Hydrogen peroxide contributes to motor dysfunction in ulcerative colitis

Ulcerative colitis (UC) affects colonic motor function, but the mechanism responsible for this motor dysfunction is not well understood. We have shown that neurokinin A (NKA) may be an endogenous neurotransmitter mediating contraction of human sigmoid colonic circular muscle (HSCCM). To elucidate factors responsible for UC motor dysfunction, we examined the role of hydrogen peroxide (H(2)O(2)) in the decrease of NKA-induced response of HSCCM. As previously demonstrated, NKA-induced contraction or Ca(2+) increase of normal muscle cells is mediated by release of Ca(2+) from intracellular stores, because it was not affected by incubation in Ca(2+)-free medium (CFM) containing 200 microM BAPTA. In UC, however, CFM reduced both cell contraction and NKA-induced Ca(2+) increase, suggesting reduced Ca(2+) release from intracellular stores. In normal Ca(2+) medium, NKA and KCl caused normal Ca(2+) signal in UC cells but reduced cell shortening. The decreased Ca(2+) signal and contraction in response to NKA or thapsigargin were partly recovered in the presence of H(2)O(2) scavenger catalase, suggesting involvement of H(2)O(2) in UC-induced dysmotility. H(2)O(2) levels were higher in UC than in normal HSCCM, and enzymatically isolated UC muscle cells contained much higher levels of H(2)O(2) than normal cells, which were significantly reduced by catalase. H(2)O(2) treatment of normal cells in CFM reproduced the reduction of NKA-induced Ca(2+) release observed in UC cells. In addition, H(2)O(2) caused a measurable, direct release of Ca(2+) from intracellular stores. We conclude that H(2)O(2) may contribute to reduction of NKA-induced Ca(2+) release from intracellular Ca(2+) stores in UC and contribute to the observed colonic motor dysfunction.     

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+).     

Regional differences in cholinergic regulation of potassium current in feline esophageal circular smooth muscle

Potassium channels are important contributors to membrane excitability in smooth muscles. There are regional differences in resting membrane potential and K(+)-channel density along the length of the feline circular smooth muscle esophagus. The aim of this study was to assess responses of K(+)-channel currents to cholinergic (ACh) stimulation along the length of the feline circular smooth muscle esophageal body. Perforated patch-clamp technique assessed K(+)-channel responses to ACh stimulation in isolated smooth muscle cells from the circular muscle layer of the esophageal body at 2 (distal)- and 4-cm (proximal) sites above the lower esophageal sphincter. Western immunoblots assessed ion channel and receptor expression. ACh stimulation produced a transient increase in outward current followed by inhibition of spontaneous transient outward currents. These ACh-induced currents were abolished by blockers of large-conductance Ca(2+)-dependent K(+) channels (BK(Ca)). Distal cells demonstrated a greater peak current density in outward current than cells from the proximal region and a longer-lasting outward current increase. These responses were abolished by atropine and the specific M(3) receptor antagonist 4-DAMP but not the M(1) receptor antagonist pirenzipine or the M(2) receptor antagonist methoctramine. BK(Ca) expression along the smooth muscle esophagus was similar, but M(3) receptor expression was greater in the distal region. Therefore, ACh can differentially activate a potassium channel (BK(Ca)) current along the smooth muscle esophagus. This activation probably occurs through release of intracellular calcium via an M(3) pathway and has the potential to modulate the timing and amplitude of peristaltic contraction along the esophagus.     

Regional differences in myosin heavy chain isoform expression and maximal shortening velocity of the rat vaginal wall smooth muscle

Contractility of the proximal and distal vaginal wall smooth muscle may play distinct roles in the female sexual response and pelvic support. The goal of this study was to determine whether differences in contractile characteristics of smooth muscle from these regions reside in differences in the expression of isoforms of myosin, the molecular motor for muscle contraction. Adult female Sprague-Dawley rats were killed on the day of estrus, and the vagina was dissected into proximal and distal segments. The Vmax at peak force was greater for tissue strips of the proximal vagina compared with that of distal (P < 0.01), although, at steady state, the Vmax for the muscle strips from the two regions was not different. Furthermore, at steady state, muscle stress was higher (P < 0.001) for distal vaginal strips (n = 5). Consistent with the high Vmax for the proximal vaginal strips, RT-PCR results revealed a higher %SM-B (P < 0.001) in the proximal vagina. A greater expression of SM-B protein (P < 0.001) was also detected by Western blotting (n = 4). Interestingly, there was no regional difference noted in SM-1/SM-2 isoforms (n = 6). The proximal vagina had a higher expression of myosin heavy chain protein (P < 0.01) and a greater percentage of smooth muscle bundles (P < 0.001). The results of this study are the first demonstration of a regional heterogeneity in Vmax and myosin isoform distribution in the vagina wall smooth muscle and confirm that the proximal vaginal smooth muscle exhibits phasic contractile characteristics compared with the distal vaginal smooth muscle, which is tonic.     

Effects of hypoxia in porcine pulmonary arterial myocytes: roles of K(V) channel and endothelin-1

Effects of acute hypoxia on intracellular Ca(2+) concentration ([Ca(2+)](i)) and cell length were recorded simultaneously in proximal and distal pulmonary (PASMCs) and femoral (FASMCs) arterial smooth muscle cells. Reducing PO(2) from normoxia to severe hypoxia (PO(2) < 10 mmHg) caused small but significant decreases in length and a reversible increase in [Ca(2+)](i) in distal PASMCs and a small decrease in length in proximal PASMCs but had no effect in FASMCs, even though all three cell types contracted significantly to vasoactive agonists. Inhibition of voltage-dependent K(+) (K(V)) channel with 4-aminopyridine produced a greater increase in [Ca(2+)](i) in distal than in proximal PASMCs. In distal PASMCs, severe hypoxia caused a slight inhibition of K(V) currents; however, it elicited further contraction in the presence of 4-aminopyridine. Endothelin-1 (10(-10) M), which itself did not alter cell length or [Ca(2+)](i), significantly potentiated the hypoxic contraction. These results suggest that hypoxia only has small direct effects on porcine PASMCs. These effects cannot be fully explained by inhibition of K(V) channels and were greatly enhanced via synergistic interactions with the endothelium-derived factor endothelin-1.     

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.     

库容性Ca2+内流参与ACh诱导的大鼠远端结肠平滑肌收缩

应用生物换能技术和Ca^2＋通道特异性阻断剂观察并记录大鼠离体远端结肠平滑肌收缩张力的变化,分析库容性Ca^2＋内流（capacitative Ca^2＋ entry,CCE）是否与ACh诱导的离体远端结肠平滑肌收缩反应有关。结果表明,以无钙的Krebs液灌流或应用EGTA螯合细胞外Ca^2＋后,高K^＋及ACh引起的远端结肠平滑肌收缩几乎完全消失。电压操纵性Ca^2＋通道阻断剂verapamil也能减弱高K^＋及ACh引起的远端结肠平滑肌收缩,其减弱的程度分别为74％和41％。在无钙的Krebs液中,5μmol／LACh可引起离体肠管瞬时性收缩,这是由肌质网（sarcoplasmic reticulum,SR）释放钙所致：然后加入10μmol／L阿托品（atropine）,并在此基础上恢复细胞外Ca^2＋（2．5mmol／L）,结肠平滑肌则出现持续性收缩,待收缩反应达峰值时,加入5μmol／L verapamil,收缩无明显变化,且该收缩反应对钙库操纵性通道（store-operated Ca^2＋ channel,socc）阻断剂La^3＋敏感,20,50和100μmol／L的La^3＋使上述收缩张力分别降低15％,23％和36％,且呈浓度依赖性,但对Cd^2＋不敏感。研究结果提示,细胞外Ca^2＋内流对高K^＋及ACh介导的离体远端结肠平滑肌持续性收缩是必需的,由ACh诱导的远端结肠平滑肌收缩至少包括SR释放钙引起的短暂性收缩及受体操纵性Ca^2＋通道（receptor-operated Ca^2＋ channel,ROCC）、电压操纵性Ca^2＋通道（voltage-operated Ca^2＋ channel,VOCC）和CCE介导的胞外Ca^2＋ 内流等途径。这将从通道水平进一步分析消化管平滑肌收缩的机制和特征,亦将为预防和控制因胃肠动力紊乱所致的消化管疾病寻求有针对性的药物干预和治疗提供理论依据。     

Mechanisms mediating the oxygen-induced vasoreactivity of the ductus arteriosus in the chicken embryo

The avian embryo provides a novel model for studying the ductus arteriosus (DA) during the transition from in ovo to ex ovo life. Here we examined the mechanisms regulating the vasoreactivity of the two morphologically distinct portions of the chicken DA (proximal and distal) in response to O(2). Oxygen-induced contraction is redox sensitive and reversed by the reducing agent dithiothreitol and the H(2)O(2) scavenger N-mercaptopropionylglycine. As in the mammalian DA, inhibiting mitochondrion-derived reactive oxygen species production with rotenone and antimycin A relaxed the O(2)-constricted DA. The contractile response to O(2) matures during hatching and is mimicked by the K(v) channel inhibitor 4-aminopyridine (4-AP) on day 19 and externally pipped (EP) embryos. Together, O(2) and 4-AP significantly increase DA tone above that observed with either alone. The O(2)-induced contraction is mediated by influx of extracellular Ca(2+) through l-type Ca(2+) and store-operated channels. Inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores play a minor role in the O(2)-induced contraction. The O(2)-induced contraction is mediated by the Rho kinase pathway, as fasudil and Y-27632 significantly relax the O(2) contracted DA. Prostaglandins E(2), F(2alpha), and D(2) produce significant contraction of the proximal DA. The O(2)-induced relaxation of the distal portion of the DA is mediated by an endothelial-derived nitric oxide/cGMP pathway. Both 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and endothelial cell removal inhibit O(2)-induced relaxation in the distal segment. Mechanisms regulating O(2)-induced contraction in chicken proximal DA are similar to those found in mammalian DA, making the chicken a useful model for studying development of this O(2)-sensitive vessel.     

Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action

Effect of ANG II was investigated in in vitro smooth muscle strips and in isolated smooth muscle cells (SMC). Among different species, rat internal and sphincter (IAS) smooth muscle showed significant and reproducible contraction that remained unmodified by different neurohumoral inhibitors. The AT(1) antagonist losartan but not AT(2) antagonist PD-123319 antagonized ANG II-induced contraction of the IAS smooth muscle and SMC. ANG II-induced contraction of rat IAS smooth muscle and SMC was attenuated by tyrosine kinase inhibitors genistein and tyrphostin, protein kinase C (PKC) inhibitor H-7, Ca(2+) channel blocker nicardipine, Rho kinase inhibitor Y-27632 or p(44/42) mitogen-activating protein kinase (MAPK(44/42)) inhibitor PD-98059. Combinations of nicardipine and H-7, Y-27632, and PD-98059 caused further attenuation of the ANG II effects. Western blot analyses revealed the presence of both AT(1) and AT(2) receptors. We conclude that ANG II causes contraction of rat IAS smooth muscle by the activation of AT(1) receptors at the SMC and involves multiple intracellular pathways, influx of Ca(2+), and activation of PKC, Rho kinase, and MAPK(44/42).     

Role of Ca(2+) fluctuations in L6 myotubes in the regulation of the hexokinase II gene.

Expression of the hexokinase (HK) II gene in skeletal muscle is upregulated by electrically stimulated muscle contraction and moderate-intensity exercise. However, the molecular mechanism by which this occurs is unknown. Alterations in intracellular Ca(2+) homeostasis accompany contraction and regulate gene expression in contracting skeletal muscle. Therefore, as a first step in understanding the exercise-induced increase in HK II, the ability of Ca(2+) to increase HK II mRNA was investigated in cultured skeletal muscle cells, namely L6 myotubes. Exposure of cells to the ionophore A-23187 resulted in an approximately threefold increase in HK II mRNA. Treatment of cells with the extracellular Ca(2+) chelator EGTA did not alter HK II mRNA, nor was it able to prevent the A-23187-induced increase. Treatment of cells with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) also resulted in an approximately threefold increase in HK II mRNA in the absence of ionophore, which was similar to the increase in HK II mRNA induced by the combination of BAPTA-AM and A-23187. In summary, a rise in intracellular Ca(2+) is not necessary for the A-23187-induced increase in HK II mRNA, and increases in HK II mRNA occur in response to treatments that decrease intracellular Ca(2+) stores. Depletion of intracellular Ca(2+) stores may be one mechanism by which muscle contraction increases HK II mRNA.     

Alteration of the increase in intracellular [Ca(2+)] in proliferating smooth muscle cells.

Proliferation of smooth muscle cells (SMC) has a role in the development of cardiovascular diseases. We investigated the alteration of contractile signals in proliferating SMC by measuring the increase in intracellular [Ca(2+)] to endothelin-1 (ET-1), noradrenaline (NA), or angiotensin II (AgII). We found that the increase in intracellular [Ca(2+)] by NA or ET-1 decreased in proliferating SMC in comparison to growth-arrested SMC. The increase in intracellular [Ca(2+)] by AgII was stable between the cells. Immunoblotting of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) which are responsible for the mobilization of Ca(2+) by those vasoactive substances revealed that expression of IP(3)R type 1 and type 2 was decreased. Expression of IP(3)R type 3 was increased. The altered Ca(2+) signaling by the cell growth might involve the expression of IP(3)R subtypes.     

Increase of L-type Ca2+ current by protease-activated receptor 2 activation contributes to augmentation of spontaneous uterine contractility in pregnant rats

We evaluated the effects of protease-activated receptor (PAR)-2 on spontaneous myometrial contraction (SMC) in isolated term pregnant myometrial strips of rat, and elucidated the cellular mechanisms of this effect using a conventional voltage-clamp method. In isometric tension measurements, trypsin and SL-NH(2), PAR-2 agonists, significantly augmented SMC in frequency and amplitude; however, boiled trypsin (BT) and LR-NH(2) had no effect on SMC. These stimulatory effects of PAR-2 agonists on SMC were nearly completely occluded by pre-application of Bay K 8644, an L-type voltage-gated Ca(2+) channel activator, thus showing the involvement of L-type voltage-gated Ca(2+) channels in PAR-2-induced augmentation of SMC. In addition, PAR-2 agonists significantly enhanced L-type voltage-gated Ca(2+) currents (I(Ca-L)), as measured by a conventional voltage-clamp method, and this increase was primarily mediated by activation of phospholipase C (PLC) and protein kinase C (PKC) via G-protein activation. Taken together, we have demonstrated that PAR-2 may actively regulate SMC during pregnancy by modulating Ca(2+) influx through L-type voltage-gated Ca(2+) channels, and that this increase of I(Ca-L) may be primarily mediated by PLC and PKC activation. These results suggest a cellular mechanism for the pathophysiological effects of PAR-2 activation on myometrial contractility during pregnancy and provide basic and theoretical information about developing new agents for the treatment of premature labor and other obstetric complications.     

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.     

Increase of [Ca(2+)]i and release of arachidonic acid via activation of M2 receptor coupled to Gi and rho proteins in oesophageal muscle

We have previously shown that acetylcholine-induced contraction of oesophageal circular muscle depends on activation of phosphatidylcholine selective phospholipase C and D, which result in formation of diacylglycerol, and of phospholipase 2 which produces arachidonic acid. Diacylglycerol and arachidonic acid interact synergistically to activate protein kinase C. We have therefore investigated the relationship between cytosolic Ca(2+) and activation of phospholipase A(2) in response to acetylcholine-induced stimulation, by measuring the intracellular free Ca(2+) ([Ca(2+)]i), muscle tension, and [3H] arachidonic acid release. Acetylcholine-induced contraction was associated with increased [Ca(2+)]i and arachidonic acid release in a dose-dependent manner. In Ca(2+)-free medium, acetylcholine did not produce contraction, [Ca(2+)]i increase, and arachidonic acid release. In contrast, after depletion of Ca(2+) stores by thapsigargin (3 microM), acetylcholine caused a normal contraction, [Ca(2+)]i increase and arachidonic acid release. The increase in [Ca(2+)]i and arachidonic acid release were attenuated by the M2 receptor antagonist methoctramine, but not by the M3 receptor antagonist p-fluoro-hexahydro siladifenidol. Increase in [Ca(2+)]i and arachidonic acid release by acetylcholine were inhibited by pertussis toxin and C3 toxin. These findings indicate that contraction and arachidonic acid release are mediated through muscarinic M2 coupled to Gi or rho protein activation and Ca(2+) influx. Acetylcholine-induced contraction and the associated increase in [Ca(2+)]i and release of arachidonic acid were completely reduced by the combination treatment with a phospholipase A(2) inhibitor dimethyleicosadienoic acid and a phospholipase D inhibitor pCMB. They increased by the action of the inhibitor of diacylglycerol kinase R59949, whereas they decreased by a protein kinase C inhibitor chelerythrine. These data suggest that in oesophageal circular muscle acetylcholine-induced [Ca(2+)]i increase and arachidonic acid release are mediated through activation of M2 receptor coupled to Gi or rho protein, resulting in the activation of phospholipase A(2) and phospholipase D to activate protein kinase C.     

Ca2+ activation of smooth muscle contraction: evidence for the involvement of calmodulin that is bound to the triton insoluble fraction even in the absence of Ca2+.

Smooth muscle contraction is activated by phosphorylation of the 20-kDa light chains of myosin catalyzed by Ca(2+)/calmodulin (CaM)-dependent myosin light chain kinase (MLCK). According to popular current theory, the CaM involved in MLCK regulation is Ca(2+)-free and dissociated from the kinase at resting cytosolic free Ca(2+) concentration ([Ca(2+)](i)). An increase in [Ca(2+)](i) saturates the four Ca(2+)-binding sites of CaM, which then binds to and activates actin-bound MLCK. The results of this study indicate that this theory requires revision. Sufficient CaM was retained after skinning (demembranation) of rat tail arterial smooth muscle in the presence of EGTA to support Ca(2+)-evoked contraction, as observed previously with other smooth muscle tissues. This tightly bound CaM was released by the CaM antagonist trifluoperazine (TFP) in the presence of Ca(2+). Following removal of the (Ca(2+))(4)-CaM-TFP(2) complex, Ca(2+) no longer induced contraction. The addition of exogenous CaM to TFP-treated tissue at a [Ca(2+)] subthreshold for contraction or even in the absence of Ca(2+) (presence of 5 mm EGTA), followed by washout of unbound CaM, restored Ca(2+)-induced contraction; this required MLCK activation, since it was blocked by the MLCK inhibitor ML-9. The data suggest, therefore, that a specific pool of cellular CaM, tightly bound to myofilaments at resting [Ca(2+)](i), or even in the absence of Ca(2+), is responsible for activation of contraction following a local increase in [Ca(2+)]. This mechanism would allow for localized changes in [Ca(2+)] in regions of the cell distant from the myofilaments to regulate distinct Ca(2+)-dependent processes without triggering a contractile response. Immobilized CaM, therefore, resembles troponin C, the Ca(2+)-binding regulatory protein of striated muscle, which is also bound to the thin filament in a Ca(2+)-independent manner.     

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.     

Inositol trisphosphate receptors in smooth muscle cells

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are a family of tetrameric intracellular calcium (Ca(2+)) release channels that are located on the sarcoplasmic reticulum (SR) membrane of virtually all mammalian cell types, including smooth muscle cells (SMC). Here, we have reviewed literature investigating IP(3)R expression, cellular localization, tissue distribution, activity regulation, communication with ion channels and organelles, generation of Ca(2+) signals, modulation of physiological functions, and alterations in pathologies in SMCs. Three IP(3)R isoforms have been identified, with relative expression and cellular localization of each contributing to signaling differences in diverse SMC types. Several endogenous ligands, kinases, proteins, and other modulators control SMC IP(3)R channel activity. SMC IP(3)Rs communicate with nearby ryanodine-sensitive Ca(2+) channels and mitochondria to influence SR Ca(2+) release and reactive oxygen species generation. IP(3)R-mediated Ca(2+) release can stimulate plasma membrane-localized channels, including transient receptor potential (TRP) channels and store-operated Ca(2+) channels. SMC IP(3)Rs also signal to other proteins via SR Ca(2+) release-independent mechanisms through physical coupling to TRP channels and local communication with large-conductance Ca(2+)-activated potassium channels. IP(3)R-mediated Ca(2+) release generates a wide variety of intracellular Ca(2+) signals, which vary with respect to frequency, amplitude, spatial, and temporal properties. IP(3)R signaling controls multiple SMC functions, including contraction, gene expression, migration, and proliferation. IP(3)R expression and cellular signaling are altered in several SMC diseases, notably asthma, atherosclerosis, diabetes, and hypertension. In summary, IP(3)R-mediated pathways control diverse SMC physiological functions, with pathological alterations in IP(3)R signaling contributing to disease.     

cAMP inhibits IP(3)-dependent Ca(2+) release by preferential activation of cGMP-primed PKG

The singular effects and interplay of cAMP- and cGMP-dependent protein kinase (PKA and PKG) on Ca(2+) mobilization were examined in dispersed smooth muscle cells. In permeabilized muscle cells, exogenous cAMP and cGMP inhibited inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of cAMP and cGMP caused synergistic inhibition that was exclusively mediated by PKG and attenuated by PKA. In intact muscle cells, low concentrations (10 nM) of isoproterenol and sodium nitroprusside (SNP) inhibited agonist-induced, IP(3)-dependent Ca(2+) release and muscle contraction via PKA and PKG, respectively. A combination of isoproterenol and SNP increased PKA and PKG activities: the increase in PKA activity reflected inhibition of phosphodiesterase 3 activity by cGMP, whereas the increase in PKG activity reflected activation of cGMP-primed PKG by cAMP. Inhibition of Ca(2+) release and muscle contraction by the combination of isoproterenol and SNP was preferentially mediated by PKG. In light of studies showing that PKG phosphorylates the IP(3) receptor in intact and permeabilized muscle cells, whereas PKA phosphorylates the receptor in permeabilized cells only, the results imply that inhibition of IP(3)-induced Ca(2+) release is mediated exclusively by PKG. The effect of PKA on agonist-induced Ca(2+) release probably reflects inhibition of IP(3) formation.     

Essential role for calcium waves in migration of human vascular smooth muscle cells

Vascular smooth muscle cell (SMC) migration is characterized by extension of the lamellipodia at the leading edge, lamellipodial attachment to substrate, and release of the rear (uropod) of the cell, all of which enable forward movement. However, little is known regarding the role of intracellular cytosolic Ca(2+) concentration ([Ca(2+)](i)) in coordinating these distinct activities of migrating SMCs. The objective of our study was to determine whether regional changes of Ca(2+) orchestrate the migratory cycle in human vascular SMCs. We carried out Ca(2+) imaging using digital fluorescence microscopy of fura-2 loaded human smooth muscle cells. We found that motile SMCs exhibited Ca(2+) waves that characteristically swept from the rear of polarized cells toward the leading edge. Ca(2+) waves were less evident in nonpolarized, stationary cells, although acute stimulation of these SMCs with the agonists platelet-derived growth factor-BB or histamine could elicit transient rise of [Ca(2+)](i). To investigate a role for Ca(2+) waves in the migratory cycle, we loaded cells with the Ca(2+) chelator BAPTA, which abolished Ca(2+) waves and significantly reduced retraction, supporting a causal role for Ca(2+) in initiation of retraction. However, lamellipod motility was still evident in BAPTA-loaded cells. The incidence of Ca(2+) oscillations was reduced when Ca(2+) release from intracellular stores was disrupted with the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin or by treatment with the inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxy-diphenyl borate or xestospongin C, implicating Ca(2+) stores in generation of waves. We conclude that Ca(2+) waves are essential for migration of human vascular SMCs and can encode cell polarity.     

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.