The frequency of calcium oscillations induced by 5-HT, ACH, and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles

Increased resistance of airways or blood vessels within the lung is associated with asthma or pulmonary hypertension and results from contraction of smooth muscle cells (SMCs). To study the mechanisms regulating these contractions, we developed a mouse lung slice preparation containing bronchioles and arterioles and used phase-contrast and confocal microscopy to correlate the contractile responses with changes in [Ca(2+)](i) of the SMCs. The airways are the focus of this study. The agonists, 5-hydroxytrypamine (5-HT) and acetylcholine (ACH) induced a concentration-dependent contraction of the airways. High concentrations of KCl induced twitching of the airway SMCs but had little effect on airway size. 5-HT and ACH induced asynchronous oscillations in [Ca(2+)](i) that propagated as Ca(2+) waves within the airway SMCs. The frequency of the Ca(2+) oscillations was dependent on the agonist concentration and correlated with the extent of sustained airway contraction. In the absence of extracellular Ca(2+) or in the presence of Ni(2+), the frequency of the Ca(2+) oscillations declined and the airway relaxed. By contrast, KCl induced low frequency Ca(2+) oscillations that were associated with SMC twitching. Each KCl-induced Ca(2+) oscillation consisted of a large Ca(2+) wave that was preceded by multiple localized Ca(2+) transients. KCl-induced responses were resistant to neurotransmitter blockers but were abolished by Ni(2+) or nifedipine and the absence of extracellular Ca(2+). Caffeine abolished the contractile effects of 5-HT, ACH, and KCl. These results indicate that (a) 5-HT and ACH induce airway SMC contraction by initiating Ca(2+) oscillations, (b) KCl induces Ca(2+) transients and twitching by overloading and releasing Ca(2+) from intracellular stores, (c) a sustained, Ni(2+)-sensitive, influx of Ca(2+) mediates the refilling of stores to maintain Ca(2+) oscillations and, in turn, SMC contraction, and (d) the magnitude of sustained airway SMC contraction is regulated by the frequency of Ca(2+) oscillations.     

Modulation of the Ca2+ sensitivity of airway smooth muscle cells in murine lung slices

To investigate the phenomenon of Ca(2+) sensitization, we developed a new intact airway and arteriole smooth muscle cell (SMC) "model" by treating murine lung slices with ryanodine-receptor antagonist, ryanodine (50 microM), and caffeine (20 mM). A sustained elevation in intracellular Ca(2+) concentration ([Ca(2+)](i)) was induced in both SMC types by the ryanodine-caffeine treatment due to the depletion of internal Ca(2+) stores and the stimulation of a persistent influx of Ca(2+). Arterioles responded to this sustained increase in [Ca(2+)](i) with a sustained contraction. By contrast, airways responded to sustained high [Ca(2+)](i) with a transient contraction followed by relaxation. Subsequent exposure to methacholine (MCh) induced a sustained concentration-dependent contraction of the airway without a change in the [Ca(2+)](i). During sustained MCh-induced contraction, Y-27632 (a Rho-kinase inhibitor) and GF-109203X (a protein kinase C inhibitor) induced a concentration-dependent relaxation without changing the [Ca(2+)](i). The cAMP-elevating agents, forskolin (an adenylyl cyclase activator), IBMX (a phosphodiesterase inhibitor), and caffeine (also acting as a phosphodiesterase inhibitor), exerted similar relaxing effects. These results indicate that 1) ryanodine-caffeine treatment is a valuable tool for investigating the contractile mechanisms of SMCs while avoiding nonspecific effects due to cell permeabilization, 2) in the absence of agonist, sustained high [Ca(2+)](i) has a differential time-dependent effect on the Ca(2+) sensitivity of airway and arteriole SMCs, 3) MCh facilitates the contraction of airway SMCs by inducing Ca(2+) sensitization via the activation of Rho-kinase and protein kinase C, and 4) cAMP-elevating agents contribute to the relaxation of airway SMCs through Ca(2+) desensitization.     

The contraction of smooth muscle cells of intrapulmonary arterioles is determined by the frequency of Ca2+ oscillations induced by 5-HT and KCl

Increased resistance of the small blood vessels within the lungs is associated with pulmonary hypertension and results from a decrease in size induced by the contraction of their smooth muscle cells (SMCs). To study the mechanisms that regulate the contraction of intrapulmonary arteriole SMCs, the contractile and Ca(2+) responses of the arteriole SMCs to 5-hydroxytrypamine (5-HT) and KCl were observed with phase-contrast and scanning confocal microscopy in thin lung slices cut from mouse lungs stiffened with agarose and gelatin. 5-HT induced a concentration-dependent contraction of the arterioles. Increasing concentrations of extracellular KCl induced transient contractions in the SMCs and a reduction in the arteriole luminal size. 5-HT induced oscillations in [Ca(2+)](i) within the SMCs, and the frequency of these Ca(2+) oscillations was dependent on the agonist concentration and correlated with the extent of sustained arteriole contraction. By contrast, KCl induced Ca(2+) oscillations that occurred with low frequencies and were preceded by small, localized transient Ca(2+) events. The 5-HT-induced Ca(2+) oscillations and contractions occurred in the absence of extracellular Ca(2+) and were resistant to Ni(2+) and nifedipine but were abolished by caffeine. KCl-induced Ca(2+) oscillations and contractions were abolished by the absence of extracellular Ca(2+) and the presence of Ni(2+), nifedipine, and caffeine. Arteriole contraction was induced or abolished by a 5-HT(2)-specific agonist or antagonist, respectively. These results indicate that 5-HT, acting via 5-HT(2) receptors, induces arteriole contraction by initiating Ca(2+) oscillations and that KCl induces contraction via Ca(2+) transients resulting from the overfilling of internal Ca(2+) stores. We hypothesize that the magnitude of the sustained intrapulmonary SMC contraction is determined by the frequency of Ca(2+) oscillations and also by the relaxation rate of the SMC.     

Endothelin-induced contraction of bronchiole and pulmonary arteriole smooth muscle cells is regulated by intracellular Ca2+ oscillations and Ca2+ sensitization

Endothelin-1 (ET) induces increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) sensitization, and contraction of both bronchiole and pulmonary arteriole smooth muscle cells (SMCs) and may play an important role in the pathophysiology of asthma and pulmonary hypertension. However, because it remains unclear how changes in [Ca(2+)](i) and the Ca(2+) sensitivity regulate SMC contraction, we have studied mouse lung slices with phase-contrast and confocal microscopy to correlate the ET-induced contraction with the changes in [Ca(2+)](i) and Ca(2+) sensitivity of bronchiole and arteriole SMCs. In comparison with acetylcholine (ACh) or serotonin (5-HT), ET induced a stronger and long-lasting contraction of both bronchioles and arterioles. This ET-induced contraction was associated with prominent asynchronous Ca(2+) oscillations that were propagated as Ca(2+) waves along the SMCs. These Ca(2+) oscillations were mediated by cyclic intracellular Ca(2+) release and required external Ca(2+) for their maintenance. Importantly, as the frequency of the Ca(2+) oscillations increased, the extent of contraction increased. ET-induced contraction was also associated with an increase in Ca(2+) sensitivity. In "model" slices in which the [Ca(2+)](i) was constantly maintained at an elevated level by pretreatment of slices with caffeine and ryanodine, the addition of ET increased bronchiole and arteriole contraction. These results indicate that ET-induced contraction of bronchiole and arteriole SMCs is regulated by the frequency of Ca(2+) oscillations and by increasing the sensitivity of the contractile machinery to Ca(2+).     

Airway contractility and smooth muscle Ca(2+) signaling in lung slices from different mouse strains.

To investigate the hypothesis that altered Ca2+ signaling in airway smooth muscle cells (SMCs) is responsible for airway hyperreactivity, we compared, with the use of confocal and phase-contrast microscopy, the airway contractility and Ca2+ changes in SMCs induced by acetylcholine (ACh) in lung slices from different mouse strains (A/J, Balb/C, and C3H/ HeJ). The airways from each mouse strain displayed a concentration-dependent contraction to ACh. The contractile response of the airways of the C3H/HeJ mice was found, in contrast to earlier studies, to be much greater and faster than that of A/J and Balb/C mice. This difference in airway reactivity can be, in part, attributable to halothane, a volatile anesthetic that was previously used during in vivo measurements of airway reactivity but found here to significantly alter the ACh contractile response of airways in lung slices. The ACh-induced Ca2+ response of the airway SMCs in all of the various mouse strains was also concentration dependent. The magnitude of the initial Ca2+ increase and the frequency of the subsequent Ca2+ oscillations induced by ACh increased with ACh concentration. However, no differences in the Ca2+ responses to ACh could be distinguished between the mouse strains. These results suggest that the mechanism responsible for airway hyperreactivity in different mouse strains resides with the Ca2+ sensitivity of the contractile apparatus of the SMCs rather than with the Ca2+ signaling itself.     

ATP stimulates Ca2+ oscillations and contraction in airway smooth muscle cells of mouse lung slices

In airway smooth muscle cells (SMCs) from mouse lung slices, > or =10 microM ATP induced Ca2+ oscillations that were accompanied by airway contraction. After approximately 1 min, the Ca2+ oscillations subsided and the airway relaxed. By contrast, > or =0.5 microM adenosine 5'-O-(3-thiotriphosphate) (nonhydrolyzable) induced Ca2+ oscillations in the SMCs and an associated airway contraction that persisted for >2 min. Adenosine 5'-O-(3-thiotriphosphate)-induced Ca2+ oscillations occurred in the absence of external Ca2+ but were abolished by the phospholipase C inhibitor U-73122 and the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin. Adenosine, AMP, and alpha,beta-methylene ATP had no effect on airway caliber, and the magnitude of the contractile response induced by a variety of nucleotides could be ranked in the following order: ATP = UTP > ADP. These results suggest that the SMC response to ATP is impaired by ATP hydrolysis and mediated via P2Y(2) or P2Y(4) receptors, activating phospholipase C to release Ca2+ via the inositol 1,4,5-trisphosphate receptor. We conclude that ATP can serve as a spasmogen of airway SMCs and that Ca2+ oscillations in SMCs are required to sustain airway contraction.     

Drosophila Pkd2 is haploid-insufficient for mediating optimal smooth muscle contractility

Humans heterozygous for PKD1 or PKD2 develop autosomal dominant polycystic kidney disease, a common genetic disorder characterized by renal cyst formation and extrarenal complications such as hypertension and vascular aneurysms. Cyst formation requires the somatic inactivation of the wild type allele. However, it is unknown whether this recessive mechanism applies to life-threatening vascular aneurysms, which could involve weakening of the endothelial lining or surrounding vascular smooth muscle cells (SMCs). Drosophila Pkd2 at 33E3 (Pkd2) encodes a PKD2 family of Ca(2+)-activated Ca(2+)-permeable cation channels. We show here that loss-of-function Pkd2 mutations severely reduced the contractility of the visceral SMCs, which was restored by expressing wild type Pkd2 cDNA via a muscle-specific Gal4 driver. The effect of Pkd2 mutations alone on the skeletal muscle was minimal but was exacerbated by ryanodine-induced perturbation of intracellular Ca(2+) stores. Consistent with this, Pkd2 interacted strongly with a ryanodine receptor mutation, causing a synergistic reduction of larval body wall contraction rate that is normally regulated through Ca(2+) oscillation during excitation-contraction coupling in the skeletal muscle. These results suggest that PKD2 cooperates with the ryanodine receptor to promote optimal muscle contractility through intracellular Ca(2+) homeostasis. Further genetic analysis indicated that Pkd2 is strongly haploinsufficient for normal SMC contractility. Since Ca(2+) homeostasis is a conserved mechanism for optimal muscle performance, our results raise the possibility that inactivation of just one PKD2 copy is sufficient to compromise vascular SMC contractility and function in PKD2 heterozygous patients, thus explaining their increased susceptibility to hypertension and vascular aneurysms.     

Role of intracellular Ca(2+) release in histamine-induced depolarization in rabbit middle cerebral artery

The role of Ca(2+) mobilization from intracellular stores and Ca(2+)-activated Cl(-) channels in caffeine- and histamine-induced depolarization and contraction of the rabbit middle cerebral artery has been studied by recording membrane potential and isometric force. Caffeine induced a transient contraction and a transient followed by sustained depolarization. The transient depolarization was abolished by ryanodine, DIDS, and niflumic acid, suggesting involvement of Ca(2+)-activated Cl(-) channels. Histamine-evoked transient contraction in Ca(2+)-free solution was abolished by ryanodine or by caffeine-induced depletion of Ca(2+) stores. Ryanodine slowed the development of depolarization induced by histamine in Ca(2+)-containing solution but did not affect its magnitude. In arteries treated with 1 mM Co(2+), histamine elicited a transient depolarization and contraction, which was abolished by ryanodine. DIDS and niflumic acid reduced histamine-evoked depolarization and contraction. Histamine caused a sustained depolarization and contraction in low-Cl(-) solution. These results suggest that Ca(2+) mobilization from ryanodine-sensitive stores is involved in histamine-induced initial, but not sustained, depolarization and contraction. Ca(2+)-activated Cl(-) channels contribute mainly to histamine-induced initial depolarization and less importantly to sustained depolarization, which is most likely dependent on activation of nonselective cation channels.     

Non-Selective Cation Channels Mediate Chloroquine-Induced Relaxation in Precontracted Mouse Airway Smooth Muscle

Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs) or by inactivating voltage-dependent L-type Ca²⁺ channels (VDLCCs). In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs) precontracted with acetylcholine (ACH). In the presence of nifedipine (10 µM), ACH induced cytosolic Ca²⁺ elevation and cell shortening in single airway smooth muscle cells (ASMCs), and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca²⁺-free conditions, and, following a restoration of Ca²⁺, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs) were blocked by chloroquine. Pyrazole 3 (Pyr3), an inhibitor of transient receptor potential C3 (TRPC3) channels, partially inhibited ACH-induced contraction, intracellular Ca²⁺ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.     

Nitric oxide induces airway smooth muscle cell relaxation by decreasing the frequency of agonist-induced Ca2+ oscillations

Nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, but the underlying mechanism is not well understood. Consequently, we investigated the effects of NO on airway SMC contraction, Ca²⁺ signaling, and Ca²⁺ sensitivity in mouse lung slices with phase-contrast and confocal microscopy. Airways that were contracted in response to the agonist 5-hydroxytryptamine (5-HT) transiently relaxed in response to the NO donor, NOC-5. This NO-induced relaxation was enhanced by zaprinast or vardenafil, two selective inhibitors of cGMP-specific phosphodiesterase-5, but blocked by ODQ, an inhibitor of soluble guanylyl cyclase, and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Simultaneous measurements of airway caliber and SMC [Ca²⁺]_i revealed that airway contraction induced by 5-HT correlated with the occurrence of Ca²⁺ oscillations in the airway SMCs. Airway relaxation induced by NOC-5 was accompanied by a decrease in the frequency of these Ca²⁺ oscillations. The cGMP analogues and selective PKG activators 8Br-cGMP and 8pCPT-cGMP also induced airway relaxation and decreased the frequency of the Ca²⁺ oscillations. NOC-5 inhibited the increase of [Ca²⁺]_i and contraction induced by the photolytic release of inositol 1,4,5-trisphosphate (IP₃) in airway SMCs. The effect of NO on the Ca²⁺ sensitivity of the airway SMCs was examined in lung slices permeabilized to Ca²⁺ by treatment with caffeine and ryanodine. Neither NOC-5 nor 8pCPT-cGMP induced relaxation in agonist-contracted Ca²⁺-permeabilized airways. Consequently, we conclude that NO, acting via the cGMP–PKG pathway, induced airway SMC relaxation by predominately inhibiting the release of Ca²⁺ via the IP₃ receptor to decrease the frequency of agonist-induced Ca²⁺ oscillations.     

Thrombin-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells

Thrombin is a procoagulant inflammatory agonist that can disrupt the endothelium-lumen barrier in the lung by causing contraction of endothelial cells and promote pulmonary cell proliferation. Both contraction and proliferation require increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this study, we compared the effect of thrombin on Ca(2+) signaling in human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells. Thrombin increased the [Ca(2+)](cyt) in both cell types; however, the transient response was significantly higher and recovered quicker in the PASMC, suggesting different mechanisms may contribute to thrombin-mediated increases in [Ca(2+)](cyt) in these cell types. Depletion of intracellular stores with cyclopiazonic acid (CPA) in the absence of extracellular Ca(2+) induced calcium transients representative of those observed in response to thrombin in both cell types. Interestingly, CPA pretreatment significantly attenuated thrombin-induced Ca(2+) release in PASMC; this attenuation was not apparent in PAEC, indicating that a PAEC-specific mechanism was targeted by thrombin. Treatment with a combination of CPA, caffeine, and ryanodine also failed to abolish the thrombin-induced Ca(2+) transient in PAEC. Notably, thrombin-induced receptor-mediated calcium influx was still observed in PASMC after CPA pretreatment in the presence of extracellular Ca(2+). Ca(2+) oscillations were triggered by thrombin in PASMC resulting from a balance of extracellular Ca(2+) influx and Ca(2+) reuptake by the sarcoplasmic reticulum. The data show that thrombin induces increases in intracellular calcium in PASMC and PAEC with a distinct CPA-, caffeine-, and ryanodine-insensitive release existing only in PAEC. Furthermore, a dynamic balance between Ca(2+) influx, intracellular Ca(2+) release, and reuptake underlie the Ca(2+) transients evoked by thrombin in some PASMC. Understanding of such mechanisms will provide an important insight into thrombin-mediated vascular injury during hypertension.     

Different roles of ryanodine receptors and inositol (1,4,5)-trisphosphate receptors in adrenergically stimulated contractions of small arteries

The functions of ryanodine receptors (RyRs) and inositol (1,4,5)-trisphosphate receptors [Ins(1,4,5)P(3)Rs] in adrenergically activated contractions of pressurized rat mesenteric small arteries were investigated. Caffeine (20 mM) but not phenylephrine (PE; 10 microM) facilitated the depletion of smooth muscle sarcoplasmic reticulum (SR) Ca(2+) stores by ryanodine (40 microM). In ryanodine-treated SR-depleted arteries, 1) Ca(2+) sparks were absent, 2) low concentrations of PE failed to elicit either vasoconstriction or normal asynchronous propagating Ca(2+) waves, and 3) high [PE] induced abnormally slow oscillatory contractions (vasomotion) and synchronous Ca(2+) oscillations. In ryanodine-treated SR-depleted arteries denuded of endothelium, high [PE] induced steady contraction and steady elevation of intracellular [Ca(2+)]. In contrast, 2-aminoethyl diphenylborate (2-APB), a putative blocker of Ins(1,4,5)P(3)Rs, produced opposite effects to ryanodine: 1) Ca(2+) sparks were present; 2) Ca(2+) waves were absent; 3) caffeine-releasable Ca(2+) stores were intact; and 4) PE, even at high concentrations on endothelial-denuded arteries, failed to elicit contraction, asynchronous Ca(2+) waves, or synchronous Ca(2+) oscillations or maintained elevated [Ca(2+)]. We conclude that 1) Ins(1,4,5)P(3)Rs are essential for adrenergically induced asynchronous Ca(2+) waves and the associated steady vasoconstriction, 2) RyRs are not appreciably opened during adrenergic activation (because PE did not facilitate the development of the effects of ryanodine), and 3) Ins(1,4,5)P(3)Rs are not essential for Ca(2+) sparks. This provides an explanation of the fact that adrenergic stimulation decreases the frequency of Ca(2+) sparks (previously reported) while simultaneously increasing the frequency of asynchronous propagating Ca(2+) waves; different SR Ca(2+)-release channels are involved.     

Src modulates serotonin-induced calcium signaling by regulating phosphatidylinositol 4,5-bisphosphate

We tested the hypothesis that, in airway smooth muscle cells, stimulation of G-protein-coupled receptors by contractile agonists activates Src kinase and that this kinase modulates cell contractility and Ca(2+) signaling by affecting the levels of the phospholipase C substrate phosphatidylinositol 4,5-bisphosphate (PIP(2)). Stimulation of cultured rat tracheal smooth muscle cells with serotonin (5-HT) induced an increase in Src activity, Ca(2+) mobilization, and contraction (decrease in cell area). 5-HT-evoked cell contraction was reduced by a specific inhibitor of Src family kinases, 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1). Peak Ca(2+) responses to 5-HT were attenuated by PP1 and an anti-Src-blocking antibody and augmented by expression of constitutively activated Y529F Src. Sustained phases of Ca(2+) responses to 5-HT and Ca(2+) influx resulting from emptying of Ca(2+) stores in the endoplasmic reticulum by thapsigargin were also decreased after PP1 treatment. PP1 significantly reduced the turnover of inositol phosphates produced on 5-HT stimulation and the amount of PIP(2) in the Triton X-100-insoluble lipid fraction. Overall, these data demonstrate that, in rat tracheal smooth muscle cells, Src kinase modulates 5-HT-evoked cell contractility and Ca(2+) signaling by regulating PIP(2) levels and Ca(2+) influx.     

Functional role of ryanodine-sensitive Ca2+ stores in acidic pH-induced contraction in Wistar Kyoto rat aorta

Acidic pH induced a contraction in the isolated aorta from Wistar Kyoto rat. The magnitude of contraction was dependent upon the degree of extracellular acidification. The maximum level of contraction observed at pH 6.5 was 84.6 +/- 3.4% of the 64.8 mM KCl-induced contraction. To investigate the role of extracellular as well as intracellular Ca(2+) in acidic pH-induced contraction (APIC), we changed the extracellular pH in the presence of EGTA. Sustained contraction induced by acidic pH in the presence of extracellular Ca(2+) was completely abolished in the presence of EGTA, while a transient but significant contraction was still observed. Ryanodine, a selective ryanodine receptor blocker and cyclopiazonic acid (CPA), an inhibitor of sarco-/endoplasmic reticulum Ca(2+) ATPase, abolished the transient contraction, when pH was decreased in Ca(2+)-free solution. On the other hand, neither xestospongin C, a selective inositol-1,4,5-trisphosphate receptor antagonist nor U-73122, a phospholipase C inhibitor showed this effect. These results suggest the involvement of Ca(2+) release from ryanodine-/CPA-sensitive store of sarcoplasmic reticulum (SR). In normal Ca(2+)-containing solution, ryanodine and CPA did not alter the maximum level of APIC. However, they significantly decreased the rate of rise of APIC. U-73122, suppressed the maximum contraction induced by acidic pH without affecting the rate of rise of APIC, while xestospongin C and U-73343, an inactive analogue of U-73122, had no effect on both parameters of APIC. From these results, it is concluded that acidic pH induces Ca(2+) release from the ryanodine-/CPA-sensitive store of SR and that release provides supportive effect on initiating rapid transient contraction, but not on the sustained contraction, which is entirely due to Ca(2+) influx.     

Thyroid hormone-induced overexpression of functional ryanodine receptors in the rabbit heart

Modifications in the Ca(2+)-uptake and -release functions of the sarcoplasmic reticulum (SR) may be a major component of the mechanisms underlying thyroid state-dependent alterations in heart rate, myocardial contractility, and metabolism. We investigated the influence of hyperthyroid state on the expression and functional properties of the ryanodine receptor (RyR), a major protein in the junctional SR (JSR), which mediates Ca(2+) release to trigger muscle contraction. Experiments were performed using homogenates and JSR vesicles derived from ventricular myocardium of euthyroid and hyperthyroid rabbits. Hyperthyroidism, with attendant cardiac hypertrophy, was induced by the injection of L-thyroxine (200 microg/kg body wt) daily for 7 days. Western blotting analysis using cardiac RyR-specific antibody revealed a significant increase (>50%) in the relative amount of RyR in the hyperthyroid compared with euthyroid rabbits. Ca(2+)-dependent, high-affinity [(3)H]ryanodine binding was also significantly greater ( approximately 40%) in JSR from hyperthyroid rabbits. The Ca(2+ )sensitivity of [(3)H]ryanodine binding and the dissociation constant for [(3)H]ryanodine did not differ significantly between euthyroid and hyperthyroid hearts. Measurement of Ca(2+)-release rates from passively Ca(2+)-preloaded JSR vesicles and assessment of the effect of RyR-Ca(2+)-release channel (CRC) blockade on active Ca(2+)-uptake rates revealed significantly enhanced (>2-fold) CRC activity in the hyperthyroid, compared with euthyroid, JSR. These results demonstrate overexpression of functional RyR in thyroid hormone-induced cardiac hypertrophy. Relative abundance of RyR may be responsible, in part, for the changes in SR Ca(2+) release, cytosolic Ca(2+) transient, and cardiac systolic function associated with thyroid hormone-induced cardiac hypertrophy.     

Functional coupling of Ca(2+) channels to ryanodine receptors at presynaptic terminals. Amplification of exocytosis and plasticity

Ca(2+)-induced Ca(2+) release (CICR) enhances a variety of cellular Ca(2+) signaling and functions. How CICR affects impulse-evoked transmitter release is unknown. At frog motor nerve terminals, repetitive Ca(2+) entries slowly prime and subsequently activate the mechanism of CICR via ryanodine receptors and asynchronous exocytosis of transmitters. Further Ca(2+) entry inactivates the CICR mechanism and the absence of Ca(2+) entry for >1 min results in its slow depriming. We now report here that the activation of this unique CICR markedly enhances impulse-evoked exocytosis of transmitter. The conditioning nerve stimulation (10-20 Hz, 2-10 min) that primes the CICR mechanism produced the marked enhancement of the amplitude and quantal content of end-plate potentials (EPPs) that decayed double exponentially with time constants of 1.85 and 10 min. The enhancement was blocked by inhibitors of ryanodine receptors and was accompanied by a slight prolongation of the peak times of EPP and the end-plate currents estimated from deconvolution of EPP. The conditioning nerve stimulation also enhanced single impulse- and tetanus-induced rises in intracellular Ca(2+) in the terminals with little change in time course. There was no change in the rate of growth of the amplitudes of EPPs in a short train after the conditioning stimulation. On the other hand, the augmentation and potentiation of EPP were enhanced, and then decreased in parallel with changes in intraterminal Ca(2+) during repetition of tetani. The results suggest that ryanodine receptors exist close to voltage-gated Ca(2+) channels in the presynaptic terminals and amplify the impulse-evoked exocytosis and its plasticity via CICR after Ca(2+)-dependent priming.     

Ginger attenuates acetylcholine-induced contraction and Ca2+ signalling in murine airway smooth muscle cells

Asthma is a chronic disease characterized by inflammation and hypersensitivity of airway smooth muscle cells (ASMCs) to different spasmogens. The past decade has seen increased use of herbal treatments for many chronic illnesses. Ginger (Zingiber officinale) is a common food plant that has been used for centuries in treating respiratory illnesses. In this study, we report the effect of its 70% aqueous methanolic crude extract (Zo.Cr) on acetylcholine (ACh)-induced airway contraction and Ca(2+) signalling in ASMCs using mouse lung slices. Airway contraction and Ca(2+) signalling, recorded via confocal microscopy, were induced with ACh, either alone or after pretreatment of slices with Zo.Cr and (or) verapamil, a standard Ca(2+) channel blocker. ACh (10 micromol/L) stimulated airway contraction, seen as decreased airway diameter, and also stimulated Ca(2+) transients (sharp rise in [Ca(2+)]i) and oscillations in ASMCs, seen as increased fluo-4-induced fluorescence intensity. When Zo.Cr (0.3-1.0 mg/mL) was given 30 min before ACh administration, the ACh-induced airway contraction and Ca(2+) signalling were significantly reduced. Similarly, verapamil (1 micromol/L) also inhibited agonist-induced airway contraction and Ca(2+) signalling, indicating a similarity in the modes of action. When Zo.Cr (0.3 mg/mL) and verapamil (1 micromol/L) were given together before ACh, the degree of inhibition was the same as that observed when each of these blockers was given alone, indicating absence of any additional inhibitory mechanism in the extract. In Ca(2+) -free solution, both Zo.Cr and verapamil, when given separately, inhibited Ca(2+) (10 mmol/L)-induced increase in fluorescence and airway contraction. This shows that ginger inhibits airway contraction and associated Ca(2+) signalling, possibly via blockade of plasma membrane Ca(2+) channels, thus reiterating the effectiveness of this age-old herb in treating respiratory illnesses.     

Functional triads consisting of ryanodine receptors, Ca(2+) channels, and Ca(2+)-activated K(+) channels in bullfrog sympathetic neurons. Plastic modulation of action potential

Fluorescent ryanodine revealed the distribution of ryanodine receptors in the submembrane cytoplasm (less than a few micrometers) of cultured bullfrog sympathetic ganglion cells. Rises in cytosolic Ca(2+) ([Ca(2+)](i)) elicited by single or repetitive action potentials (APs) propagated at a high speed (150 microm/s) in constant amplitude and rate of rise in the cytoplasm bearing ryanodine receptors, and then in the slower, waning manner in the deeper region. Ryanodine (10 microM), a ryanodine receptor blocker (and/or a half opener), or thapsigargin (1-2 microM), a Ca(2+)-pump blocker, or omega-conotoxin GVIA (omega-CgTx, 1 microM), a N-type Ca(2+) channel blocker, blocked the fast propagation, but did not affect the slower spread. Ca(2+) entry thus triggered the regenerative activation of Ca(2+)-induced Ca(2+) release (CICR) in the submembrane region, followed by buffered Ca(2+) diffusion in the deeper cytoplasm. Computer simulation assuming Ca(2+) release in the submembrane region reproduced the Ca(2+) dynamics. Ryanodine or thapsigargin decreased the rate of spike repolarization of an AP to 80%, but not in the presence of iberiotoxin (IbTx, 100 nM), a BK-type Ca(2+)-activated K(+) channel blocker, or omega-CgTx, both of which decreased the rate to 50%. The spike repolarization rate and the amplitude of a single AP-induced rise in [Ca(2+)](i) gradually decreased to a plateau during repetition of APs at 50 Hz, but reduced less in the presence of ryanodine or thapsigargin. The amplitude of each of the [Ca(2+)](i) rise correlated well with the reduction in the IbTx-sensitive component of spike repolarization. The apamin-sensitive SK-type Ca(2+)-activated K(+) current, underlying the afterhyperpolarization of APs, increased during repetitive APs, decayed faster than the accompanying rise in [Ca(2+)](i), and was suppressed by CICR blockers. Thus, ryanodine receptors form a functional triad with N-type Ca(2+) channels and BK channels, and a loose coupling with SK channels in bullfrog sympathetic neurons, plastically modulating AP.     

Internal Ca(2+) release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue.

Calcium ions, present inside all eukaryotic cells, are important second messengers in the transduction of biological signals. In mammalian cells, the release of Ca(2+) from intracellular compartments is required for signaling and involves the regulated opening of ryanodine and inositol-1,4,5-trisphosphate (IP3) receptors. However, in budding yeast, no signaling pathway has been shown to involve Ca(2+) release from internal stores, and no homologues of ryanodine or IP3 receptors exist in the genome. Here we show that hyperosmotic shock provokes a transient increase in cytosolic Ca(2+) in vivo. Vacuolar Ca(2+), which is the major intracellular Ca(2+) store in yeast, is required for this response, whereas extracellular Ca(2+) is not. We aimed to identify the channel responsible for this regulated vacuolar Ca(2+) release. Here we report that Yvc1p, a vacuolar membrane protein with homology to transient receptor potential (TRP) channels, mediates the hyperosmolarity induced Ca(2+) release. After this release, low cytosolic Ca(2+) is restored and vacuolar Ca(2+) is replenished through the activity of Vcx1p, a Ca(2+)/H(+) exchanger. These studies reveal a novel mechanism of internal Ca(2+) release and establish a new function for TRP channels.