The role of nitric oxide and l-type calcium channel blocker in the contractility of rabbit ileum in vitro

Nitric oxide (NO) and calcium channel blockers are two agents that can affect gastrointestinal motility. The goal of this work was to study the rabbit intestinal smooth muscle contraction response to (1) sodium nitroprusside (SNP), the NO donor, and its potential mechanism of action, and (2) nifedipine, the l-type Ca²⁺ channel blocker; to clarify the degree of participation by extra- and intracellular Ca²⁺ in smooth muscle contraction. We used standard isometric tension and intracellular micro-electrode recordings. To record the activity of the longitudinal smooth muscle of the ileum, segments of 1.5?cm length of the ileum were suspended vertically in organ baths of Krebs solution. The mechanical activity of the isolated ileal longitudinal muscle was recorded. Different substances were added, and the changes produced on spontaneous contraction were recorded. We found that SNP produced significant decrease, while nitric oxide synthase inhibitor produced significant increase in the amplitude of spontaneous contractions. Both apamin, the Ca²⁺-dependent K⁺ channel blocker, and methylene blue, the inhibitor of soluble guanylate cyclase, alone, partially decreased relaxation induced by SNP. Addition of both methylene blue and apamine together abolished the inhibitory effect produced by SNP on spontaneous contractions. Nifedipine produced significant decrease in the amplitude of spontaneous contractions. In conclusion, in longitudinal muscle of rabbit ileum, calcium channels blocker are potent inhibitors of spontaneous activity. However, both extracellular and intracellular Ca²⁺ participates in the spontaneous contractions. NO also has inhibitory effect on spontaneous activity, and this effect is mediated by cGMP generation system and Ca²⁺-dependent K⁺ channels.     

Heartwood extract of Rhus verniciflua Stokes and its active constituent fisetin attenuate vasoconstriction through calcium-dependent mechanism in rat aorta

Rhus verniciflua Stokes (RVS) exert cardiovascular protective activity by promoting blood circulation, but its active ingredients and underlying mechanism have yet to be identified. This study investigated the vascular effects of RVS, focusing on vasoconstriction and smooth muscle Ca²⁺ signaling. RVS heartwood extract attenuated contraction of aortic rings induced by the vasoconstrictors serotonin and phenylephrine, and inhibited the Ca²⁺ signaling evoked by serotonin in vascular smooth muscle cells. Subsequent activity-guided fractionation identified fisetin as an active constituent exerting a Ca²⁺ inhibitory effect. Fisetin could inhibit major Ca²⁺ mobilization pathways including extracellular Ca²⁺ influx mediated by the L-type voltage-gated Ca²⁺ channel, Ca²⁺ release from the intracellular store and store-operated Ca²⁺ entry. In accordance with Ca²⁺ inhibitory effect, fisetin attenuated vasoconstriction by serotonin and phenylephrine. These results suggest that the anticontractile effect, which is presumably mediated by inhibition of Ca²⁺ signaling, may contribute to the improvement of blood circulation by RVS.     

Differential functional properties of Ca stores in pulmonary arterial conduit and resistance myocytes

In smooth muscle cells, oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i) are controlled by inositol 1,4,5-trisphosphate (InsP₃) and ryanodine (Ry) receptors on the sarcoplasmic reticulum (SR). Here we show that these Ca²⁺ oscillations are regulated differentially by InsP₃ and Ry receptors in cells dispersed from the main trunk of the pulmonary artery (conduit myocytes) or from tertiary and quaternary arterial branches (resistance myocytes). Ry receptor antagonists inhibit either spontaneous or ATP-induced Ca²⁺ oscillations in resistance myocytes but they do not affect the oscillations in most conduit myocytes. In contrast, agents that inhibit InsP₃ production or activation of InsP₃ receptors do not alter the oscillations is resistance myocytes but block them in conduit myocytes. We have also examined the degree of overlap of Ry- and InsP₃-sensitive stores in myocytes along the pulmonary arterial tree. In conduit myocytes, depletion of Ry-sensitive stores with repeated application of caffeine in the presence of Ry or in Ca²⁺ free solutions did not prevent the ATP-induced Ca²⁺ release from InsP₃-dependent stores. However, responsiveness to ATP was completely abolished in resistance myocytes subjected to the same experimental protocol. Thus, InsP₃- and Ry-dependent stores appear to be separated in conduit myocytes but joined in resistance myocytes. These data demonstrate for the first time differential properties of intracellular Ca²⁺ stores and receptors in myocytes distributed along the pulmonary arterial tree and help to explain the distinct functional responses of large and small pulmonary vessels to vasoactive agents.     

Calcium oscillations in human mesenteric vascular smooth muscle

Phenylephrine (PE)-induced oscillatory fluctuations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) of vascular smooth muscle have been observed in many blood vessels isolated from a wide variety of mammals. Paradoxically, until recently similar observations in humans have proven elusive. In this study, we report for the first time observations of adrenergically-stimulated [Ca²⁺]_i oscillations in human mesenteric artery smooth muscle. In arterial segments preloaded with Fluo-4 AM and mounted on a myograph on the stage of a confocal microscope, we observed PE-induced oscillations in [Ca²⁺]_i, which initiated and maintained vasoconstriction. These oscillations present some variability, possibly due to compromised health of the tissue. This view is corroborated by our ultrastructural analysis of the cells, in which we found only (5 ± 2)% plasma membrane-sarcoplasmic reticulum apposition, markedly less than measured in healthy tissue from laboratory animals. We also partially characterized the oscillations by using the inhibitory drugs 2-aminoethoxydiphenyl borate (2-APB), cyclopiazonic acid (CPA) and nifedipine. After PE contraction, all drugs provoked relaxation of the vessel segments, sometimes only partial, and reduced or inhibited oscillations, except CPA, which rarely caused relaxation. These preliminary results point to a potential involvement of the sarcoplasmic reticulum Ca²⁺ and inositol 1,4,5-trisphosphate receptor (IP3R) in the maintenance of the Ca²⁺ oscillations observed in human blood vessels.     

Calcium oscillations in the olfactory nonsensory cells of the goldfish, Carassius auratus

Background

The olfactory nonsensory cells contribute to the maintenance of normal functions of the olfactory epithelium (OE). Specifically, the ciliated nonsensory cells of teleosts play important roles in the odorant detection by OE in aqueous environment. Their cilia show strong beating activities and cause water flow at the OE surface, making the detection of odorants by OE more efficient. Because intracellular Ca²⁺ level has been reported to play an important role in ciliary beating, the ciliary beating activity may be regulated by intracellular Ca²⁺ dynamics of these ciliated nonsensory cells.

Methods

We performed Ca²⁺ imaging experiments to analyze the Ca²⁺ dynamics in acutely dissociated OE cells of the goldfish. Furthermore, we examined the contribution of the Ca²⁺ dynamics to the ciliary beating frequency (CBF) at the surface of the intact OE.

Results

Olfactory nonsensory cells showed both spontaneous intracellular Ca²⁺ oscillations and propagating intercellular Ca²⁺ waves. Application of 2-aminoethoxydiphenylborate (2-APB), which antagonizes IP₃-induced Ca²⁺ release from intracellular stores suppressed these Ca²⁺ oscillations. Furthermore, 2-APB application to the intact OE lamellae resulted in the decrease of CBF at the surface of the OE.

Conclusions

These results indicate that spontaneous intracellular calcium oscillations persistently up-regulate the ciliary beating at the surface of the OE in teleosts.

General significance

Ciliary beating activity at the surface of OE can be regulated by the Ca²⁺ dynamics of olfactory nonsensory cells. Because this ciliary movement causes inflow of external fluid into the nostril, this regulation is suggested to influence the efficiency of odorant detection by OE.     

Ca2+ Sparks Act as Potent Regulators of Excitation-Contraction Coupling in Airway Smooth Muscle

Ca²⁺ sparks are short lived and localized Ca²⁺ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca²⁺-activated K⁺ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca²⁺ channels. But in many smooth muscles from a variety of organs, Ca²⁺ sparks can additionally activate Ca²⁺-activated Cl⁻ channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca²⁺ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca²⁺ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca²⁺ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca²⁺ channels, resulting in an increase in global [Ca²⁺]_i and cell contraction. Therefore, Ca²⁺ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.     

Mathematical Modelling of Ca2+ Oscillations in Airway Smooth Muscle Cells

The action of different agonists such as acetylcholine on the membrane of airway smooth muscle cells may induce cytosolic Ca²⁺ oscillations which can be a part of the Ca²⁺ signalling pathway, eventually leading to cell contraction. The aim of the present study is to present a mathematical model of the possible effect of the initial Ca²⁺ distribution within the cell on the form and frequency of induced Ca²⁺ oscillations. It takes into account intracellular Ca²⁺ stores such as sarcoplasmic reticulum and cytosolic proteins as well as Ca²⁺ exchange across the plasma membrane. We are able to demonstrate a closer agreement of model predictions with observed Ca²⁺ traces for a significantly wider range of parameter values, as was previously reported. We show also that the total cellular Ca²⁺ content is an important system parameter especially because of the content in sarcoplasmic reticulum. At a total Ca²⁺ increase of about 20%, the oscillation frequency increases by 25%; also, damped oscillations become sustained. Cases are indicated in which such a situation could occur.     

Both Neurons and Astrocytes Exhibited Tetrodotoxin-Resistant Metabotropic Glutamate Receptor-Dependent Spontaneous Slow Ca2+ Oscillations in Striatum

The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson''s disease. The group I mGluRs are known to modulate the intracellular Ca²⁺ signaling. To characterize Ca²⁺ signaling in striatal cells, spontaneous cytoplasmic Ca²⁺ transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca²⁺ transients (referred to as slow Ca²⁺ oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca²⁺ oscillation. Depletion of the intracellular Ca²⁺ store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca²⁺ oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. The slow Ca²⁺ oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca²⁺ oscillation may involve in the neuron-glia interaction in the striatum.     

A quantitative model for linking Na+/Ca2+ exchanger to SERCA during refilling of the sarcoplasmic reticulum to sustain [Ca2+] oscillations in vascular smooth muscle

We have developed a quantitative model for the creation of cytoplasmic Ca²⁺ gradients near the inner surface of the plasma membrane (PM). In particular we simulated the refilling of the sarcoplasmic reticulum (SR) via PM–SR junctions during asynchronous [Ca²⁺]_i oscillations in smooth muscle cells of the rabbit inferior vena cava. We have combined confocal microscopy data on the [Ca²⁺]_i oscillations, force transduction data from cell contraction studies and electron microscopic images to build a basis for computational simulations that model the transport of calcium ions from Na⁺/Ca²⁺ exchangers (NCX) on the PM to sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) pumps on the SR as a three-dimensional random walk through the PM–SR junctional cytoplasmic spaces. Electron microscopic ultrastructural images of the smooth muscle cells were elaborated with software algorithms to produce a very clear and dimensionally accurate picture of the PM–SR junctions. From this study, we conclude that it is plausible and possible for enough Ca²⁺ to pass through the PM–SR junctions to replete the SR during the regenerative Ca²⁺ release, which underlies agonist induced asynchronous Ca²⁺ oscillations in vascular smooth muscle.     

Guard of Delinquency? A Role of Microglia in Inflammatory Neurodegenerative Diseases of the CNS

Previous studies have shown that cGMP-dependent protein kinase (PKG) act on several targets in the contractile pathway to reduce intracellular Ca²⁺ and/or augment RhoA-regulated myosin light chain phosphatase (MLCP) activity and cause muscle relaxation. Recent studies have identified a novel protein M-RIP that associates with MYPT1, the regulatory subunit of MLCP. Herein, we examine whether PKG enhance MLCP activity downstream of Ca²⁺ and RhoA via phosphorylation of M-RIP in gastric smooth muscle cells. Treatment of permeabilized muscle cells with 10 μM Ca²⁺ caused an increase in MLC₂₀ phosphorylation and muscle contraction, but had no effect on Rho kinase activity. Activators of PKG (GSNO or cGMP) decreased MLC₂₀ phosphorylation and contraction in response to 10 μM Ca²⁺, implying existence of inhibitory mechanism independent of Ca²⁺ and RhoA. The effect of PKG on Ca²⁺-induced MLC₂₀ phosphorylation was attenuated by M-RIP siRNA. Both GSNO and 8-pCPT-cGMP induced phosphorylation of M-RIP; phosphorylation was accompanied by an increase in the association of M-RIP with MYPT1 and MLCP activity. Taken together, these results provide evidence that PKG induces phosphorylation of M-RIP and enhances its association with MYPT1 to augment MLCP activity and MLC₂₀ dephosphorylation and inhibits muscle contraction, downstream of Ca²⁺- or RhoA-dependent pathways.     

Optical induction of muscle contraction at the tissue scale through intrinsic cellular amplifiers

The smooth muscle cell is the principal component responsible for involuntary control of visceral organs, including vascular tonicity, secretion, and sphincter regulation. It is known that the neurotransmitters released from nerve endings increase the intracellular Ca²⁺ level in smooth muscle cells followed by muscle contraction. We herein report that femtosecond laser pulses focused on the diffraction‐limited volume can induce intracellular Ca²⁺ increases in the irradiated smooth muscle cell without neurotransmitters, and locally increased intracellular Ca²⁺ levels are amplified by calcium‐induced calcium‐releasing mechanisms through the ryanodine receptor, a Ca²⁺ channel of the endoplasmic reticulum. The laser‐induced Ca²⁺ increases propagate to adjacent cells through gap junctions. Thus, ultrashort‐pulsed lasers can induce smooth muscle contraction by controlling Ca²⁺, even with optical stimulation of the diffraction‐limited volume. This optical method, which leads to reversible and reproducible muscle contraction, can be used in research into muscle dynamics, neuromuscular disease treatment, and nanorobot control. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

TPC2 Proteins Mediate Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)- and Agonist-evoked Contractions of Smooth Muscle

Agonists such as those acting at muscarinic receptors are thought to induce contraction of smooth muscle primarily through inositol 1,4,5-trisphosphate production and release of Ca²⁺ from sarcoplasmic reticulum. However, the additional Ca²⁺-mobilizing messengers cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) may also be involved in this process, the former acting on the sarcoplasmic reticulum, the latter acting on lysosome-related organelles. In this study, we provide the first systematic analysis of the capacity of inositol 1,4,5-trisphosphate, cADPR, and NAADP to cause contraction in smooth muscle. Using permeabilized guinea pig detrusor and taenia caecum, we show that all three Ca²⁺-mobilizing messengers cause contractions in both types of smooth muscle. We demonstrate that cADPR and NAADP play differential roles in mediating contraction in response to muscarinic receptor activation, with a sizeable role for NAADP and acidic calcium stores in detrusor muscle but not in taenia caecum, underscoring the heterogeneity of smooth muscle signal transduction systems. Two-pore channel proteins (TPCs) have recently been shown to be key components of the NAADP receptor. We show that contractile responses to NAADP were completely abolished, and agonist-evoked contractions were reduced and now became independent of acidic calcium stores in Tpcn2^−/− mouse detrusor smooth muscle. Our findings provide the first evidence that TPC proteins mediate a key NAADP-regulated tissue response brought about by agonist activation of a cell surface receptor.     

SKA-31-induced activation of small-conductance calcium-activated potassium channels decreased modulation of detrusor smooth muscle function in a rat model of obesity

Journal of Bioenergetics and Biomembranes - Increased excitability and contractility of detrusor smooth muscle (DSM) cells are associated with overactive bladder (OAB), which is often induced by...     

Increase in nuclear calcium in smooth muscle cells exposed to oxidized low density lipoprotein

Vascular smooth muscle cells respond with an increase in intracellular Ca²⁺ within seconds after exposure to oxidized low density lipoprotein (oxLDL). This has been suggested to represent a signaling response that may have implications for gene expression. If so, oxLDL may induce increases in nuclear Ca²⁺ in smooth muscle cells in response to oxLDL. Aortic smooth muscle cells were exposed to 100 μg/ml oxLDL. Large, rapid increases in [Ca²⁺]_i were observed using fluo-3 as an indicator dye to detect intracellular Ca²⁺ on the stage of a confocal micro-scope. This was also confirmed using ratiometric imaging of indo signals. These elevations appeared to be localized to the nuclear region of the cell. DNA staining of the cells confirmed its localization to the nuclear / perinuclear region of the cell. Our data demonstrate that oxLDL induces a nuclear localized elevation in Ca²⁺i that may have important implications for nuclear function.     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

Identification of new functions of Ca2+ release from intracellular stores in central nervous system

Ca²⁺ release from intracellular stores regulates muscle contraction and a vast array of cell functions, but its role in the central nervous system (CNS) has not been completely elucidated. A new method of blocking IP₃ signaling by artificially expressing IP₃ 5-phosphatase has been used to clarify the functions of intracellular Ca²⁺ mobilization in CNS. Here I review two of such functions: the activity-dependent synaptic maintenance mechanism and the regulation of neuronal growth by spontaneous Ca²⁺ oscillations in astrocytes. These findings add new bases for better understanding CNS functions and suggest the presence of as yet unidentified neuronal and glial functions that are regulated by Ca²⁺ store-dependent Ca²⁺ signaling.     

Accelerated communication the direct contractile effect of gastrin releasing peptide on isolated gastric smooth muscle cells of the guinea pig

Smooth muscle cells isolated from the gastric muscle layers of the guinea pig were used to determine whether gastrin releasing peptide (GRP) can cause contraction by exerting a direct action on muscle cells. In addition, the inhibitory effect of 8-( N,N-diethylamino )-octyl-3,4,5-trimethoxybenzoate hydrochloride ( TMB-8 ), an inhibitor of intracellular Ca²⁺ release, and verapamil, a Ca²⁺ channel blocker, on the GRP-induced contraction of gastric smooth muscle cells were examined. GRP elicited a contractile response of gastric muscle cells in a dose-dependent manner. The ED₅₀ was 13 pM. TMB-8 significantly inhibited the contractile effect of GRP in gastric muscle cells. These results demonstrate the direct action of GRP on the gastric smooth muscle cells of the guinea pig, and the importance of Ca²⁺-release from intracellular calcium stores in the contractile response to GRP.