Theoretical and experimental investigation of calcium-contraction coupling in airway smooth muscle

We investigated theoretically and experimentally the Ca²⁺-contraction coupling in rat tracheal smooth muscle. [Ca²⁺]_i, isometric contraction and myosin light chain (MLC) phosphorylation were measured in response to 1 mM carbachol. Theoretical modeling consisted in coupling a model of Ca²⁺-dependent MLC kinase (MLCK) activation with a four-state model of smooth muscle contractile apparatus. Stimulation resulted in a short-time contraction obtained within 1 min, followed by a long-time contraction up to the maximal force obtained in 30 min. ML-7 and Wortmannin (MLCK inhibitors) abolished the contraction. Chelerythrine (PKC inhibitor) did not change the short-time, but reduced the long-time contraction. [Ca²⁺ _i responses of isolated myocytes recorded during the first 90 s consisted in a fast peak, followed by a plateau phase and, in 28% of the cells, superimposed Ca²⁺ oscillations. MLC phosphorylation was maximal at 5 s and then decreased whereas isometric contraction followed a Hill-shaped curve. The model properly predicts the time course of MLC phosphorylation and force of the short-time response. With oscillating Ca²⁺ signal, the predicted force does not oscillate. According to the model, the amplitude of the plateau and the frequency of oscillations encode for the amplitude of force, whereas the peak encodes for force velocity. The long-time phase of the contraction, associated with a second increase in MLC phosphorylation, may be explained, at least partially, by MLC phosphatase (MLCP) inhibition, possibly via PKC inhibition.     

Focal increases in [Ca]i may account for apparent low Ca sensitivity in swine carotid artery

Estimates of [Ca²⁺]_i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca²⁺]_i with indicators such as aequorin or Fura-2. We investigated whether focal in increases in [Ca²⁺]_i could impair such measures of [Ca²⁺]_i sensitivity. Stimulation of swine carotid artery with 10 μM histamine increased aequorin estimated [Ca²⁺]_i, Fura-2 estimated [Ca²⁺]_i and Ca²⁺ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca²⁺]_i homogeneity). Subsequent inhibition of Na⁺/Ca²⁺ exchange by replacement of Na⁺ in the PSS with choline⁺ significantly increased aequorin-estimated [Ca²⁺]_i but only minimally increased Fura-2 estimated [Ca²⁺]_i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca²⁺] inhomogeneity. Addition of 100 μM histamine to tissues in the choline⁺ buffer initially increased both aequorin and Fura-2 estimated [Ca²⁺]_i but after 10 min exposure both of the [Ca²⁺]_i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca²⁺ sensitivity, but the aequorin/Fura-2 ratio remained elevated and uncharged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca²⁺]_i, and thus can cause misleading assessments of Ca²⁺ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca²⁺]_i which can be evaluated with the aequorin/Fura-2 ratio.     

Regulation of endothelial cell gap formation and barrier dysfunction: Role of myosin light chain phosphorylation

Endothelial cell (EC) contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. We tested the hypothesis that phosphorylation of regulatory myosin light chains (MLC) by Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) is critical to EC barrier dysfunction elicited by thrombin. Thrombin stimulated a rapid (<15 sec) increase in [Ca²⁺]_i which preceded maximal MLC phosphorylation (60 sec) with a 6 to 8-fold increase above constitutive levels of phosphorylated MLC. Dramatic cellular shape changes indicative of contraction and gap formation were observed at 5 min with maximal increases in albumin permeability occurring by 10 min. Neither the Ca²⁺ ionophore, A23187, nor phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), alone or in combination, produced MLC phosphorylation. The combination was synergistic, however, in stimulating EC contraction/gap formation and barrier dysfunction (3 to 4-fold increase). Down-regulation or inhibition of PKC activity attenuated thrombin-induced MLC phosphorylation (～40% inhibition) and both thrombin- and PMA-induced albumin clearance (～50% inhibition). Agents which augmented [cAMP]_i partially blocked thrombin-induced MLC phosphorylation (～50%) and completely inhibited both thrombin- and PMA-induced EC permeability (100% inhibition). Furthermore, cAMP produced significant reduction in the basal levels of constitutive MLC phosphorylation. Finally, MLCK inhibition (with either ML-7 or KT 5926) or Ca²⁺/calmodulin antagonism (with either trifluoperazine or W-7) attenuated thrombin-induced MLC phosphorylation and barrier dysfunction. These results suggest a model wherein EC contractile events, gap formation and barrier dysfunction occur via MLCK-dependent and independent mechanisms and are significantly modulated by both PKC and cAMP-dependent protein kinase A activities. © 1995 Wiley-Liss, Inc.     

An Examination of the Role of Increased Cytosolic Free Ca2+Concentrations in the Inhibition of mRNA Translation

Mobilization of Ca²⁺sequestered by the endoplasmic reticulum (ER) produces the phosphorylation of initiation factor (eIF) 2, whereas an increase in cytosolic free Ca²⁺([Ca²⁺]_i) due to plasmalemmal Ca²⁺influx increases the phosphorylation of elongation factor (eEF) 2. In nucleated mammalian cells, depletion of ER Ca²⁺stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca²⁺]_iper se causes slowing of peptide chain elongation is lacking. L-type Ca²⁺channel activity of GH₃pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca²⁺]_ion eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca²⁺, resting [Ca²⁺]_ivalues were high (154–255 nM) and eEF-2 was phosphorylated. The Ca²⁺channel antagonist, nisoldipine, lowered [Ca²⁺]_iand reduced eEF-2 phosphorylation by half but had no effect on amino acid incorporation. The Ca²⁺channel agonist, Bay K 8644, produced sustained elevations of [Ca²⁺]_ithat were associated with 25–50% increases in eEF-2 phosphorylation, but no changes in protein synthetic rates occurred. Larger Ca²⁺influxes were achievable with either 25 mM KCl or KCl plus Bay K 8644. These treatments further increased eEF-2 phosphorylation (50–100% above control) and inhibited leucine incorporation by 20–70% but ATP content was reduced by 25–50% and total cell-associated Ca²⁺contents rose by 3- to 13-fold. eIF-2α was not phosphorylated during these treatments. Addition of low concentrations of ionomycin, which do not lower ATP content, was associated with complex changes in [Ca²⁺]_ithat resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF-2α, not eEF-2. It is concluded that changes in [Ca²⁺]_ioccurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.     

Signal transduction and protein phosphorylation in smooth muscle contraction

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, whereas striated muscles display a proportionality of generated force to the [Ca²⁺]_i level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca²⁺]_i. Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca²⁺]_i and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca²⁺-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.     

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses¹. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation². This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency⁴. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively⁵. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.Contraction of smooth muscle is generally regulated by the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) plus sensitivity to Ca^2+, of the contractile elements in response to changes in the environment surrounding the cell⁶. [Ca²⁺]_i is determined by the combination of the movement of Ca²⁺ through plasma membrane ligand or voltage gated Ca²⁺ channels and the release and uptake of Ca²⁺ from internal stores. Cytosolic Ca²⁺ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction⁸. However, the sensitivity of this pathway to Ca²⁺ can be regulated by the MLC phosphatase (MLCP)⁹. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.Here, we present a method to evaluate changes in [Ca²⁺]_i over time in isolated, perfused lymphatics in order to study Ca²⁺-dependent and Ca²⁺-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca²⁺]_i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca²⁺]_i was determined by loading lymphatics with the ratiometric, Ca²⁺-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.     

Calcium Requirement for α-MSH Action on Melanophores: Studies with Forskolin

Abstract

α-MSH-induced pigment dispersion in melanophores shows an absolute requirement for extracellular Ca²⁺. To localize Ca²⁺ sites involved in the mechanism of action of α-MSH we studied the effects of Ca²⁺ deprivation on α-MSH and forskolin-induced melanophore responses. In an in vitro melanophore system employing ventral tailfins of Xenopus tadpoles, melanophore responses were assayed in terms of pigment dispersion and the phosphorylation state of a 53 kDa melanophore-specific protein. In the same melanophore system α-MSH has been shown to specifically increase the phosphorylation of this 53 kDa protein.

Forskolin induces a dose-dependent pigment dispersion (EC₅₀ 7 × 10^?7 M). In contrast to the dispersion induced by α-MSH forskolin-induced dispersion does not require extracellular Ca²⁺. Moreover, in a Ca²⁺-free medium melanophores with permanently activated MSH-receptors aggregate, but can be redispersed by the addition of forskolin. Forskolin increases 53 kDa phosphorylation in a dosedependent manner. Maximal stimulation with forskolin (10^?5 M) is four-fold and equals maximal 53 kDa phosphorylation obtainable with α-MSH. The MSH-induced increase in 53 kDa phosphorylation is inhibited by Ca²⁺ deprivation, whereas the forskolin-induced increase is unaffected. Our results suggest that α-MSH and forskolin stimulate melanophores through a common pathway and confirm that cAMP is a second messenger in α-MSH action in this system. We conclude that the Ca²⁺ sites in the mechanism of α-MSH action on melanophores precede adenylate cyclase activation.     

CFTR Regulation of Intracellular Calcium in Normal and Cystic Fibrosis Human Airway Epithelia

In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl⁻ secretion to secretagogues acting via cAMP. Using a Ca²⁺ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca²⁺] _i via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase in [Ca²⁺] _i in normal (16HBE14o⁻ cell line and primary lung culture) and in cystic fibrosis (CFTE29o⁻ cell line) human airway epithelia. The potency order of nucleotides on [Ca²⁺] _i variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca²⁺] _i response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o⁻ cells, the forskolin-induced [Ca²⁺] _i response increased with increasing temperature. In CFTE29o⁻ cells, forskolin had no effect on [Ca²⁺] _i at body temperature-forskolin-induced [Ca²⁺] _i response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca²⁺] _i response. Together, these results demonstrate that once activated, CFTR regulates [Ca²⁺] _i by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia. Received: 3 April 2000/Revised: 30 June 2000     

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.     

Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus

KCl has long been used as a convenient stimulus to bypass G protein-coupled receptors (GPCR) and activate smooth muscle by a highly reproducible and relatively "simple" mechanism involving activation of voltage-operated Ca²⁺ channels that leads to increases in cytosolic free Ca²⁺ ([Ca²⁺]_i), Ca²⁺-calmodulin-dependent myosin light chain (MLC) kinase activation, MLC phosphorylation and contraction. This KCl-induced stimulus-response coupling mechanism is a standard tool-set used in comparative studies to explore more complex mechanisms generated by activation of GPCRs. One area where this approach has been especially productive is in studies designed to understand Ca²⁺ sensitization, the relationship between [Ca²⁺]_i and force produced by GPCR agonists. Studies done in the late 1980s demonstrated that a unique relationship between stimulus-induced [Ca²⁺]_i and force does not exist: for a given increase in [Ca²⁺]_i, GPCR activation can produce greater force than KCl, and relaxant agents can produce the opposite effect to cause Ca²⁺ desensitization. Such changes in Ca²⁺ sensitivity are now known to involve multiple cell signaling strategies, including translocation of proteins from cytosol to plasma membrane, and activation of enzymes, including RhoA kinase and protein kinase C. However, recent studies show that KCl can also cause Ca²⁺ sensitization involving translocation and activation of RhoA kinase. Rather than complicating the Ca²⁺ sensitivity story, this surprising finding is already providing novel insights into mechanisms regulating Ca²⁺ sensitivity of smooth muscle contraction. KCl as a "simple" stimulus promises to remain a standard tool for smooth muscle cell physiologists, whose focus is to understand mechanisms regulating Ca²⁺ sensitivity. K⁺ depolarization; cell signaling; signal transduction; contraction     

Mitochondrial Free [Ca] Increases during ATP/ADP Antiport and ADP Phosphorylation: Exploration of Mechanisms

ADP influx and ADP phosphorylation may alter mitochondrial free [Ca²⁺] ([Ca²⁺]_m) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca²⁺]_m is affected by H₂PO₄⁻ (P_i), Mg²⁺, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca²⁺]_m, membrane potential, redox state, matrix volume, pH_m, and O₂ consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg²⁺ and P_i. Energized mitochondria showed a dose-dependent increase in [Ca²⁺]_m after adding CaCl₂ equivalent to 20, 114, and 485 nM extramatrix free [Ca²⁺] ([Ca²⁺]_e); this uptake was attenuated at higher buffer Mg²⁺. Adding ADP transiently increased [Ca²⁺]_m up to twofold. The ADP effect on increasing [Ca²⁺]_m could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg²⁺ or P_i. Oligomycin largely reduced the increase in [Ca²⁺]_m by ADP compared to control, and [Ca²⁺]_m did not return to baseline. Carboxyatractyloside prevented the ADP-induced [Ca²⁺]_m increase. Adding CaCl₂ had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM [Ca²⁺]_e. These data suggest that matrix ADP influx and subsequent phosphorylation increase [Ca²⁺]_m largely due to the interaction of matrix Ca²⁺ with ATP, ADP, P_i, and cation buffering proteins in the matrix.     

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     

Role of Rho in Ca(2+)-insensitive contraction and paxillin tyrosine phosphorylation in smooth muscle

We investigatedwhether Rho activation is required for Ca²⁺-insensitivepaxillin phosphorylation, myosin light chain (MLC) phosphorylation, andcontraction in tracheal muscle. Tyrosine-phosphorylated proteins havebeen implicated in the Ca²⁺-insensitive contractileactivation of smooth muscle tissues. The contractile activation oftracheal smooth muscle increases tyrosine phosphorylation of thecytoskeletal proteins paxillin and focal adhesion kinase. Paxillin isimplicated in integrin-mediated signal transduction pathways thatregulate cytoskeletal organization and cell motility. In fibroblastsand other nonmuscle cells, paxillin tyrosine phosphorylation depends onthe activation of Rho and is inhibited by cytochalasin, an inhibitor ofactin polymerization. In permeabilized muscle strips, we found that AChinduced Ca²⁺-insensitive contraction, MLC phosphorylation,and paxillin tyrosine phosphorylation. Ca²⁺-insensitivecontraction and MLC phosphorylation induced by ACh were inhibited by C3transferase, an inhibitor of Rho activation; however, C3 transferasedid not inhibit paxillin tyrosine phosphorylation. Ca²⁺-insensitive paxillin tyrosine phosphorylation was alsonot inhibited by the Rho kinase inhibitor Y-27632, by cytochalasin D,or by the inhibition of MLC phosphorylation. We conclude that, intracheal smooth muscle, Rho mediates Ca²⁺-insensitivecontraction and MLC phosphorylation but that Rho is not required forCa²⁺-insensitive paxillin tyrosine phosphorylation.Paxillin phosphorylation also does not require actomyosin activation,nor is it inhibited by the actin filament capping agent cytochalasin D.

     

Suppression of Rad leads to arrhythmogenesis via PKA-mediated phosphorylation of ryanodine receptor activity in the heart

Ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of the L-type Ca²⁺ channel (LTCC). The aim was to demonstrate the role of Rad in intracellular calcium homeostasis by cardiac-Specific dominant-negative suppression of Rad. Transgenic (TG) mice overexpressing dominant-negative mutant Rad (S105N Rad TG) were generated. To measure intracellular Ca²⁺ concentration ([Ca²⁺]_i), we recorded [Ca²⁺]_i transients and Ca²⁺ sparks from isolated cardiomyocytes using confocal microscopy. The mean [Ca²⁺]_i transient amplitude was significantly increased in S105N Rad TG cardiomyocytes, compared with control littermate mouse cells. The frequency of Ca²⁺ sparks was also significantly higher in TG cells than in control cells, although there were no significant differences in amplitude. The sarcoplasmic reticulum Ca²⁺ content was not altered in the S105N Rad TG cells, as assessed by measuring caffeine-induced [Ca²⁺]_i transient. In contrast, phosphorylation of Ser²⁸⁰⁹ on the cardiac ryanodine receptor (RyR2) was significantly enhanced in TG mouse hearts compared with controls. Additionally, the Rad-mediated RyR2 phosphorylation was regulated via a direct interaction of Rad with protein kinase A (PKA).     

Glucagon receptor mediates calcium signaling by coupling to Gαq/11 and Gαi/o in HEK293 cells

Glucagon induces intracellular Ca²⁺ ([Ca²⁺]_i) elevation by stimulating glucagon receptor (GCGR). Such [Ca²⁺]_i signaling plays important physiological roles, including glycogenolysis and glycolysis in liver cells and the survival of β-cells. Previous studies indicated that phospholipase C (PLC) might be involved in glucagon-mediated [Ca²⁺]_i response. Other studies also debated whether cAMP accumulation mediated by GCGR/Gα_s coupling contributes to [Ca²⁺]_i elevation. But the exact mechanisms remain uncertain. In the present study, we found that glucagon induces [Ca²⁺]_i elevation in HEK293 cells expressing GCGR. Removing extracellular Ca²⁺ did not affect glucagon-stimulated [Ca²⁺]_i response. But depleting the intracellular Ca²⁺ store by thapsigargin completely inhibited glucagon-induced [Ca²⁺]_i response. Experiments with forskolin and adenylyl cyclase inhibtor revealed that cAMP is not the cause of [Ca²⁺]_i response. Further studies with Gα_q/11 RNAi and pertussis toxin (PTX) indicated that both Gα_q/11 and Gα_i/o are involved. Combination of Gα_q/11 RNAi and Gα_i/o inhibition almost completely abolished glucagon-induced [Ca²⁺]_i signaling.     

Calmodulin and the regulation of smooth muscle contraction

Calmodulin, the ubiquitous and multifunctional Ca²⁺-binding protein, mediates many of the regulatory effects of Ca²⁺, including the contractile state of smooth muscle. The principal function of calmodulin in smooth muscle is to activate crossbridge cycling and the development of force in response to a [Ca²⁺]_i transientvia the activation of myosin light-chain kinase and phosphorylation of myosin. A distinct calmodulin-dependent kinase, Ca²⁺/calmodulin-dependent protein kinase II, has been implicated in modulation of smooth-muscle contraction. This kinase phosphorylates myosin light-chain kinase, resulting in an increase in the calmodulin concentration required for half-maximal activation of myosin light-chain kinase, and may account for desensitization of the contractile response to Ca²⁺. In addition, the thin filament-associated proteins, caldesmon and calponin, which inhibit the actin-activated MgATPase activity of smooth-muscle myosin (the cross-bridge cycling rate), appear to be regulated by calmodulin, either by the direct binding of Ca²⁺/calmodulin or indirectly by phosphorylation catalysed by Ca²⁺/calmodulin-dependent protein kinase II. Another level at which calmodulin can regulate smooth-muscle contraction involves proteins which control the movement of Ca²⁺ across the sarcolemmal and sarcoplasmic reticulum membranes and which are regulated by Ca²⁺/calmodulin, e.g. the sarcolemmal Ca²⁺ pump and the ryanodine receptor/Ca²⁺ release channel, and other proteins which indirectly regulate [Ca²⁺]_i via cyclic nucleotide synthesis and breakdown, e.g. NO synthase and cyclic nucleotide phosphodiesterase. The interplay of such regulatory mechanisms provides the flexibility and adaptability required for the normal functioning of smooth-muscle tissues.     

Heat shock protein 20-mediated force suppression in forskolin-relaxed swine carotid artery

Increases in cyclic nucleotide levels induce smooth muscle relaxation by deactivation [reductions in myosin regulatory light chain (MRLC) phosphorylation (e.g., by reduced [Ca²⁺])] or force suppression (reduction in force without reduction in MRLC phosphorylation). Ser¹⁶-heat shock protein 20 (HSP20) phosphorylation is the proposed mediator of force suppression. We evaluated three potential hypotheses whereby Ser¹⁶-HSP20 phosphorylation could regulate smooth muscle force: 1) a threshold level of HSP20 phosphorylation could inactivate a thin filament as a whole, 2) phosphorylation of a single HSP20 could fully inactivate a small region of a thin filament, or 3) HSP20 phosphorylation could weakly inhibit myosin binding at either the thin- or thick-filament level. We tested these hypotheses by analyzing the dependence of force on Ser¹⁶-HSP20 phosphorylation in swine carotid media. First, we determined that swine HSP20 has a second phosphorylation site at Ser¹⁵⁷. Ser¹⁵⁷-HSP20 phosphorylation values were high and did not change during contractile activation or forskolin-induced relaxation. Forskolin significantly increased Ser¹⁶-HSP20 phosphorylation. The relationship between Ser¹⁶-HSP20 phosphorylation and force remained linear and was shifted downward in partially activated muscles relaxed with forskolin. Neither forskolin nor nitroglycerin induced actin depolymerization as detected using the F/G-actin ratio method in smooth muscle homogenates. These results suggest that force suppression does not occur in accordance with the first hypothesis (inactivation of a thin filament as a whole). Our data are more consistent with the second and third hypotheses that force suppression is mediated by full or partial inhibition of local myosin binding at the thin- or thick-filament level. cAMP; cGMP; nitric oxide; vascular smooth muscle     

Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: Is resting calcium responsible?

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca²⁺-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca²⁺ levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting [Ca²⁺]_i. Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca²⁺ transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca²⁺]_i, in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation.     

Forskolin and Phorbol Myristate Acetate Inhibit Intracellular Ca2+ Mobilization Induced by Amitriptyline and Bradykinin in Rat Frontocortical Neurons

Abstract— Regulations of the increase in intracellular Ca²⁺concentration ([Ca²⁺]_i) and inositol 1, 4, 5-trisphosphate (IP₃) production by increasing intracellular cyclic AMP (cAMP) levels or activating protein kinase C (PKC) were studied in rat frontocortical cultured neurons. Amitriptyline (AMI; 1 mM), a trìcyclic antidepressant, and bradykinin (BK; 1 μM) stimulated IP₃ production and caused transient [Ca²⁺]_i increases. Pretreatment with forskolin (100mkUM, 15 min) decreased the AMI-and BK-induced [Ca²⁺]_i increases by 33 and 48%, respectively. However, this treatment had no effect on the AMI-and BK-induced IP₃ productions. Dibutyryl-cAMP (2 mM, 15 min) also decreased the AMI-and BK-induced [Ca²⁺]ⁱ increases by 23 and 47%, respectively. H-8 (30 μM), an inhibitor of protein kinase A (PKA), attenuated the ability of forskolin to inhibit the AMI-and BK-induced [Ca²⁺]_i increases, suggesting that the activation of cAMP/PKA was involved in these inhibitory effects of forskolin. On the other hand, forskolin treatment had no effect on 20 mM caffeine-, 10 μM glutamate-, or 50 mM K⁺-induced [Ca²⁺]_i increases. Pretreatment with phorbol 12-myristate 13-acetate (PMA; 100 nM, 90 min) decreased both the AMI-induced [Ca²⁺]_i increases and the IP₃ production by 31 and 25%, respectively. H-7 (200 μM), an inhibitor of PKC, inhibited the ability of PMA to attenuate the [Ca²⁺]_i increases. PMA also inhibited the BK-induced IP₃ production and the [Ca²⁺]_i increases. Taken together, these results suggest that activation of cAMP/ PKA may inhibit the IP₃-mediated Ca²⁺ release from internal stores; on the other hand, activation of PKC may inhibit the phosphatidylinositol 4,5-bisphosphate breakdown and consequently reduce the [Ca²⁺]_i increases or inhibit independently both responses. PKA and PKC may differently regulate the phosphatidylinositol-Ca²⁺ signaling in rat frontocortical cultured neurons.