Interactions between membrane potential and intracellular calcium concentration in vascular smooth muscle

The intracellular calcium concentration is a major determinant of vascular tone. In the steady state it is regulated mainly by membrane potential. At the same time, several mechanisms regulating the calcium concentration, including the membrane potential, are influenced by the intracellular calcium concentration itself. There are thus multiple possible positive and negative feedback loops involved in calcium regulation. This review gives a brief overview of the different mechanisms involved, including calcium-dependent ion channels, exchangers, and ATPases, and discusses their role in agonist-mediated responses, in relation primarily to studies on the portal vein and mesenteric small arteries.     

Tyrosine kinase inhibitors suppress prostaglandin F2alpha-induced phosphoinositide hydrolysis, Ca2+ elevation and contraction in iris sphincter smooth muscle

We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F2alpha- and carbachol-induced inositol-1,4,5-trisphosphate (IP3) production, [Ca2+]i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F2alpha and carbachol induced contraction in a concentration-dependent manner with EC50 values of 0.92 x 10(-9) and 1.75 x 10(-8) M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F2alpha, but not those evoked by carbachol, on IP3 accumulation, [Ca2+]i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F2alpha analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 microM) markedly reduced (by 67%) prostaglandin F2alpha-stimulated increase in [Ca2+]i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC50 of 82 microM and increased IP3 generation in a concentration-dependent manner with an EC50 of 90 microM. The effects of vanadate were abolished by genistein (10 microM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F2alpha- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca2+]i evoked by prostaglandin F2alpha. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F2alpha receptor activation and increases in intracellular Ca2+ mobilization and identifying the tyrosine-phosphorylated substrates will provide important information about the role of protein tyrosine kinase in the mechanism of smooth muscle contraction, as well as about the mechanism of the intraocular pressure lowering effect of the prostaglandin in glaucoma patients.     

Tumor necrosis factor alpha potentiates the increase in cytosolic free calcium induced by bradykinin in guinea-pig tracheal smooth muscle cells

The ability to respond to bradykinin (BK) by a bronchospasm is one of the most intriguing characteristics observed in asthmatic patients but not in healthy subjects. The molecular basis of this sensitivity is not yet known. Therefore, we studied the effect of BK, and its putative modulation by tumor necrosis factor alpha (TNF alpha), on cytosolic free Ca2+ concentration ([Ca2+]i) in fura-2-loaded tracheal smooth muscle cells in culture. BK induced a concentration-dependent rise in [Ca2+]i at concentrations between 10(-13) and 10(-11) M which was mediated via BK receptors of the B2 subtype. The net increase in [Ca2+]i induced by 10(-12)M BK was 478 +/- 52 nM. Pre-treatment of the cells with TNF alpha (10 ng/ml) for 24 h significantly potentiated this net increase in [Ca2+]i to 956 +/- 154 nM. The presence of anti-TNF alpha antibodies inhibited this potentiation. These results show that TNF alpha is able to interact with airway smooth muscle cells which suggests the existence of receptors for TNF alpha on these cells. The study, in vivo, of such interaction should help to further elucidate the mechanisms involved in airway hyperresponsiveness.     

Intracellular alkalinization by NH4Cl increases cytosolic Ca2+ level and tension in the rat aortic smooth muscle

Intracellular pH (pHi) is elucidated to be an important regulator of various cell functions, but the role of pHi in smooth muscle contraction remains to be clarified. The purpose of the present study is to examine the effects of cell alkalinization by exposure to NH4Cl on cytosolic Ca2+ level ([Ca2+]i) and on muscle tone. We attempted simultaneous measurements of both [Ca2+]i and contractile force in rat isolated thoracic aorta from which the endothelium was removed. NH4Cl (10-80 mM) increased both [Ca2+]i and muscle tone in the presence of external Ca2+. These responses were reproducible. The removal of Ca2+ from the nutrient solution partially inhibited the rise in [Ca2+]i and the smooth muscle contraction induced by NH4Cl. In addition, the Ca2+ channel blocker verapamil also partially attenuated the responses to NH4Cl. The NH4Cl-induced responses were gradually reduced as NH4Cl was repeatedly added in a Ca(2+)-free solution. Norepinephrine (NE, 1 microM) induced a transient increase in [Ca2+]i and sustained contraction in the absence of external Ca2+, and the subsequent application of NE had little effect on [Ca2+]i. After internal Ca2+ stores were depleted by exposure to NE, the subsequent application of NH4Cl induced increases in [Ca2+]i and tension of the aorta in a Ca(2+)-free solution. These results suggest that NH4Cl mainly evokes Ca2+ release from the internal Ca2+ stores that are not linked with adrenergic alpha-receptor and causes Ca2+ influx through voltage-dependent Ca2+ channels in the vascular smooth muscle.     

Ultrastructure, calcium accumulation, and contractile response in smooth muscle

After removing extracellular Ca2+ with [ethylene bis(oxyethylenenitrilo)]tetraacetic acid, we found that the guinea pig vas deferens (VD) was mechanically responsive to electrical stimulation for a significantly greater length of time than was guinea pig taenia coli (TC). An obvious explanation for these findings is that the VD has more intracellular calcium available for contraction than does the TC. To determine if this explanation is plausible, the volume of internal storage structures within the two muscles was compared. It was found that the volumes of potential sequestering structures in the VD and TC are not significantly different. Next, the affinities of the storage structures for calcium were compared. The VD was found to accumulate approximately twice as much 45Ca as did the TC, as determined by 45Ca autoradiography. Calcium-45 was present to a greater extent in association with surface vesicles, sarcoplasmic reticulum (SR), and mitochondria than in the unassociated state within the cytoplasmic matrix. Based on the results of these experiments, we suggest that the VD and the TC of the guinea pig differ in the affinity of their storage sites for calcium.     

Correlations of PDE-4 inhibition between enzymes of smooth muscle and inflammatory cell sources

The sensitivities of PDE-4 enzymes from smooth muscle and inflammatory cell sources from different species to a range of structurally diverse compounds were compared. All inflammatory cell PDE-4 sources displayed good crosscorrelations in their sensitivity to inhibition by these compounds. Similarly, PDE-4 enzymes from smooth muscle sources were well-correlated; however, there was no crosscorrelation between PDE-4 from smooth muscle sources and those of inflammatory cell sources, possibly reflecting differences in subcellular location of enzymes as well as subtype expression. The present study concludes that PDE-4 preparations from smooth muscle sources as well as those from inflammatory cell sources may be used to model the potential smooth muscle cell relaxing properties and anti-inflammatory properties of a compound in relation to human asthma.     

Coronary arterial smooth muscle contraction by a substance released from platelets: evidence that it is thromboxane A2

When human platelets are aggregated by thrombin, material is released that rapidly contracts strips of spirally cut porcine coronary artery. Prevention of the contraction by indomethacin suggested mediation by a prostaglandin. The contraction produced by aggregating platelets was unlike those produced by prostaglandins E2, F2alpha, G2, or H2, but resembled that evoked by thromboxane A2, which is formed by platelets during aggregation.     

Effects of polyamines and calcium and sodium ions on smooth muscle cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase

Degenerative changes of the spinal column have long been and continue to be confused with the presence of spinal distress and pain. All parts of the spine undergo degenerative changes as we age. The purpose of this chapter is to describe the degenerative process and its clinical consequences. The disc degenerative process will be discussed; its consequences on the facet joint and osteophyte formation are considered. The prevalence of disc degeneration, the role of physically demanding work and leisure and the interference of spinal deformity is clarified. A section particularly important for the clinician deals with the clinical consequences of the degenerative process in disc herniation, degenerative spondylolisthesis, spondylolysis and stenosis. This chapter tries to put the degenerative changes of the spine into the context of a normal ageing process.     

Phosphorylation of a twitchin-related protein controls catch and calcium sensitivity of force production in invertebrate smooth muscle

"Catch" is a condition of prolonged, high-force maintenance at resting intracellular Ca2+ concentration ([Ca2+]) and very low energy usage, occurring in invertebrate smooth muscles, including the anterior byssus retractor muscle (ABRM) of Mytilus edulis. Relaxation from catch is rapid on serotonergic nerve stimulation in intact muscles and application of cAMP in permeabilized muscles. This release of catch occurs by protein kinase A-mediated phosphorylation of a high (approximately 600 kDa) molecular mass protein, the regulator of catch. Here, we identify the catch-regulating protein as a homologue of the mini-titin, twitchin, based on (i) a partial cDNA of the purified isolated protein showing 77% amino acid sequence identity to the kinase domain of Aplysia californica twitchin; (ii) a polyclonal antibody to a synthetic peptide in this sequence reacting with the phosphorylated catch-regulating protein band from permeabilized ABRM; and (iii) the similarity of the amino acid composition and molecular weight of the protein to twitchin. In permeabilized ABRM, at all but maximum [Ca2+], phosphorylation of twitchin results in a decreased calcium sensitivity of force production (half-maximum at 2.5 vs. 1.3 microM calcium). At a given submaximal force, with equal numbers of force generators, twitchin phosphorylation increased unloaded shortening velocity approximately 2-fold. These data suggest that aspects of the catch state exist not only at resting [Ca2+], but also at higher submaximal [Ca2+]. The mechanism that gives rise to force maintenance in catch probably operates together, to some extent, with that of cycling myosin crossbridges.     

The relationship between osmotic stress and calcium elevation: in vitro and in vivo rat lens models

Recent evaluations by the U.S. General Accounting Office and the National Alliance for the Mentally Ill of reemployment efforts of the federal-state vocational rehabilitation program found that services offered by state vocational rehabilitation agencies do not produce long-term earnings for clients with emotional or physical disabilities. This paper examines reasons for these poor outcomes and the implications of recent policy reform recommendations. Congress must decide whether to take action at the federal level to upgrade programs affecting persons with severe mental illnesses or to continue to rely on state decision making. The federal-state program largely wastes an estimated $490 million annually on time-limited services to consumers with mental illnesses. Rechanneled into a variety of innovative and more appropriate integrated services models, the money could buy stable annual vocational rehabilitation funding for 62,000 to 90,000 consumers with severe mental illnesses. Larger macrosystem problems involve the dynamics of the labor market that limit job opportunities and the powerful work disincentives for consumers with severe disabilities now inherent in Social Security Disability Insurance, Supplemental Security Income, Medicare, and Medicaid.     

Calponin phosphatase from smooth muscle: a possible role of type 1 protein phosphatase in smooth muscle relaxation

Smooth muscle myosin bound phosphatase (MBP) purified from chicken gizzard, which is a holoenzyme of type 1 delta protein phosphatase and dephosphorylated intact myosin, catalyzed the dephosphorylation of calponin phosphorylated by protein kinase C (PK-C). The Km of MBP for calponin was 0.6 microM and the Vmax was 350 nmol/min/mg. All of the multiple sites of phosphorylation by PK-C of calponin were completely dephosphorylated by MBP. Functionally, calponin dephosphorylated by MBP recovered its inhibitory effect on the actin-activated Mg(2+)-ATPase activity of myosin. Therefore, these results suggest that a type 1 delta protein phosphatase causes relaxation of smooth muscle by the dephosphorylation not only of myosin but also of calponin.     

Oxytocin receptor-mediated activation of phosphoinositidase C and elevation of cytosolic calcium in the gonadotrope-derived alphaT3-1 cell line

Gonadotropes synthesize and secrete LH and FSH under the control of GnRH, which acts via phosphoinositidase C (PIC)-linked G protein coupled receptors. Additionally, gonadotropin released from the pituitary is influenced by oxytocin, a peptide that has been shown to play a role in generation of the preovulatory LH surge. Although oxytocin receptors are present in the pituitary, studies have identified their presence on lactotropes but not on gonadotropes, raising the question of which cells act as the direct target of oxytocin in gonadotrope regulation. In this study, we examined effects of oxytocin on alphaT3-1 cells, a gonadotrope-derived cell line. Oxytocin, vasopressin, and vasotocin each stimulated accumulation of [3H]inositol phosphates in cells prelabeled with [3H]inositol, indicating activation of PIC. The rank order of potency (oxytocin > vasotocin > vasopressin) and sensitivity to inhibition by oxytocin and vasopressin receptor antagonists, revealed the effect to be mediated by oxytocin-selective receptors. Like other PIC activators, these nonapeptides caused biphasic (spike-plateau) increases in the cytosolic Ca2+. The spike response to oxytocin and GnRH were both retained in Ca2+-free medium, reflecting mobilization of intracellular Ca2+, and were comparably reduced by thapsigargin, implying mobilization of Ca2+ from a shared thapsigargin-sensitive intracellular pool. Brief stimulation with oxytocin, vasopressin, or vasotocin prevented subsequent Ca2+ responses to oxytocin, but not to GnRH, suggesting that the oxytocin receptor undergoes rapid homologous desensitization and reinforcing the interpretation that the nonapeptides act via the same receptor type. Oxytocin did not increase Ca2+ in cells stimulated with GnRH, whereas GnRH caused a spike Ca2+ increase even in the presence of oxytocin, implying that different mechanisms of desensitization (Ca2+ pool depletion and receptor uncoupling) are operating for two distinct PIC-coupled receptors in these cells. The demonstration that oxytocin acts directly via PIC-linked, oxytocin-selective receptors to increase cytosolic Ca2+ in a gonadotrope-derived cell line is consistent with the possibility that oxytocin has a comparable effect on nonimmortalized gonadotropes.     

Interaction between growth factors and kinins in arterial smooth muscle cells

Bradykinin receptors are present on vascular smooth muscle cells; however, the regulation and biological function of these receptors is unclear. To address these questions the interaction between growth factors and kinins in cultured arterial smooth muscle cells has been examined. Based upon the data a hypothesis is presented that platelet-derived growth factor (PDGF) upregulates cell surface bradykinin B2 receptors on arterial smooth muscle cells. The biological effect of the increase in B2 receptors is currently unclear but under certain conditions they may enhance mitogenesis. These mitogenic effects however, are strongly opposed by the effects of bradykinin acting via a B1-type of receptor which mediates potent inhibition of growth factor-induced mitogenesis.     

Myoblast fusion is not a prerequisite for the appearance of calcium current, calcium release, and contraction in rat skeletal muscle cells developing in culture

During in vitro development of rat skeletal muscle cells, contraction and calcium currents progressively appear after fusion of myoblasts. To investigate whether muscle-specific functions are expressed in the absence of myoblast fusion, rat neonatal muscle cells were cultured in a differentiation medium under conditions that are well known to inhibit fusion: prolonged culture in a low-calcium medium or treatment with cytochalasin B. We have demonstrated that the fusion-arrested cells expressed differentiative properties in L-type calcium current, transient release of calcium ions from internal stores in response to caffeine and depolarizing agents, and contraction elicited by depolarization. Properties and potential-dependence of L-type calcium currents were similar to that in control fused cells, but T-type calcium currents were not observed, while both types coexist in myotubes. Properties of calcium transients and voltage dependence of contraction suggested that the excitation-contraction mechanisms were well established. However, comparing to well-developed myotubes at the same time of culture, the characteristics of calcium transients and contraction of fusion-arrested cells were closer to those of younger myotubes, which can be interpreted in terms of a delay in maturation of excitation-contraction coupling and contractile machinery. All these observations demonstrate that myoblast fusion is not necessary for triggering the establishment of calcium transport and release and contractile functions of rat muscle cells developing in culture. The appearance of muscle-specific functions is consistent with previous results demonstrating that the fusion-arrested cells express muscle-specific proteins and structures.     

Oscillations of receptor-operated cationic current and internal calcium in single guinea-pig ileal smooth muscle cells

In single cells isolated from guinea-pig ileal smooth muscle, held under voltage clamp at -40 mV or -50 mV by patch pipette in the whole-cell recording mode, carbachol (CCh) evoked an oscillatory inward cationic current. The frequency of current oscillations increased with increasing CCh concentration. CCh-evoked current oscillations were followed very closely by oscillations in intracellular free Ca2+ estimated from the Indo-1 signal, and were abolished by inclusion of EGTA in the pipette solution. Ryanodine and heparin, but not nifedipine, blocked the generation of current oscillations. CCh-evoked current oscillations were abolished upon withdrawal of extracellular calcium and restored upon its reintroduction. Inclusion of GTP[gamma S] in the pipette solution caused the generation of an oscillatory inward current, which was blocked by ryanodine. The present results are consistent with the hypothesis that CCh-evoked cationic current is gated by activation of a G protein and is steeply dependent on [Ca2+]i, fluctuations in the release of Ca2+ from stores during carbachol's action produce oscillations in [Ca2+]i which cause similar oscillations in the cationic current.     

Regulation of force and shortening velocity by calcium and myosin phosphorylation in chemically skinned smooth muscle

The phosphatase inhibitor okadaic acid (OA) was used to study the relationship between [Ca2+], rates of phosphorylation/dephosphorylation and the mechanical properties of smooth muscle fibres. Force/velocity relationships were determined with the isotonic quick release technique in chemically skinned guinea-pig taenia coli muscles at 22 degrees C. In the maximally thiophosphorylated muscle neither OA (10 microM) nor Ca2+ (increase from pCa 9.0 to pCa 4.5) influenced the force-velocity relationship. When the degree of activation was altered by varying [Ca2+] in the presence of 0.5 microM calmodulin, both force and the maximal shortening velocity (Vmax) were altered. At pCa 5.75, at which force was about 35% of the maximal at pCa 4.5, Vmax was 55% of the maximal value. When OA was introduced into fibres at pCa 6.0, force was increased from less than 5% to 100% of the maximal force obtained in pCa 4.5. The relationship between the degree of myosin light chain phosphorylation and force was similar in the two types of activation; varied [OA] at constant [Ca2+] and at varied [Ca2+]. The relation between force and Vmax when the degree of activation was altered with OA was almost identical to that obtained with varied [Ca2+]. The results show that Ca2+ and OA do not influence force or Vmax in the maximally phosphorylated state and suggest that the level of myosin light chain phosphorylation is the major factor determining Vmax. The finding that the relationship between force and Vmax was similar when activation was altered with OA and Ca2+ suggests, however, that alterations in the absolute rates of phosphorylation and dephosphorylation at a constant phosphorylation level do not influence the mechanical properties of the skinned smooth muscle fibres.