane 45Ca2+ influx and intracellular calcium mobilization in cultured rabbit aortic smooth muscle cells. In calcium containing buffer, endothelin-1 induced a concentration-dependent 45Ca2+ efflux response over the range of 10 pM to 100 nM with an EC50 of approximately 60 pM. Maximum endothelin-stimulated 45Ca2+ efflux was not affected by the absence of extracellular calcium or the presence of 1 microM verapamil. Endothelin-1 did not induce transplasmalemmal 45Ca2+ uptake at times up to 30 min. These findings suggest that an alteration in intracellular calcium handling, rather than extracellular calcium influx, is responsible for the endothelin-stimulated increase in intracellular calcium concentration in rabbit aortic smooth muscle cells.     

Membrane mechanisms of regulating Ca ion concentration in smooth muscle cells

The aim of this review is to summarize some current concepts on the membrane mechanisms of energy-dependent Ca2+ transport in the smooth muscles. The emphasis is placed on the properties and mechanisms of regulation of plasma membrane and endoplasmic reticulum calcium pumps, sarcolemmal sodium/calcium exchanger and mitochondrial Ca2+ transport.     

Components of cholinergic excitation coupling with smooth muscle contraction in the guinea pig taenia coli]

In concentrations of 10(-9)-10(-7) g/ml acetylcholine increased the tone of the smooth muscles of the longitudinal band of the large intestine of a guinea pig, increasing the permeability of the cellular membranes for the entering flux of 45Ca2+. In concentrations of 10(-6) g/ml and over acetylcholine caused a release of the membranous calcium and in the concentrations of 10(-5)-10(-3) g/ml markedly increased the permeability of the membranes of the smooth muscle cells for the 22Na+ ions causing depolarization and an increase in the frequency of the action potentials. It is supposed that the coupling of the cholinergic stimulus with the end effect (muscle contraction) included 3 components: intensification of the entrance of Ca2+ into the smooth muscle cells, release of the membrane calcium and adhesion mechanism.     

Ca-ATPase of human myometrium plasma membranes

We determined and characterized the Mg2+-dependent, Ca2+-stimulated ATPase (Ca-ATPase) activity in cell plasma membranes from the myometrium of pregnant women, and compared these characteristics to those of the active Ca2+-transport already demonstrated in this tissue. Similarly to the Ca2+-transport system, the Ca2+-ATPase is Mg2+-dependent, stimulated by calmodulin, and inhibited by vanadate. The Km for Ca2+ activation is 0.40 microM, very similar to that found for active calcium transport, i.e. 0.25 microM. Consequently, this Ca2+-ATPase can be responsible for the active calcium transport across the plasma membranes of smooth muscle cells.     

Metabolic considerations and cellular mechanism related to calcium's antihypertensive effects

Whether maintenance of normal calcium homeostasis can afford protection against the development of hypertension in humans has emerged as a controversial area of both clinical and basic cardiovascular disease research. The data that have provoked this debate are derived from epidemiological reports, human studies, animal investigations, and cellular research. Ten published reports have identified an association between greater dietary calcium consumption and lower blood pressure in humans. In both humans and experimental animals with hypertension, several end-organ defects have been identified that are consistent with an inability to maintain external calcium balance. With the provision of supplemental dietary calcium, both humans and experimental models with high blood pressure have reduced their blood pressure. A variety of membrane-associated defects of Ca2+-ATPase-dependent calcium transport have been identified in cells derived from multiple organs of both the hypertensive animal and human. These abnormalities of cellular calcium handling could account for the failure of the hypertensive subject to appropriately defend its calcium balance. More important, they provide a theoretical mechanism by which calcium, interacting with calmodulin, might favorably modify vascular smooth muscle function and, thereby, peripheral vascular resistance.     

Na+/K+-ATPase regulation in Dahl salt-sensitive and salt-resistant rats

Circulating Na+/K+-ATPase inhibitors have been implicated in volume-expanded forms of hypertension. Inhibition of vascular smooth muscle cell Na+/K+-ATPase has been demonstrated to elevate intracellular Ca2+ levels and enhance contractility, thus providing a mechanism of raised peripheral resistance. In cells chronically subjected to Na+/K+-ATPase inhibition, however, new Na+/K+-ATPase molecules are synthesized, which then restore the intracellular milieu to preinsult conditions. Restoral of the preinsult intracellular milieu in vascular smooth muscle cells would then be expected to lead to the reduction of muscle cell contractility and peripheral resistance. Thus circulating Na+/K+-ATPase inhibitors may not be effective in eliciting chronic forms of hypertension unless the target cell "homeostatic response" is impaired. We demonstrate an apparent such impairment in Dahl salt-sensitive rats, a genetic model of salt-sensitive hypertension.     

Localization of phospholamban in smooth muscle using immunogold electron microscopy

Phospholamban, the putative regulator of the Ca2+-ATPase in cardiac sarcoplasmic reticulum, was immunolocalized in canine visceral and vascular smooth muscle. Gently disrupted tissues were labeled with an affinity-purified phospholamban polyclonal antibody and indirect immunogold, using preembedding techniques. The sarcoplasmic reticulum of smooth muscle cells was specifically labeled with patches of immunogold distributed in a nonuniform fashion, while the sarcolemma did not appear to contain any phospholamban. The outer nuclear envelopes were also observed to be heavily labeled with the affinity-purified phospholamban polyclonal antibody. These findings suggest that phospholamban may play a role in the regulation of cytoplasmic and intranuclear calcium levels in smooth muscle cells.     

Mechanisms and consequences of Na+,K+-pump regulation by insulin and leptin.

Hormonal control of the Na+,K+-pump modulates membrane potential in mammalian cells, which in turn drives ion coupled transport processes and maintains cell volume and osmotic balance. Na+,K+-pump regulation is particularly important in the musculoskeletal, cardiovascular and renal systems. Decreased Na+,K+-pump activity can result in a rise in intracellular Na+ concentrations which in turn increase Na+/Ca2+ exchange, thereby raising intracellular Ca2+ levels. In cardiac and skeletal muscle, this could interfere with normal contractile activity. Similarly, in vascular smooth muscle the result would be resistance to vasodilation. Inhibition of the Na+,K+-pump can also reduce the driving force for renal tubular Na+ reabsorption, elevating Na+ excretion. By virtue of decreasing the membrane potential, thus allowing more efficient depolarization of nerve endings and by increasing intracellular Ca2+, inhibition of the Na+,K+-pump can increase nervous tone. The ability of insulin to stimulate the Na+,K+-pump in various cells and tissues, and the physiological significance thereof, have been well documented. Much less is known about the effect of leptin on the Na+,K+-pump. We have shown that leptin inhibits Na+,K+-pump function in 3T3-L1 fibroblasts. Defects in insulin and leptin action are associated with diabetes and obesity, respectively, both of which are commonly associated with cardiovascular complications. In this review we discuss the mechanisms of Na+,K+-pump regulation by insulin and leptin and highlight how, when they fail, they may contribute to the pathophysiology of hypertension associated with diabetes and obesity.