Morphofunctional study of 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA)‐induced differentiation of U937 cells under exposure to a 6 mT static magnetic field

This study deals with the morphofunctional influence of 72 h exposure to a 6 mT static magnetic field (SMF) during differentiation induced by 50 ng/ml 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA) in human leukaemia U937 cells. The cell morphology of U937 cells was investigated by optic and electron microscopy. Specific antibodies and/or molecules were used to label CD11c, CD14, phosphatidylserine, F‐actin and to investigate the distribution and activity of lysosomes, mitochondria and SER. [Ca²⁺]_i was evaluated with a spectrophotometer. The degree of differentiation in SMF‐exposed cells was lower than that of non‐exposed cells, the difference being exposure time‐dependent. SMF‐exposed cells showed cell shape and F‐actin modification, inhibition of cell attachment, appearance of membrane roughness and large blebs and impaired expression of specific macrophagic markers on the cell surface. The intracellular localization of SER and lysosomes was only partially affected by exposure. A significant localization of mitochondria with an intact membrane potential at the cell periphery in non‐exposed, TPA‐stimulated cells was observed; conversely, in the presence of SMF, mitochondria were mainly localised near the nucleus. In no case did SMF exposure affect cell viability. The sharp intracellular increase of [Ca²⁺]_i could be one of the causes of the above‐described changes. Bioelectromagnetics 30:352–364, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of 6 mT static magnetic field on the bcl-2, bax, p53 and hsp70 expression in freshly isolated and in vitro aged human lymphocytes

An increasing number of evidence indicates that static magnetic fields (SMFs) are capable of altering apoptosis, mainly through modulation of Ca²⁺ influx. Here we present data that suggest apoptotic-related gene expression as an alternative pathway, through which exposure to 6 milliTesla (mT) SMF can interfere with apoptosis. Exposure to 6 mT SMF affects the apoptotic rate (spontaneous and drug-induced) and [Ca²⁺]_i in isolated human lymphocytes; the aged cells are more susceptible to exposure than fresh ones. The exposure to 6 mT exerted a protective effect on chemical or physical-induced apoptosis, irrespective of the age of the cells.The investigation of the gene expression of bcl-2, bax, p53 and hsp70 in freshly isolated and in culture-aged human lymphocytes indicates that these genes are modulated by SMF exposure in the experimental conditions used, in a gene-, age- and time-dependent manner. The exposure of isolated lymphocytes to SMF for up to 24 h modulated increased bax and p53 and decreased hsp70, and bcl-2. The amount of increment and/or decrement of the proteins varied for each gene examined and was independent of the apoptotic inducers. Finally, the same stress applied to freshly isolated or aged lymphocytes resulted in different modulation of bcl-2, bax and hsp70.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Calcium,protease activation,and cytoskeleton remodeling underlie growth cone formation and neuronal regeneration

The cytoarchitecture, synaptic connectivity, and physiological properties of neurons are determined during their development by the interactions between the intrinsic properties of the neurons and signals provided by the microenvironment through which they grow. Many of these interactions are mediated and translated to specific growth patterns and connectivity by specialized compartments at the tips of the extending neurites: the growth cones (GCs). The mechanisms underlying GC formation at a specific time and location during development, regeneration, and some forms of learning processes, are therefore the subject of intense investigation. Using cultured Aplysia neurons we studied the cellular mechanisms that lead to the transformation of a differentiated axonal segment into a motile GC. We found that localized and transient elevation of the free intracellular calcium concentration ([Ca²⁺] _i ) to 200–300 M induces GC formation in the form of a large lamellipodium that branches up into growing neurites. By using simultaneous on-line imaging of [Ca²⁺] _i and of intraaxonal proteolyticactivity, we found that the elevated [Ca²⁺] _i activate proteases in the region in which a GC is formed. Inhibition of the calcium-activated proteases prior to the local elevation of the [Ca²⁺] _i blocks the formation of GCs. Using retrospective immunofluorescent methods we imaged the proteolysis of the submembrane spectrin network, and the restructuring of the cytoskeleton at the site of GC formation. The restructuring of the actin and microtubule network leads to local accumulation of transported vesicles, which then fuse with the plasma membrane in support of the GC expansion.     

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes

Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca²⁺ release in the cell cortex. Here, we show that this Ca²⁺ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca²⁺ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca²⁺ release during meiotic maturation. The Ca²⁺ wave was inhibited by the classical antagonists of the InsP₃-linked Ca²⁺ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca²⁺ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP₃, despite the massive release of Ca²⁺, implying that exocytosis of the cortical granules requires not only a Ca²⁺ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca²⁺ signals and in regulating cortical granule exocytosis.     

Permeability of a cell junction and the local cytoplasmic free ionized calcium concentration: A study with aequorin

Summary A technique is devised to determine the spatial distribution of the free ionized cytoplasmic calcium concentration ([Ca²⁺] _i ) inside a cell:Chironomus salivary gland cells are loaded with aequorin, and the Ca²⁺-dependent light emission of the aequorin is scanned with an image-intensifier/television system. With this technique, the [Ca²⁺] _i is determined simultaneously with junctional electrical coupling when Ca²⁺ is microinjected into the cells, or when the cells are exposed to metabolic inhibitors, Ca-transporting ionophores, or Ca-free medium. Ca microinjections elevating the [Ca²⁺] _i the junctional locale produce depression of junctional membrane conductance. When the [Ca²⁺] _i elevation is confined to the vicinity of one cell junction, the conductance of that junction alone is depressed; other junctions of the same cell are not affected. The depression sets in as the [Ca²⁺] _i rises in the junctional locale, and reverses after the [Ca²⁺] _i falls to baseline. When the [Ca²⁺] _i elevation is diffuse throughout the cell, the conductances of all junctions of the cell are depressed. The Ca injections produce no detectable [Ca²⁺] _i elevations in cells adjacent to the injected one; the Ca-induced change in junctional membrane permeability seems fast enough to block appreciable transjunctional flow of Ca²⁺. Control injections of Cl^– or K⁺ do not affect junctional conductance. The Ca injections that elevate [Ca²⁺] _i sufficiently to depress junctional conductance also produce under the usual conditions an increase in nonjunctional membrane conductance and, hence, depolarization. But injections that elevate [Ca²⁺] _i at the junction while largely avoiding nonjunctional membrane cause depression of junctional conductance with little or no depolarization. Moreover, elevations of [Ca²⁺] _i in cells clamped near resting potential produce the depression, too. On the other hand, complete depolarization in K medium does not produce the depression, unless accompanied by [Ca²⁺] _i elevation. Thus, the depolarization is neither necessary nor sufficient for depression of junctional conductance. Treatment with cyanide, dinitrophenol and ionophores X537 A or A23187 produces diffuse elevation of [Ca²⁺] _i associated with depression of nunctional conductance. Prolonged exposure to Ca-free medium leads to fluctuation in [Ca²⁺] _i where rise and fall of [Ca²⁺] _i correlate respectively with fall and rise in junctional conductance.     

A Prototypic Intracellular Calcium Antagonist, TMB-8, Protects Cultured Cerebellar Granule Cells Against the Delayed, Calcium-Dependent Component of Glutamate Neurotoxicity

Abstract: The effect(s) of a prototypic intracellular Ca²⁺ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free-Ca²⁺ levels ([Ca²⁺]_i) that was dependent on the extracellular concentration of Ca²⁺ ([Ca²⁺]_o). In addition, this increase in [Ca²⁺]_i correlated with a decrease in cell viability that was also dependent on [Ca²⁺]_o. Glutamate-induced toxicity, quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an “early” Na⁺/Cl^?-dependent component observed within minutes of glutamate exposure, and a “delayed” Ca²⁺-dependent component (ED₅₀～50 µM) that coincided with progressive degeneration of granule cells 4–24 h after a brief (5–15 min) exposure to 100 µM glutamate. Quantitative analysis of cell viability and morphological observations identify a “window” in which TMB-8 (at >100 µM) protects granule cells from the Ca²⁺-dependent, but not the Na⁺/Cl^?-dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca²⁺]_i. These findings suggest that Ca²⁺ release from a TMB-8-sensitive intracellular store may be a necessary step in the onset of glutamate-induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate-evoked increases in [Ca²⁺]_i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca²⁺ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca²⁺]_i and glutamate-induced neurotoxicity.     

Latrunculin A depolarizes starfish oocytes

Depolymerization of the actin cytoskeleton may liberate Ca²⁺ from InsP₃-sensitive stores in some cell types, including starfish oocytes, while inhibiting Ca²⁺ influx in others. However, no information is available on the modulation of membrane potential (V_m) by actin. The present study was aimed to ascertain whether the widely employed actin depolymerizing drug, latrunculin A (Lat A), affects V_m in mature oocytes of the starfish Astropecten aranciacus. Lat A induced a membrane depolarization which was mimicked by cytochalasin D, another popular actin disruptor, and prevented by jasplakinolide, a stabilizer of the actin network. Lat A-elicited depolarization consisted in a positive shift in V_m which reached the threshold of activation of voltage-gated Ca²⁺ channels (VGCC), thus triggering an action potential. Lat A-promoted depolarization lacked the action potential in Ca²⁺-free sea water, while it was abolished upon removal of external Na⁺. Moreover, membrane depolarization was prevented by pre-injection of BAPTA and heparin, but not ryanodine. These data indicate that Lat A induces a membrane depolarization by releasing Ca²⁺ from InsP₃Rs. The Ca²⁺ signal in turn activates a Ca²⁺-dependent Na⁺ entry, which causes the positive shift in V_m and stimulates the VGCC.     

Altered intracellular calcium fluxes in pancreatic cancer induced diabetes mellitus: Relevance of the S100A8 N‐terminal peptide (NT‐S100A8)

After isolating NT‐S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca²⁺]_i oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT‐S100A8. In NT‐S100A8 stimulated β‐TC6 (insulinoma cell line) culture medium, insulin and [Ca²⁺] were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca²⁺]_i oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT‐S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT‐S100A8 induced a rapid insulin release and enhanced β‐TC6 [Ca²⁺]_i oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT‐S100A8, [Ca²⁺] in β‐TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT‐S100A8 did not counteract insulin induced phosphorylation of the insulin receptor, Akt and IκB‐α, but it independently activated Akt and NF‐κB signaling in PC cells. In conclusion, NT‐S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF‐κB signaling, NT‐S100A8 enhances [Ca²⁺]_i oscillations and insulin release, probably by inducing Ca²⁺ influx from the extracellular space, but it does not interfere with insulin signaling. J. Cell. Physiol. 226: 456–468, 2011. © 2010 Wiley‐Liss, Inc.     

Regulation of Intracellular Ca2+ by CFTR in Chinese Hamster Ovary Cells

In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl⁻ secretion. As recently proposed, beside its role of Cl⁻ channel, CFTR may regulate the activity of other channels such as a Ca²⁺-activated Cl⁻ channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers, that CFTR can act as a regulator of intracellular [Ca²⁺] _i ([Ca²⁺] _i ), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca²⁺] _i in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with an adenovirus-LacZ (CHO-LacZ). The transient [Ca²⁺] _i increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 μm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca²⁺] _i in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 μm) or reactive blue-2. (100 μm), and with hexokinase (0.28 U/mg) inhibited the [Ca²⁺] _i response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation by ATP released from the cell and regulation of [Ca²⁺] _i by CFTR in CHO epithelial cell membranes. Received: 5 April 1999/Revised: 28 June 1999     

The Role of Calcium in the Volume Regulation of Rat Lacrimal Acinar Cells

Earlier studies have suggested a role for Ca²⁺ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca²⁺-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca²⁺ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd³⁺ inhibited RVD, suggesting that Ca²⁺ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca²⁺] _i during exposure to hypotonic solutions. The absence of any changes in [Ca²⁺] _i resulted from the buffering of cytosolic Ca²⁺ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca²⁺ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca²⁺] _i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca²⁺-activated K⁺ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca²⁺ in controlling cell volume in lacrimal acinar cells. Received: 20 February 1998/Revised: 1 May 1998     

Intracellular calcium oscillations in a T-cell line after exposure to extremely-low-frequency magnetic fields with variable frequencies and flux densities

Low-frequency magnetic fields (MF) can increase the cytosolic calcium concentration ([Ca²⁺]_i) in lymphocytes in the same manner as a physiological stimulus such as antibodies directed towards the CD3 complex. In this study, MF with various frequencies and flux densities were used, while [Ca²⁺]_i changes were recorded using microfluorometry with fura-2 as a probe. The applied sinusoidal MF induced oscillatory changes of [Ca²⁺], in the leukemic cell line Jurkat in a manner similar to that seen with stimulation by antibodies. The response at 0.15 mT was over a frequency range from 5 to 100 Hz, with a fairly broad peak having its maximum at 50 Hz. The result of testing increasing flux densities at 50 Hz was a threshold response with no effect below 0.04 mT and a plateau at 0.15 mT. On the basis of the characteristic calcium pattern resulting from an applied MF, we suggest that MF influence molecular events in regular signal transduction pathways of T cells. © 1995 Wiley-Liss, Inc.     

The pattern of exposure to static magnetic field of nurses involved in activities related to contrast administration into patients diagnosed in 1.5 T MRI scanners

Static magnetic fields (SMFs) and time-varying electromagnetic fields exposure is necessary to obtain the diagnostic information regarding the structure of patient's tissues, by the use of magnetic resonance imaging (MRI) scanners. A diagnostic procedure may also include the administration of pharmaceuticals called contrast, which are to be applied to a patient during the examination. The nurses involved in administering contrast into a patient during the pause in examination are approaching permanently active magnets of MRI scanners and are exposed to SMF. There were performed measurements of spatial distribution of SMF in the vicinity of MRI magnets and parameters of personal exposure of nurses (i.e. individual exposimetric profiles of variability in time of SMF affecting the nurse who is performing tasks in the vicinity of magnet, characterized by statistical parameters of recorded magnetic flux density affecting the nurse). The SMF exposure in the vicinity of various MRI magnets depends on both magnetic flux density of the main field B ₀ (applicable to a patient) and the construction of the scanner, but the most important factor determining the workers' exposure is the work practice. In the course of a patient's routine examination in scanners of B ₀ = 1.5 T, the nurses are present over ～0.4–2.9 min in SMF exceeding 0.03% of B ₀ (i.e. 0.5 mT), but only sometimes they are present in SMF exceeding 5% of B ₀ (i.e. 75 mT). When patients need more attention because of their health status/condition, the nurses' exposure may be significantly longer – it may even exceed 10 min and 30% of B ₀ (i.e. 500 mT). We have found that the level of exposure of nurses to SMF may vary from < 5% of the main field (a median value: 0.5–1.5%; inter-quartile range: 0.04–8.8%; max value: 1.3–12% of B ₀) when a patient is moved from the magnets bore before contrast administration, up to the main field level (B ₀) when a patient stays in the magnets bore and nurse is crawling into the bore.     

Effect of static magnetic field on morphology and growth metabolism of Flavobacterium sp. m1-14

Increasing evidence shows that static magnetic fields (SMFs) can affect microbial growth metabolism, but the specific mechanism is still unclear. In this study, we have investigated the effect of moderate-strength SMFs on growth and vitamin K₂ biosynthesis of Flavobacterium sp. m1-14. First, we designed a series of different moderate-strength magnetic field intensities (0, 50, 100, 150, 190 mT) and exposure times (0, 24, 48, 72, 120 h). With the optimization of static magnetic field intensity and exposure time, biomass and vitamin K₂ production significantly increased compared to control. The maximum vitamin K₂ concentration and biomass were achieved when exposed to 100 mT SMF for 48 h; compared with the control group, they increased by 71.3% and 86.8%, respectively. Interestingly, it was found that both the cell viability and morphology changed significantly after SMF treatment. Second, the adenosine triphosphate (ATP) and glucose-6-phosphate dehydrogenase (G6PDH) metabolism is more vigorous after exposed to 100 mT SMF. This change affects the cell energy metabolism and fermentation behavior, and may partially explain the changes in bacterial biomass and vitamin K₂ production. The results show that moderate-strength SMFs may be a promising method to promote bacterial growth and secondary metabolite synthesis.

     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca²⁺]_i was strictly dependent on extracellular Ca²⁺ (Ca²⁺_e); similarly, in NG108-15 cells, a component of the elevations in [Ca²⁺]_i was Ca²⁺_e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca²⁺_e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn²⁺. Using ¹⁹F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn²⁺ concentration ([Zn²⁺]_i). In buffer containing 200 µM EGTA to prevent the Ca²⁺_e-dependent elevations in [Ca²⁺]_i, the [Zn²⁺]_i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn²⁺]_i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn²⁺]_i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn²⁺]_i to 3.99 ± 0.68 nM. No Zn²⁺ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca²⁺]_i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca²⁺]_i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca²⁺]_i differed significantly from either pretreatment levels or buffer-treated controls.     

Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

Static magnetic field effect on microcirculation,direct versus baroreflex-mediated approach

We compared in conscious rabbits, sedated using pentobarbital intravenous (i.v.) infusion (5 mg kg^{? 1} h^{? 1}), the effect of a static magnetic field (SMF), generated by Nd₂–Fe₁₄–B magnets, on microcirculation during its 40 min local exposure to the microvascular network in cutaneous tissue [20 sham exposure and 20 SMF (0.25 T) exposure runs] or to sinocarotid baroreceptors [14 sham exposure and 14 SMF (0.35 T) exposure runs]. Mean femoral artery blood pressure (BP), heart rate (HR), arterial baroreflex sensitivity (BRS), assessed from HR and BP responses to i.v. bolus of nitroprusside and phenylephrine, and microcirculatory blood flow, using microphotoelectric plethysmography (MPPG), were simultaneously monitored. SMF significantly increased microcirculation on a 17.8% in microvascular and on a 23.3% in baroreceptor exposure series. In baroreceptor exposure series, SMF significantly decreased BP, increased heart rate variability, BRS and sodium nitroprusside (NO-donor) i.v. bolus microcirculatory vasodilatory effect. These suggest augmentation of the arterial baroreflex capacity support NO-dependent vasodilation, by increased sensitivity of vessels to NO, to be a new physiological mechanism of BP buffering and microcirculatory control. A significant positive correlation was also found between increase in BRS and in MPPG (r = 0.66, p < 0.009), indicating baroreflex participation in the regulation of the microcirculation and its enhancement after SMF exposure. Both direct and baroreflex-mediated approaches demonstrate SMF significant vasodilatory effect with potential clinical implication in macro- and microcirculatory disorders.     

Spinorphin inhibits membrane depolarization- and capsaicin-induced intracellular calcium signals in rat primary nociceptive dorsal root ganglion neurons in culture

Abstract

Objective: Spinorphin is a potential endogenous antinociceptive agent although the mechanism(s) of its analgesic effect remain unknown. We conducted this study to investigate, by considering intracellular calcium concentrations as a key signal for nociceptive transmission, the effects of spinorphin on cytoplasmic Ca²⁺ ([Ca²⁺]_i) transients, evoked by high-K⁺ (30?mM) depolariasation or capsaicin, and to determine whether there were any differences in the effects of spinorphin among subpopulation of cultured rat dorsal root ganglion (DRG) neurons. Methods: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium sensitive dye fura-2 AM (1?µM). Intracellular calcium responses in individual DRG neurons were quantified using standard fura-2 based ratiometric calcium imaging technique. All data were analyzed by using unpaired t test, p?<?0.05 defining statistical significance. Results: Here we found that spinorphin inhibited cytoplasmic Ca²⁺ ([Ca²⁺]_i) transients, evoked by depolarization and capsaicin selectively in medium and small cultured rat DRG neurons. Spinorphin (10–300?µM) inhibited the Ca²⁺ signals in concentration dependant manner in small- and medium diameter DRG neurons. Capsaicin produced [Ca²⁺]_i responses only in small- and medium-sized DRG neurons, and pre-treatment with spinorphin significantly attenuated these [Ca²⁺]_i responses. Conclusion: Results from this study indicates that spinorphin significantly inhibits [Ca²⁺]_i signaling, which are key for the modulation of cell membrane excitability and neurotransmitter release, preferably in nociceptive subtypes of this primary sensory neurons suggesting that peripheral site is involved in the pain modulating effect of this endogenous agent.     

Mechanosensitive Calcium Entry and Mobilization in Renal A6 Cells

Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca²⁺ signal in response to osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior to a transient increase in [Ca²⁺] _i and the cells remained swollen after [Ca²⁺] _i had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca²⁺] after a delay of 22 sec. Both the RVD and [Ca²⁺] _i response to hypotonicity were inhibited in a Ca²⁺-free bathing solution and by gadolinium (10 μm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa) inside a cell-attached patch-pipette induced a similar global increase in [Ca²⁺] _i as occurred after hypotonic shock. A stretch-sensitive [Ca²⁺] _i increase was also observed in a Ca²⁺-free bathing solution, provided the patch-pipette contained Ca²⁺. The mechanosensitive [Ca²⁺] _i response was by gadolinium (10 μm) or Ca²⁺-free pipette solutions, even when Ca²⁺ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca²⁺] _i response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful amplification system linked to intracellular Ca²⁺ release mechanisms. Received: 3 August 1998/Revised: 19 November 1998