Signal Transduction in Smooth Muscle: Selected Contribution: NO released to flow reduces myogenic tone of skeletal muscle arterioles by decreasing smooth muscle Ca2+ sensitivity

To clarify the contribution of intracellularCa²⁺ concentration([Ca²⁺]_i)-dependent and -independentsignaling mechanisms in arteriolar smooth muscle (aSM) to modulation ofarteriolar myogenic tone by nitric oxide (NO), released in response toincreases in intraluminal flow from the endothelium, changes in aSM[Ca²⁺]_i and diameter of isolated rat gracilismuscle arterioles (pretreated with indomethacin) were studied byfluorescent videomicroscopy. At an intraluminal pressure of 80 mmHg, [Ca²⁺]_i significantly increased andmyogenic tone developed in response to elevations of extracellularCa²⁺ concentration. The Ca²⁺ channelinhibitor nimodipine substantially decreased[Ca²⁺]_i and completely inhibited myogenictone. Dilations to intraluminal flow (that were inhibited byN-nitro-L-arginine methyl ester)or dilations to the NO donorS-nitroso-N-acetyl-DL-penicillamine (that were inhibited by the guanylate cyclase inhibitor1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) were notaccompanied by substantial decreases in aSM[Ca²⁺]_i. 8-Bromoguanosine cGMP and thecGMP-specific phosphodiesterase inhibitor zaprinast significantlydilated arterioles yet elicited only minimal decreases in[Ca²⁺]_i. Thus flow-induced endothelialrelease of NO elicits relaxation of arteriolar smooth muscle by acGMP-dependent decrease of the Ca²⁺ sensitivity of thecontractile apparatus without substantial changes in thepressure-induced level of [Ca²⁺]_i.

     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents     

Regulation of intracellular Ca2+ release in corpus cavernosum smooth muscle: synergism between nitric oxide and cGMP

Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca²⁺ concentration ([Ca²⁺]_i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺, indicating release from intracellular stores. Whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (SIL) caused no change in basal [Ca²⁺]_i, the PE-induced rise of [Ca²⁺]_i was reversibly inhibited by 27 ± 7% (n = 21, P < 0.005) in rat and by 55 ± 15% (n = 9, P < 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca²⁺]_i. However, the combination of 8-bromo-cGMP with SNAP reduced the Ca²⁺ peak by 42 ± 9% (n = 22, P < 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca²⁺ release from intracellular stores. Reduction of intracellular Ca²⁺ release may contribute to relaxation of the corpus cavernosum, leading to erection. calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor     

Effects of NO donors and synthase agonists on endothelial cell uptake of L-Arg and superoxide production

It is commonly believed thatthe activity of NO synthase (NOS) solely controls NO production fromits substrates, L-Arg and O₂. The Michaelis-Menten constant(K_m) of NOS forL-Arg is in the micromolarrange; cellular levels of L-Argare much higher. However, evidence strongly suggests that cellularsupply of L-Arg may becomelimiting and lead to reduced NO and increased superoxide anion(O₂·) formation, promotingcardiovascular dysfunction. Uptake ofL-Arg into cells occursprimarily (~85%) through the actions of aNa⁺-independent, carrier-mediatedtransporter (system y⁺). We haveexamined the effects of NOS agonists (substance P, bradykinin, and ACh)and NO donors(S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport ofL-Arg into bovine aorticendothelial cells (BAEC). Our results demonstrate that NOS agonistsincrease y⁺ transporter activity.A rapidly acting NO donor initially increases L-Arg uptake; however, afterlonger exposure, L-Arg uptake is suppressed. Exposure of BAEC withoutL-Arg to substance P and aCa²⁺ ionophore (A-23187) increasedO₂· formation, which was blockedwith concurrent presence ofL-Arg or the NOS antagonistN-nitro-L-arginine methyl ester.We conclude that factors including NO itself controly⁺ transport function and theproduction of NO and O₂·.

     

Rat cerebellar granule cells are protected from glutamate-induced excitotoxicity by S-nitrosoglutathione but not glutathione

In cultured rat cerebellar granule cells, glutamate or N-methyl-D-aspartate (NMDA) activation of the NMDA receptor caused a sustained increase in cytosolic Ca²⁺ levels ([Ca²⁺]_i), reactive oxygen species (ROS) generation, and cell death (respective EC₅₀ values for glutamate were 12, 30, and 38 µM) but no increase in caspase-3 activity. Removal of extracellular Ca²⁺ blocked all three glutamate-induced effects, whereas pretreatment with an ROS scavenger inhibited glutamate-induced cell death but had no effect on the [Ca²⁺]_i increase. This indicates that glutamate-induced cell death is attributable to [Ca²⁺]_i increase and ROS generation, and the [Ca²⁺]_i increase precedes ROS generation. Apoptotic cell death was not seen until 24 h after exposure of cells to glutamate. S-nitrosoglutathione abolished glutamate-induced ROS generation and cell death, and only a transient [Ca²⁺]_i increase was seen; similar results were observed with another nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine, but not with glutathione, which suggests that the effects were caused by NO. The transient [Ca²⁺]_i increase and the abolishment of ROS generation induced by glutamate and S-nitrosoglutathione were still seen in the presence of an ROS scavenger. Glial cells, which were present in the cultures used, showed no [Ca²⁺]_i increase in the presence of glutamate, and glutamate-induced granule cell death was independent of the percentage of glial cells. In conclusion, NO donors protect cultured cerebellar granule cells from glutamate-induced cell death, which is mediated by ROS generated by a sustained [Ca²⁺]_i increase, and glial cells provide negligible protection against glutamate-induced excitotoxicity. cytosolic calcium concentration; N-methyl-D-aspartate; reactive oxygen species     

Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion

We previously reported that human growth hormone (hGH) increases cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and proliferation in pancreatic -cells (Sjöholm Å, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, and Berggren PO. J Biol Chem 275: 21033–21040, 2000) and that the hGH-induced rise in [Ca²⁺]_i involves Ca²⁺-induced Ca²⁺ release facilitated by tyrosine phosphorylation of ryanodine receptors (Zhang Q, Kohler M, Yang SN, Zhang F, Larsson O, and Berggren PO. Mol Endocrinol 18: 1658–1669, 2004). Here we investigated the tyrosine kinases that convey the hGH-induced rise in [Ca²⁺]_i and insulin release in BRIN-BD11 -cells. hGH caused tyrosine phosphorylation of Janus kinase (JAK)2 and c-Src, events inhibited by the JAK2 inhibitor AG490 or the Src kinase inhibitor PP2. Although hGH-stimulated rises in [Ca²⁺]_i and insulin secretion were completely abolished by AG490 and JAK2 inhibitor II, the inhibitors had no effect on insulin secretion stimulated by a high K⁺ concentration. Similarly, Src kinase inhibitor-1 and PP2, but not its inactive analog PP3, suppressed [Ca²⁺]_i elevation and completely abolished insulin secretion stimulated by hGH but did not affect responses to K⁺. Ovine prolactin increased [Ca²⁺]_i and insulin secretion to a similar extent as hGH, effects prevented by the JAK2 and Src kinase inhibitors. In contrast, bovine GH evoked a rise in [Ca²⁺]_i but did not stimulate insulin secretion. Neither JAK2 nor Src kinase inhibitors influenced the effect of bovine GH on [Ca²⁺]_i. Our study indicates that hGH stimulates rise in [Ca²⁺]_i and insulin secretion mainly through activation of the prolactin receptor and JAK2 and Src kinases in rat insulin-secreting cells. c-Src; growth hormone receptor; prolactin receptor; Ca²⁺-induced Ca²⁺ release     

Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA

The involvement of cAMP- andCa²⁺-mediated pathways in theactivation of tyrosine hydroxylase (TH) gene expression by nicotine wasexamined in PC-12 cells. ExtracellularCa²⁺ and elevations inintracellular Ca²⁺ concentration([Ca²⁺]_i)were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in[Ca²⁺]_iwas inhibited by blockers of either L-type or N-type, and to a lesserextent P/Q-, but not T-type, voltage-gatedCa²⁺ channels. With continualnicotine treatment,[Ca²⁺]_ireturned to basal levels within 3-4 min. After a lag of~5-10 min, there was a smaller elevation in[Ca²⁺]_ithat persisted for 6 h and displayed different responsiveness toCa²⁺ channel blockers. This secondphase of elevated[Ca²⁺]_iwas blocked by an inhibitor of store-operatedCa²⁺ channels, consistent with theobserved generation of inositol trisphosphate.1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine,prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor2',5'-dideoxyadenosine (DDA) prevented thenicotine-elicited phosphorylation of cAMP response element bindingprotein. DDA also blocked the elevation of TH mRNA only when addedafter the initial transient rise in [Ca²⁺]_iand not after 1 h. This study reveals that several temporal phases areinvolved in the induction of TH gene expression by nicotine, each ofthem with differing requirements forCa²⁺ and cAMP.

     

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

The plasmalemmal Na⁺-K⁺-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca²⁺. However, the ability of PTX to directly increase cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via Na⁺ pump channels and to initiate Ca²⁺ overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca²⁺]_i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3–100 nM) produced a graded increase in [Ca²⁺]_i that was dependent on extracellular Ca²⁺. The increase in [Ca²⁺]_i initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC₅₀ < 1 µM. The elevation in [Ca²⁺]_i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca²⁺]_i occurs via the Na⁺ pump. Subsequent to the rise in [Ca²⁺]_i, PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca²⁺ or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca²⁺ overload. necrosis; vital dyes; membrane blebs; time-lapse video microscopy; fura-2     

Enhanced response to caffeine and 4-chloro-m-cresol in malignant hyperthermia-susceptible muscle is related in part to chronically elevated resting [Ca2+]i

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic syndrome caused by exposure to halogenated volatile anesthetics and/or depolarizing muscle relaxants. We have measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) using double-barreled, Ca²⁺-selective microelectrodes in myoballs prepared from skeletal muscle of MH-susceptible (MHS) and MH-nonsusceptible (MHN) swine. Resting [Ca²⁺]_i was approximately twofold in MHS compared with MHN quiescent myoballs (232 ± 35 vs. 112 ± 11 nM). Treatment of myoballs with caffeine or 4-chloro-m-cresol (4-CmC) produced an elevation in [Ca²⁺]_i in both groups; however, the concentration required to cause a rise in [Ca²⁺]_i elevation was four times lower in MHS than in MHN skeletal muscle cells. Incubation of MHS cells with the fast-complexing Ca²⁺ buffer BAPTA reduced [Ca²⁺]_i, raised the concentration of caffeine and 4-CmC required to cause an elevation of [Ca²⁺]_i, and reduced the amount of Ca²⁺ release associated with exposure to any given concentration of caffeine or 4-CmC to MHN levels. These results suggest that the differences in the response of MHS skeletal myoballs to caffeine and 4-CmC may be mediated at least in part by the chronic high resting [Ca²⁺]_i levels in these cells. calcium homeostasis; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid     

Regulation of cytoplasmic calcium levels by two nitric oxide receptors

We examined the effects of dissolved nitric oxide (NO) gas oncytoplasmic calcium levels ([Ca²⁺]_i) in C6glioma cells under anoxic conditions. The maximum elevation (27 ± 3 nM) of [Ca²⁺]_i was reached at 10 µM NO. Asecond application of NO was ineffective if the first was >0.5 µM.The NO donor diethylamine/NO mimicked the effects of NO. Acute exposureof the cells to low calcium levels was without effect on the NO-evokedresponse. Thapsigargin (TG) increased [Ca²⁺]_iand was less effective if cells were pretreated with NO. Hemoglobin inhibited the effects of NO at a molar ratio of 10:1. 8-Bromo-cGMP waswithout effect on the NO-evoked response. If cells were pretreated withTG or exposed chronically to nominal amounts of calcium, NO decreased[Ca²⁺]_i. The results suggest that C6 gliomacells have two receptors for NO. One receptor (NO_A)elevates [Ca²⁺]_i and resides on theendoplasmic reticulum (ER). The other receptor (NO_B)decreases [Ca²⁺]_i and resides on theplasmalemma or the ER. The latter receptor dominates when the level ofcalcium within intracellular stores is diminished.

     

Dual effects of n-alcohols on fluid secretion from guinea pig pancreatic ducts

Ethanol strongly augments secretin-stimulated, but not acetylcholine (ACh)-stimulated, fluid secretion from pancreatic duct cells. To understand its mechanism of action, we examined the effect of short-chain n-alcohols on fluid secretion and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in guinea pig pancreatic ducts. Fluid secretion was measured by monitoring the luminal volume of isolated interlobular ducts. [Ca²⁺]_i was estimated using fura-2 microfluorometry. Methanol and ethanol at 0.3–10 mM concentrations significantly augmented fluid secretion and induced a transient elevation of [Ca²⁺]_i in secretin- or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated ducts. However, they failed to affect fluid secretion and [Ca²⁺]_i in unstimulated and ACh-stimulated ducts. In contrast, propanol and butanol at 0.3–10 mM concentrations significantly reduced fluid secretion and decreased [Ca²⁺]_i in unstimulated ducts and in ducts stimulated with secretin, DBcAMP, or ACh. Both stimulatory and inhibitory effects of n-alcohols completely disappeared after their removal from the perfusate. Propanol and butanol inhibited the plateau phase, but not the initial peak, of [Ca²⁺]_i response to ACh as well as the [Ca²⁺]_i elevation induced by thapsigargin, suggesting that they inhibit Ca²⁺ influx. Removal of extracellular Ca²⁺ reduced [Ca²⁺]_i in duct cells and completely abolished secretin-stimulated fluid secretion. In conclusion, there is a distinct cutoff point between ethanol (C2) and propanol (C3) in their effects on fluid secretion and [Ca²⁺]_i in duct cells. Short-chain n-alcohols appear to affect pancreatic ductal fluid secretion by activating or inhibiting the plasma membrane Ca²⁺ channel. intracellular calcium; acetylcholine     

Cellular Responses to Mechanical Stress: Invited Review: Effects of flow on vascular endothelial intracellular calcium signaling of rat aortas ex vivo

To study the effects of flow on in situendothelial intracellular calcium concentration([Ca²⁺]_i) signaling, rat aortic rings wereloaded with fura 2, mounted on a tissue flow chamber, and divided intocontrol and flow-pretreated groups. The latter was perfused with bufferat a shear stress of 50 dyns/cm² for 1 h. Endothelial[Ca²⁺]_i responses to ACh or shear stresseswere determined by ratio image analysis. Moreover, ACh-induced[Ca²⁺]_i elevation responses were measured ina calcium-free buffer, or in the presence of SKF-96365, to elucidatethe role of calcium influx in the flow effects. Our results showed that1) ACh increased endothelial[Ca²⁺]_i in a dose-dependent manner, and theseresponses were incremented by flow-pretreatment; 2) thedifferences in ACh-induced [Ca²⁺]_i elevationbetween control and flow-pretreated groups were abolished by SKF-96365or by Ca²⁺-free buffer; and 3) in the presenceof 10⁵ M ATP, shear stress induced dose-dependent[Ca²⁺]_i elevation responses that were notaltered by flow-pretreatment. In conclusion, flow-pretreatment augmentsthe ACh-induced endothelial calcium influx in rat aortas ex vivo.

     

Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells

Thrombin and related protease-activated receptors 1, 2, 3, and 4 (PAR1–4) play a multifunctional role in many types of cells including endothelial cells. Here, using RT-PCR and immunofluorescence staining, we showed for the first time that PAR1–4 are expressed on primary human brain microvascular endothelial cells (HBMEC). Digital fluorescence microscopy and fura 2 were used to monitor intracellular Ca²⁺ concentration ([Ca²⁺]_i) changes in response to thrombin and PAR1-activating peptide (PAR1-AP) SFFLRN. Both thrombin and PAR1-AP induced a dose-dependent [Ca²⁺]_i rise that was inhibited by pretreatment of HBMEC with the phospholipase C inhibitor U-73122 and the sarco(endo)plasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin. Thrombin induced transient [Ca²⁺]_i increase, whereas PAR1-AP exhibited sustained [Ca²⁺]_i rise. The PAR1-AP-induced sustained [Ca²⁺]_i rise was significantly reduced in the absence of extracellular calcium or in the presence of an inhibitor of store-operated calcium channels, SKF-96365. Restoration of extracellular Ca²⁺ to the cells that were initially activated by PAR1-AP in the absence of extracellular Ca²⁺ resulted in significant [Ca²⁺]_i rise; however, this effect was not observed after thrombin stimulation. Pretreatment of the cells with a low thrombin concentration (0.1 nM) prevented [Ca²⁺]_i rise in response to high thrombin concentration (10 nM), but pretreatment with PAR1-AP did not prevent subsequent [Ca²⁺]_i rise to high PAR1-AP concentration. Additionally, treatment with thrombin decreased transendothelial electrical resistance in HBMEC, whereas PAR1-AP was without significant effect. These findings suggest that, in contrast to thrombin, stimulation of PAR1 by untethered peptide SFFLRN results in stimulation of store-operated Ca²⁺ influx without significantly affecting brain endothelial barrier functions. store-operated calcium influx; desensitization; transendothelial electrical resistance; digital imaging     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca²⁺ concentration([Ca²⁺]_i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm²). Before application of shear, cells were treatedwith two Ca²⁺ channel inhibitors or various blockers ofintracellular Ca²⁺ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe [Ca²⁺]_i response, neithergadolinium nor nifedipine, an L-type channel Ca²⁺ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca²⁺ chelator, or thapsigargin,which empties intracellular Ca²⁺ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP₃-mediated intracellular Ca²⁺release is required for modulating flow-induced responses in MC3T3-E1 cells.

     

VCAM-1-induced inwardly rectifying K(+) current enhances Ca(2+) entry in human THP-1 monocytes

Hyperpolarization in human leukemia THP-1 monocytes adherent tovascular cell adhesion molecule (VCAM)-1 is due to an induction ofinwardly rectifying K⁺ currents(I_ir) (Colden-Stanfield M and Gallin EK,Am J Physiol Cell Physiol 275: C267-C277, 1998).We determined whether the VCAM-1-induced hyperpolarization issufficient to augment the increase in intracellular free calcium([Ca²⁺]_i) produced by Ca²⁺ storedepletion with thapsigargin (TG) and readdition of external CaCl₂ in fura 2-loaded THP-1 monocytes. Whereas there was a2.1-fold increase in [Ca²⁺]_i in monocytesbound to glass for 5 h in response to TG and CaCl₂ addition, adherence to VCAM-1 produced a 5-fold increase in[Ca²⁺]_i. Depolarization of monocytes adherentto VCAM-1 by I_ir blockade or exposure to high[K⁺] abolished the enhancement of the peak[Ca²⁺]_i response. In monocytes bound toglass, hyperpolarization of the membrane potential with valinomycin, aK⁺ ionophore, to the level of hyperpolarization seen incells adherent to VCAM-1 produced similar changes in peak[Ca²⁺]_i. Adherence of monocytes to E-selectinproduced a similar peak [Ca²⁺]_i to cellsbound to glass. Thus monocyte adherence to the physiological substrateVCAM-1 produces a hyperpolarization that is sufficient to enhanceCa²⁺ entry and may impact Ca²⁺-dependentmonocyte function.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Mechanical signals and mechanosensitive modulation of intracellular [Ca(2+)] in smooth muscle

We testedthe hypothesis that strain is the primary mechanical signal in themechanosensitive modulation of intracellular Ca²⁺concentration ([Ca²⁺]_i) in airway smoothmuscle. We found that [Ca²⁺]_i wassignificantly correlated with muscle length during isotonic shorteningagainst 20% isometric force (F_iso). When the isotonic loadwas changed to 50% F_iso, data points from the 20 and 50% F_iso experiments overlapped in thelength-[Ca²⁺]_i relationship. Similarly, datapoints from the 80% F_iso experiments clustered near thosefrom the 50% F_iso experiments. Therefore, despite 2.5- and4-fold differences in external load, [Ca²⁺]_idid not deviate much from the length-[Ca²⁺]_irelation that fitted the 20% F_iso data. Maximal inhibition of sarcoplasmic reticular (SR) Ca²⁺ uptake by 10 µMcyclopiazonic acid (CPA) did not significantly change[Ca²⁺]_i in carbachol-induced isometriccontractions and isotonic shortening. CPA also did not significantlychange myosin light-chain phosphorylation or force redevelopment whencarbachol-activated muscle strips were quickly released from optimallength (L_o) to 0.5 L_o. These results are consistent with thehypothesis and suggest that SR Ca²⁺ uptake is not theunderlying mechanism.

     

Elevated resting [Ca(2+)](i) in myotubes expressing malignant hyperthermia RyR1 cDNAs is partially restored by modulation of passive calcium leak from the SR

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered in susceptible individuals by inhalation anesthetics and depolarizing skeletal muscle relaxants. This syndrome has been linked to a missense mutation in the type 1 ryanodine receptor (RyR1) in more than 50% of cases studied to date. Using double-barreled Ca²⁺ microelectrodes in myotubes expressing wild-type RyR1 (_WTRyR1) or RyR1 with one of four common MH mutations (_MHRyR1), we measured resting intracellular Ca²⁺ concentration ([Ca²⁺]_i). Changes in resting [Ca²⁺]_i produced by several drugs known to modulate the RyR1 channel complex were investigated. We found that myotubes expressing any of the _MHRyR1s had a 2.0- to 3.7-fold higher resting [Ca²⁺]_i than those expressing _WTRyR1. Exposure of myotubes expressing _MHRyR1s to ryanodine (500 µM) or (2,6-dichloro-4-aminophenyl)isopropylamine (FLA 365; 20 µM) had no effects on their resting [Ca²⁺]_i. However, when myotubes were exposed to bastadin 5 alone or to a combination of ryanodine and bastadin 5, the resting [Ca²⁺]_i was significantly reduced (P < 0.01). Interestingly, the percent decrease in resting [Ca²⁺]_i in myotubes expressing _MHRyR1s was significantly greater than that for _WTRyR1. From these data, we propose that the high resting myoplasmic [Ca²⁺]_i in _MHRyR1 expressing myotubes is due in part to a related structural conformation of _MHRyR1s that favors "passive" calcium leak from the sarcoplasmic reticulum. ryanodine; FLA 365; bastadin 5; resting intracellular calcium concentration; sarcoplasmic reticulum     

Polyunsaturated fatty acids mobilize intracellular Ca2+ in NT2 human teratocarcinoma cells by causing release of Ca2+ from mitochondria

In a variety of disorders, overaccumulation of lipid in nonadipose tissues, including the heart, skeletal muscle, kidney, and liver, is associated with deterioration of normal organ function, and is accompanied by excessive plasma and cellular levels of free fatty acids (FA). Increased concentrations of FA may lead to defects in mitochondrial function found in diverse diseases. One of the most important regulators of mitochondrial function is mitochondrial Ca²⁺ ([Ca²⁺]_m), which fluctuates in coordination with intracellular Ca²⁺ ([Ca²⁺]_i). Polyunsaturated FA (PUFA) have been shown to cause [Ca²⁺]_i mobilization albeit by unknown mechanisms. We have found that PUFA but not monounsaturated or saturated FA cause [Ca²⁺]_i mobilization in NT2 human teratocarcinoma cells. Unlike the [Ca²⁺]_i response to the muscarinic G protein-coupled receptor agonist carbachol, PUFA-mediated [Ca²⁺]_i mobilization in NT2 cells is independent of phospholipase C and inositol-1,4,5-trisphospate (IP₃) receptor activation, as well as IP₃-sensitive internal Ca²⁺ stores. Furthermore, PUFA-mediated [Ca²⁺]_i mobilization is inhibited by the mitochondria uncoupler carboxyl cyanide m-chlorophenylhydrozone. Direct measurements of [Ca²⁺]_m with X-rhod-1 and ⁴⁵Ca²⁺ indicate that PUFA induce Ca²⁺ efflux from mitochondria. Further studies show that ruthenium red, an inhibitor of the mitochondrial Ca²⁺ uniporter, blocks PUFA-induced Ca²⁺ efflux from mitochondria, whereas inhibitors of the mitochondrial permeability transition pore cyclosporin A and bongkrekic acid have no effect. Thus PUFA-gated Ca²⁺ release from mitochondria, possibly via the Ca²⁺ uniporter, appears to be the underlying mechanism for PUFA-induced [Ca²⁺]_i mobilization in NT2 cells. arachidonic acid; mitochondrial Ca²⁺ uniporter; G protein-coupled receptor; IP₃ receptor