In this chapter, current topics in the year 1996 about smooth muscle with regards to Ca2+ storage, Ca2+ release and reuptake, and Ca2+ regulation of contraction are discussed. Ca2+ is mobilized from the endoplasmic reticulum, the physiologically important Ca2+ reservoir in vascular smooth muscle, through two Ca2+-release channels: inositol trisphosphate and the ryanodine receptor. The characteristics, function, and control of these two receptors are summarized. Furthermore, evidence for a role of the nucleus as a potential Ca2+ storage site is presented and the role of mitochondria and Ca2+ extrusion systems are discussed. Also, an overview of the current understanding of the mechanism of contraction and relaxation of smooth muscle is given and the modulation of the Ca2+ sensitivity of the force is discussed.

The gene family of organellar-type Ca2+ transport ATPases consists of three members. SERCA1 is expressed exclusively in fast skeletal muscle; SERCA2 is ubiquitously expressed, whereas SERCA3 is considered to be mainly expressed in cells of the hematopoietic lineage and in some epithelial cells. In the brain, the organellar-type Ca2+ transport ATPases are almost exclusively transcribed from the SERCA2 gene. Four different SERCA2 mRNAs have been described (classes 1–4). However, unlike in nonneuronal cells, which express the class 1, 2, and 3 splice variants, the main SERCA2 mRNA in the brain is the class 4 messenger. Similar to classes 2 and 3, the class 4 codes for the ubiquitously expressed SERCA2b protein. Recently, we have reported the distribution of the SERCA isoforms in the brain (Baba-Aissa et al., 1996a,b). SERCA2b was present in most neurons of all investigated brain regions. The highest levels were found in the Purkinje neurons of the cerebellum and in the pyramidal cells of the hippocampus. Interestingly, SERCA3 and SERCA2a are coexpressed along with SERCA2b in the Purkinje neurons, but are weakly expressed in the other brain regions if present at all. Since these three protein isoforms have a different affinity for Ca2+, their possible roles in relation to Ca2+ stores in neurons are discussed.

Activation of cells by hormones, growth factors or neurotransmitters leads to an increased production of inositol trisphosphate (InsP3) and, after activation of the InsP3 receptor (InsP3R), to Ca2+ release from intracellular Ca2+ stores. The release of intracellular Ca2+ is characterised by a graded response when submaximal doses of agonists are used. The basic phenomenon, called "quantal Ca2+ release", is that even the maintained presence of a submaximal dose of agonist or of InsP3 for long time periods (up to 20 min) provokes only a partial release of Ca2+. This partial, or quantal, release phenomenon is due to the fact that the initially very rapid InsP3-induced Ca2+ release eventually develops into a much slower release phase. Physiologically, quantal release allows the Ca2+ stores to function as increment detectors and to induce local Ca2+ responses. The basic mechanism for quantal release of Ca2+ is presently not known. Possible mechanisms to explain the quantal behaviour of InsP3- induced Ca2+ release include the presence of InsP3Rs with varying sensitivities for InsP3, heterogeneous InsP3R distribution, intrinsic inactivation of the InsP3Rs, and regulation of the InsP3Rs by Ca2+ store content. This article reviews critically the evidence for the various mechanisms and evaluates their functional importance. A Ca2+-mediated conformational change of the InsP3R is most likely the key feature of the mechanism for quantal Ca2+ release, but the exact mode of operation remains unclear. It should also be pointed out that in intact cells more than one mechanism can be involved.

Changes in cytosolic Ca2+ concentration ([Ca2+]i) and in membrane potential were monitored in single A7r5 smooth-muscle cells during spontaneous spiking and after arginine vasopressin stimulation. Spontaneous Ca2+ oscillations, which were associated with the occurrence of action potentials, occurred in about 90% of the confluent monolayers investigated. This spontaneous activity was synchronized amongst all the cells of the monolayer, indicating that the cells were electrically coupled. Arginine vasopressin stimulation produced a [Ca2+]i rise that was about 5 times higher than the amplitude of the spontaneous Ca2+ oscillations and resulted in a subsequent cessation of spontaneous electrical activity and associated Ca2+ spiking, which persisted after [Ca2+]i returned to baseline. Individual cells in the monolayer responded to arginine vasopressin with a different latency. Agonist-induced Ca2+ waves within one cell propagated much more slowly than spontaneous [Ca2+]i rises. We conclude that agonist-induced [Ca2+]i increases in an electrically coupled cell monolayer can be asynchronous.

We have investigated whether reducing agents and substances that interfere with glutathione metabolism would affect the histamine-induced rises in internal Ca2+ concentration ([Ca2+]i) in indo-1-loaded HeLa cells. Individual cells responded to 1 μM histamine with either baseline or sinusoidal Ca2+ oscillations, a single Ca2+ peak or a maintained elevation of the [Ca2+]i. Only a few cells did not respond. The sulphydryl reducing agent dithiothreitol (5 mM) did not affect these responses to histamine. A 24-h preincubation with 1 mM dl-buthionine (SR)-sulphoximine, which reduces the cellular glutathione content to less than 20% of its control value, affected neither these histamine responses, nor the [Ca2+]i rises after application of 2 μM thapsigargin. We conclude that oxidation of critical sulphydryl groups is not required for the normal response to histamine and also that glutathione plays no role in agonistinduced Ca2+ signalling in HeLa cells.

There is no consensus about the different types of Ca2+ transport processes in the endoplasmic reticulum that are targeted by the sulphydryl reagent thimerosal. We have therefore investigated how thimerosal affects the various Ca2+ transport processes in permeabilized A7r5 smooth-muscle cells, using an unidirectional 45Ca2+ flux technique. Thimerosal up to a concentration of 32 μM did not have an effect on the passive 45Ca2+ leak from the stores, while higher concentrations increased this aspecific leak. Thimerosal inhibited the endoplasmic reticulum Ca2+ pump with an EC50 of 9 μM. Thimerosal exerted a biphasic effect on the Ca2+ release induced by inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with a stimulation of the release at thimerosal concentrations below 10 μM, and an inhibitory effect at higher concentrations. Thimerosal (2.5–250 μM) did not exert an effect on the specific binding of [3H]Ins(1,4,5)P3 to its receptor, indicating that it probably did not act at the level of the binding site. This finding contrasts with the effect of the closely related sulphydryl reagent parachloromercuriphenylsulphonate, which, at high concentrations, inhibited [3H]Ins(1,4,5)P3 binding. The effects of thimerosal were largely prevented by the sulphydryl reducing agent dithiothreitol (3 mM). We conclude that thimerosal concentrations ranging from 0.32 to 1 μM can stimulate the Ins(1,4,5)P3-induced Ca2+ release without inhibiting the Ca2+ pumps or without increasing the passive Ca2+ permeability of the endoplasmic reticulum.

The effect of differentiation on the RNA processing of the PMCA1 gene encoding a plasma-membrane Ca2+ pump and of the SERCA2 gene encoding a sarco(endo)plasmic reticulum Ca2+ pump was studied in the myogenic BC3H1 cell line. A differentiation stage-dependent change in the RNA processing was observed for both genes. Proliferating myoblasts only expressed the non-muscle mRNA isoform whereas in differentiated cells muscle-specific processing became activated. The switch to muscle-specific RNA processing for both the PMCA1 and SERCA2 genes was found to be linked to the myogenic conversion of the BC3H1 cells. Our results furthermore indicated that the myogenic RNA processing could be reversed for both types of Ca2+ pumps since the expression of the PMCA1 and SERCA2 muscle-specific messengers was rapidly down-regulated by cycloheximide treatment.

BACKGROUND: The development of any society depends on proper planning in various fields such as population and birth control. Fertility control is designed to create a level of population growth appropriate to the resources available and to ensure a good life. Receiving information and education is one of the basic strategies to change the attitude toward fertility and awareness in most people in society. Therefore, this study was conducted to determine the effect of education on knowledge of fertility counseling and attitudes toward fertility control in health workers. MATERIALS AND METHODS: The present study was a randomized clinical trial with a control group that was conducted in the presence of 107 health workers of health centers and community health centers working in Mashhad in 2017. The research units were randomly divided into three groups (webinar training, group discussion training, and control). Research tools were researcher-made questionnaires on attitudes toward fertility and measuring healthy reproductive awareness that all study participants completed at the beginning of the study and 2 weeks after the intervention. Data analysis was performed by the Chi-square, one-way analysis of variance, and independent t-test using the SPSS software version 16. RESULTS: The results of this study showed that at the beginning of the study, all three groups were homogeneous in terms of quantitative and qualitative demographic variables including age, education, work experience, type of employment, and number of family members. The results of Kruskal–Wallis test showed that before the intervention, the three groups did not have a statistically significant difference in terms of mean scores of awareness about fertility counseling (P = 0.77) and attitude toward fertility control (P = 0.523), but this relationship was significant after the interventions. Furthermore, the results of Mann–Whitney intragroup test showed that the scores before and after the intervention were significant in both educational groups (P = 0.001). CONCLUSION: Considering the importance of healthy fertility counseling and the important place of education in promoting awareness and attitude toward healthy fertility, it is recommended to use active educational methods to promote the awareness and attitude of health workers to provide healthy fertility services to couples.

We compared the changes of the cytoplasmic Ca2+ concentration ([Ca2+]i), as measured with the fluorescent Ca2+ indicator fura-2, and the force development in intact smooth muscle of the tonic anococcygeus (AC) and the phasic vas deferens (VD) of the mouse, during activation by K+ depolarization and by agonists. Resting [Ca2+]i was observed to be 33% lower in AC (80 nM) than in VD (115 nM), while the Ca2+ threshold for contraction was found to be about 120 nM in AC and 160 nM in VD. For a similar [Ca2+]i increase, the agonist stimulation induced a higher force development than the K+ depolarization in both muscle types. During prolonged depolarization, the force/calcium ratio increased in AC but strongly declined in VD. This decline of the force/calcium ratio in VD during depolarization was partially reversed by lowering [Ca2+]o. Our results indicate that the Ca2+ threshold for force development was about 150% of the resting [Ca2+]i in both cell types. The resting [Ca2+]i was lower in the tonic AC than in the phasic VD. Agonist-induced sensitization to Ca2+ occurred in both muscle types. The tonic and phasic smooth muscles essentially differed in the respective modulation of their Ca2+ sensitivity during contraction. The desensitization to Ca2+ was specific for phasic muscle, in which it occurred as an early, time- and Ca(2+)-dependent process that was partially reversible.

Fluorescence energy transfer has been used to study the interaction of various phospholipids with the erythrocyte (Ca2+ + Mg2+)-ATPase. The fluorescence energy transfer between tryptophan residues of the (Ca2+ + Mg2+)-ATPase purified from erythrocytes and pyrene-labelled analogues of phosphatidylcholine (Pyr-PC), phosphatidylinositol (Pyr-PI), phosphatidylinositol 4-phosphate (Pyr-PIP), phosphatidylinositol 4,5-bisphosphate (Pyr-PIP2), phosphatidylglycerol (Pyr-PG) and phosphatidic acid (Pyr-PA) was measured. A positive correlation was found between the number of negative charges on the phospholipids (PIP2 greater than PIP greater than PA greater than PI = PG greater than PC) and the potency of their pyrene-labelled analogues to act as quantum acceptors in fluorescence energy transfer from the tryptophan residues of the (Ca2+ + Mg2+)-ATPase. This is the first time that a physical interaction between PIP/PIP2 and an intrinsic membrane protein has been demonstrated. The dependence of the energy transfer on the number of negative charges of the phospholipids closely resembles the previously demonstrated charge dependence of the enzymatic activity of the (Ca2+ + Mg2+)-ATPase (Missiaen, L., Raeymaekers, L., Wuytack, F., Vrolix, M., Desmet, H. and Casteels, R. (1989) Biochem. J. 263, 687-694). It is concluded that the stimulation of the (Ca2+ + Mg2+)-ATPase activity by negatively charged phospholipids is based on a binding of these lipids to the (Ca2+ + Mg2+)-ATPase and that the negative charges are a major modulatory factor for this interaction.

We will demonstrate that compound 4880 and ruthenium red inhibit the smooth-muscle plasma-membrane Ca2+ pump by counteracting the stimulant effect of negatively charged phospholipids. Both substances did not affect the purified enzyme re-activated by pure phosphatidylcholine or phosphatidylinositol and measured in the absence of calmodulin, indicating that under these conditions they did not have a direct effect on the ATPase protein. Ruthenium red and compound 4880 however inhibited the (Ca2+ + Mg2+)-ATPase in the presence of phosphatidylinositol 4-phosphate and especially phosphatidylinositol 4,5-biphosphate. The K0.5 for inhibition was 25 μM ruthenium red and 9 μg/ml of compound 4880. The inhibition by ruthenium red developed slowly with half maximal inhibition occurring after about 75 s while that by compound 4880 developed immediately within the time required for mixing. The efficacy of ruthenium red increased as the concentration of the acidic phospholipid increased, while no such cooperativity was observed for compound 4880. Ruthenium red reduced the Vmax for Ca2+ without affecting the affinity for Ca2+, while compound 4880 decreased both parameters. In conclusion, although ruthenium red and compound 4880 affect the ATPase differently, both substances most likely inhibit the plasma-membrane Ca2+ pump by counteracting the stimulation by negatively charged phospholipids.

Studies of intact smooth muscle have suggested that its anomalous aerobic lactate production may reflect an intracellular compartmentation of glycolytic enzyme cascades designed to support specific exergonic processes. In particular, we have postulated a membrane-associated glycolytic cascade that preferentially supports the ATP requirements of membrane functions. We tested this hypothesis by using a smooth muscle plasma membrane fraction (PMV) purified for calcium pump activity. We show that glycolytic enzymes are endogenous in PMV and can produce NADH, ATP, and lactate from fructose 1,6-diphosphate in the presence of glycolytic cofactors. This glycolytic cascade can fuel the calcium pump despite the presence of an ATP trap that eliminated calcium uptake fueled by exogenously added ATP. This plasma membrane glycolytic cascade is coupled to calcium pump function in a tissue with both oxidative and glycolytic metabolism. Thus coupling of metabolic cascades with the specific processes they subserve may be a more general feature of cellular organization than was previously thought.

The sections in this article are: 1 Ionic Content 1.1 Chemical Analysis 1.1.1 Total Tissue Content 1.1.2 Extracellular Space 1.1.3 Cellular Ion Content 1.2 Tissue Tracer Content 1.3 Electron Microscopy and X-Ray Microanalysis 1.4 Ion-Sensitive Microelectrodes 1.5 Optical Methods 2 Ion Movements 2.1 Passive Ion Movements 2.2 Active Transport 2.2.1 Sodium-potassium Pump 2.2.2 Calcium Pumps in Plasmalemma and Endoplasmic Reticulum 2.3 Exchange Mechanism 2.3.1 Sodium-sodium Exchange 2.3.2 Sodium-calcium Exchange and its Role in Calcium Extrusion 2.3.3 Chloride-bicarbonate Exchange 2.3.4 Sodium-Hydrogen Exchange

The purified calmodulin dependent (Ca2++Mg2+)-ATPase (CaMg ATPase) from porcine antral smooth muscle transports Ca2+ after reconstitution in lipid vesicles indicating that this enzyme is indeed a Ca2+-transport ATPase. For CaMg ATPase reconstituted in asolectin vesicles a good correlation was found between the time course of Ca2+ accumulation and the corresponding changes in CaMg ATPase activity. The ATPase activity was stimulated 8-fold by A23187, which further indicates a tight coupling between ATP hydrolysis and Ca2+ transport. Asolectin vesicles with incorporated enzyme accumulated Ca2+ with a ratio approaching one Ca2+ ion transported for each ATP hydrolyzed. For CaMg ATPase reconstituted in phosphatidylcholine vesicles on the other hand, Ca2+ transport and CaMg ATPase were poorly coupled as is shown by the approximately 3.5 fold stimulation by A23187. The activity of the CaMg ATPase when reconstituted in asolectin vesicles was stimulated 1.25 fold by calmodulin while in phosphatidylcholine a value of 4.25 was obtained. The CaMg ATPase activity of the enzyme reconstituted either in asolectin or phosphatidylcholine was, after its stimulation by A23187, still further stimulated by detergent by a factor of 5.

The effects of three Ca-antagonists: diltiazem, nicardipine and flunarizine have been studied on excitatory junction potentials (e.j.p.s), force development and efflux of transmitter during stimulation of perivascular nerves in the rabbit ear artery.