Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

Thyroid hormone inhibits biliary growth in bile duct-ligated rats by PLC/IP(3)/Ca(2+)-dependent downregulation of SRC/ERK1/2

The role of the thyroid hormone agonist 3,3',5 L-tri-iodothyronine (T3) on cholangiocytes is unknown. We evaluated the in vivo and in vitro effects of T3 on cholangiocyte proliferation of bile duct-ligated (BDL) rats. We assessed the expression of ₁-, ₂-, ₁-, and ₂-thyroid hormone receptors (THRs) by immunohistochemistry in liver sections from normal and BDL rats. BDL rats were treated with T3 (38.4 µg/day) or vehicle for 1 wk. We evaluated 1) biliary mass and apoptosis in liver sections and 2) proliferation in cholangiocytes. Serum-free T3 levels were measured by chemiluminescence. Purified BDL cholangiocytes were treated with 0.2% BSA or T3 (1 µM) in the absence/presence of U-73122 (PLC inhibitor) or BAPTA/AM (intracellular Ca²⁺ chelator) before measurement of PCNA protein expression by immunoblots. The in vitro effects of T3 (1 µM) on 1) cAMP, IP₃, and Ca²⁺ levels and 2) the phosphorylation of Src Tyr139 and Tyr530 (that, together, regulate Src activity) and ERK1/2 of BDL cholangiocytes were also evaluated. ₁-, ₂-, ₁-, and ₂-THRs were expressed by bile ducts of normal and BDL rats. In vivo, T3 decreased cholangiocyte proliferation of BDL rats. In vitro, T3 inhibition of PCNA protein expression was blocked by U-73122 and BAPTA/AM. Furthermore, T3 1) increased IP₃ and Ca²⁺ levels and 2) decreased Src and ERK1/2 phosphorylation of BDL cholangiocytes. T3 inhibits cholangiocyte proliferation of BDL rats by PLC/IP₃/Ca²⁺-dependent decreased phosphorylation of Src/ERK1/2. Activation of the intracellular signals triggered by T3 may modulate the excess of cholangiocyte proliferation in liver diseases. cholestasis; cholangiopathies; hyperplasia; intrahepatic biliary epithelium; mitosis     

Inhibition of Galphaq-dependent PLC-beta1 activity by PKG and PKA is mediated by phosphorylation of RGS4 and GRK2

In smooth muscle of the gut, G_q-coupled receptor agonists activate preferentially PLC-1 to stimulate phosphoinositide (PI) hydrolysis and inositol 1,4,5-trisphosphate (IP₃) generation and induce IP₃-dependent Ca²⁺ release. Inhibition of Ca²⁺ mobilization by cAMP- (PKA) and cGMP-dependent (PKG) protein kinases reflects inhibition of PI hydrolysis by both kinases and PKG-specific inhibitory phosphorylation of IP₃ receptor type I. The mechanism of inhibition of PLC-1-dependent PI hydrolysis has not been established. Neither G_q nor PLC-1 was directly phosphorylated by PKA or PKG in gastric smooth muscle cells. However, both kinases 1) phosphorylated regulator of G protein signaling 4 (RGS4) and induced its translocation from cytosol to plasma membrane, 2) enhanced ACh-stimulated association of RGS4 and G_q·GTP and intrinsic G_q·GTPase activity, and 3) inhibited ACh-stimulated PI hydrolysis. RGS4 phosphorylation and inhibition of PI hydrolysis were blocked by selective PKA and PKG inhibitors. Expression of RGS4(S52A), which lacks a PKA/PKG phosphorylation site, blocked the increase in GTPase activity and the decrease in PI hydrolysis induced by PKA and PKG. Blockade of PKA-dependent effects was only partial. Selective phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which contains a RGS domain, by PKA augmented ACh-stimulated GRK2:G_q·GTP association; both effects were blocked in cells expressing GRK2(S685A), which lacks a PKA phosphorylation site. Inhibition of PI hydrolysis induced by PKA was partly blocked in cells expressing GRK2(S685A) and completely blocked in cells coexpressing GRK2(S685A) and RGS4(S52A) or G_q(G188S), a G_q mutant that binds GRK2 but not RGS4. The results demonstrate that inhibition of PLC-1-dependent PI hydrolysis by PKA is mediated via stimulatory phosphorylation of RGS4 and GRK2, leading to rapid inactivation of G_q·GTP. PKG acts only via phosphorylation of RGS4. regulators of G protein signaling; G protein-coupled receptor kinase 2; phospholipase C; cAMP-dependent protein kinase; cGMP-dependent protein kinase     

Production of IL-1beta, hydrogen peroxide, and nitric oxide by colonic mucosa decreases sigmoid smooth muscle contractility in ulcerative colitis

We have previously shown that sigmoid circular muscle cells from patients with ulcerative colitis (UC) exhibit reduced contraction and Ca²⁺ signaling in response to the neurotransmitter neurokinin A (NKA) and that IL-1 and H₂O₂ may contribute to these reduced responses in UC. In addition, we have found that nitric oxide (NO) levels were significantly increased in UC circular muscle. To establish the site of origin for IL-1, H₂O₂, and NO, we assembled an in vitro system in which normal or UC mucosa were sealed between two chambers filled with oxygenated Krebs solution. Because the mucosa consists of full-thickness mucosa and submucosa, it is expected that whatever is released into the undernatant from the submucosal side may diffuse to the circular muscle layer in the intact colon. Treatment of normal sigmoid circular muscle cells for 2 h with undernatants collected from the UC submucosal side (UCS) significantly decreased contraction induced by NKA and thapsigargin and the NKA- and caffeine-induced Ca²⁺ signal in Ca²⁺-free medium. In addition, UC mucosa released into the undernatant on its submucosal side significantly more H₂O₂, IL-1, and NO than normal mucosa. The reduction in contraction and Ca²⁺ signal induced by UCS was partially reversed by pretreatment with an IL-1 antibody or with catalase. The NO scavenger hemoglobin partially prevented UCS-induced reduction in contraction and Ca²⁺ signaling in response to NKA but not the reduced response to thapsigargin or caffeine. Sodium nitroprusside inhibited NKA but not the caffeine-induced Ca²⁺ signal. We conclude that in UC the mucosa releases IL-1, H₂O₂, and NO, which may contribute to the impaired Ca²⁺ release and altered sigmoid muscle contractility. neurokinin A; calcium; human; colon     

Regulation of TRP channel TRPM2 by the tyrosine phosphatase PTPL1

TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca²⁺-permeable channel, which mediates susceptibility to cell death following activation by oxidative stress, TNF, or -amyloid peptide. We determined that TRPM2 is rapidly tyrosine phosphorylated after stimulation with H₂O₂ or TNF. Inhibition of tyrosine phosphorylation with the tyrosine kinase inhibitors genistein or PP2 significantly reduced the increase in [Ca²⁺]_i observed after H₂O₂ or TNF treatment in TRPM2-expressing cells, suggesting that phosphorylation is important in TRPM2 activation. Utilizing a TransSignal PDZ domain array blot to identify proteins which interact with TRPM2, we identified PTPL1 as a potential binding protein. PTPL1 is a widely expressed tyrosine phosphatase, which has a role in cell survival and tumorigenesis. Immunoprecipitation and glutathione-S-transferase pull-down assays confirmed that TRPM2 and PTPL1 interact. To examine the ability of PTPL1 to modulate phosphorylation or activation of TRPM2, PTPL1 was coexpressed with TRPM2 in human embryonic kidney-293T cells. This resulted in significantly reduced TRPM2 tyrosine phosphorylation, and inhibited the rise in [Ca²⁺]_i and the loss of cell viability, which follow H₂O₂ or TNF treatment. Consistent with these findings, reduction in endogenous PTPL1 expression with small interfering RNA resulted in increased TRPM2 tyrosine phosphorylation, a significantly greater rise in [Ca²⁺]_i following H₂O₂ treatment, and enhanced susceptibility to H₂O₂-induced cell death. Endogenous TRPM2 and PTPL1 was associated in U937-ecoR cells, confirming the physiological relevance of this interaction. These data demonstrate that tyrosine phosphorylation of TRPM2 is important in its activation and function and that inhibition of TRPM2 tyrosine phosphorylation reduces Ca²⁺ influx and protects cell viability. They also suggest that modulation of TRPM2 tyrosine phosphorylation is a mechanism through which PTPL1 may mediate resistance to cell death. transient receptor potential channels; oxidative stress     

ATP stimulation of Na+/Ca2+ exchange in cardiac sarcolemmal vesicles

In cardiacsarcolemmal vesicles, MgATP stimulatesNa⁺/Ca²⁺exchange with the following characteristics:1) increases 10-fold the apparentaffinity for cytosolic Ca²⁺;2) a Michaelis constant for ATP of~500 µM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory;4) not observed in the presence of20 µM eosin alone but reinstated when vanadate is added;5) mimicked by adenosine5'-O-(3-thiotriphosphate), without the need for vanadate, but not by ,-methyleneadenosine 5'-triphosphate; and 6) notaffected by unspecific protein alkaline phosphatase but abolished by aphosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLCeffect is counteracted by phosphatidylinositol. In addition, in theabsence of ATP,L--phosphatidylinositol4,5-bisphosphate (PIP₂) was ableto stimulate the exchanger activity in vesicles pretreated with PI-PLC.This MgATP stimulation is not related to phosphorylation of thecarrier, whereas phosphorylation appeared in the phosphoinositides,mainly PIP₂, thatcoimmunoprecipitate with the exchanger. Vesicles incubated with MgATPand no Ca²⁺ show a markedsynthesis ofL--phosphatidylinositol4-monophosphate (PIP) with little production ofPIP₂; in the presence of 1 µM Ca²⁺, the net synthesis of PIP issmaller, whereas that of PIP₂increases ninefold. These results indicate thatPIP₂ is involved in the MgATPstimulation of the cardiacNa⁺/Ca²⁺exchanger through a fast phosphorylation chain: aCa²⁺-independent PIP formationfollowed by a Ca²⁺-dependentsynthesis of PIP₂.

     

Ca(2+)-dependent activation of Cl(-) currents in Xenopus oocytes is modulated by voltage

Ca²⁺-activatedCl currents (I_Cl,Ca) wereexamined using fluorescence confocal microscopy to monitorintracellular Ca²⁺ liberation evoked by flash photolysis ofcaged inositol 1,4,5-trisphosphate (InsP₃) involtage-clamped Xenopus oocytes. Currents at +40 mV exhibited asteep dependence on InsP₃ concentration([InsP₃]), whereas currents at140 mV exhibited a higher threshold and more graded relationshipwith [InsP₃]. Ca²⁺ levelsrequired to half-maximally activate I_Cl,Ca wereabout 50% larger at 140 mV than at +40 mV, and currents evokedby small Ca²⁺ elevations were reduced >25-fold. Thehalf-decay time of Ca²⁺ signals shortened at increasinglypositive potentials, whereas the decay of I_Cl,Calengthened. The steady-state current-voltage (I-V) relationshipfor I_Cl,Ca exhibited outward rectification withweak photolysis flashes but became more linear with stronger stimuli.Instantaneous I-V relationships were linear with both strongand weak stimuli. Current relaxations following voltage steps duringactivation of I_Cl,Ca decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at 160 mV. We conclude that InsP₃-mediated Ca²⁺liberation activates a single population of Clchannels, which exhibit voltage-dependent Ca²⁺ activationand voltage-independent instantaneous conductance.

     

Ca2+]i regulates trafficking of Cav1.3 (alpha1D Ca2+ channel) in insulin-secreting cells

Chronic exposure of pancreatic -cells to high concentrations of glucose impairs the insulin secretory response to further glucose stimulation. This phenomenon is referred to as glucose desensitization. It has been shown that glucose desensitization is associated with abnormal elevation of -cell basal intracellular free Ca²⁺ concentration ([Ca²⁺]_i). We have investigated the relationship between the basal intracellular free Ca²⁺ and the L-type (Ca_v1.3) Ca²⁺ channel translocation in insulin-secreting cells. Glucose stimulation or membrane depolarization induced a nifedipine-sensitive Ca²⁺ influx, which was attenuated when the basal [Ca²⁺]_i was elevated. Using voltage-clamp techniques, we found that changing [Ca²⁺]_i could regulate the amplitude of the Ca²⁺ current. This effect was attenuated by drugs that interfere with the cytoskeleton. Immunofluorescent labeling of Ca_v1.3 showed an increase in the cytoplasmic distribution of the channels under high [Ca²⁺]_i conditions by deconvolution microscopy. The [Ca²⁺]_i-dependent translocation of Ca_v1.3 channel was also demonstrated by Western blot analysis of biotinylation/NeutrAvidin-bead-eluted surface proteins in cells preincubated at various [Ca²⁺]_i. These results suggest that Ca_v1.3 channel trafficking is involved in glucose desensitization of pancreatic -cells. internalization; intracellular free calcium; glucose desensitization     

Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC₂₀) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P₁ and S1P₂ receptors. S1P activated G_q, G₁₃, and all G_i isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or G_q antibody, PLC-1 or PLC-3 antibody, and by expression of G_q or G_i minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G₁₃ or G_q minigene and abolished by expression of both. S1P stimulated Ca²⁺ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC₅₀ 1 nM). Initial contraction and MLC₂₀ phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and G_q or G antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC₂₀ phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P₂ and S1P₁ involving concurrent activation of PLC-1 and PLC-3 via G_q and G_i, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca²⁺ release and MLCK-mediated MLC₂₀ phosphorylation, and 2) sustained contraction exclusively mediated by S1P₂ involving activation of RhoA via G_q and G₁₃, resulting in Rho kinase- and PKC-dependent MLC₂₀ phosphorylation. muscle contraction; signal transduction     

Deglycosylation of the beta1-subunit of the BK channel changes its biophysical properties

Large-conductance Ca²⁺-activated potassium (BK) channels are composed of pore-forming -subunits and auxiliary -subunits. The -subunits are widely expressed in many cell types, whereas the -subunits are more tissue specific and influence diverse aspects of channel function. In the current study, we identified the presence of the smooth muscle-specific 1-subunit in murine colonic tissue using Western blotting. The native 1-subunits migrated in SDS-PAGE as two molecular mass bands. Enzymatic removal of N-linked glycosylations from the 1-subunit resulted in a single band that migrated at a lower molecular mass than the native 1-subunit bands, suggesting that the native 1-subunit exists in either a core glycosylated or highly glycosylated form. We investigated the functional consequence of deglycosylating the 1-subunit during inside-out single-channel recordings. During inside-out single-channel recordings, with N-glycosidase F in the pipette solution, the open probability (P_o) and mean open time of BK channels increased in a time-dependent manner. Deglycosylation of BK channels did not affect the conductance but shifted the steady-state voltage of activation toward more positive potentials without affecting slope when Ca²⁺ concentration was <1 µM. Treatment of myocytes lacking the 1-subunits of the BK channel with N-glycosidase F had no effect. These data suggest that glycosylations on the 1-subunit in smooth muscle cells can modify the biophysical properties of BK channels. peptide N-glycosidase F; large-conductance Ca²⁺-activated K⁺ channels; N-linked glycosylation; single-channel recording; auxiliary subunit     

Permeant but not impermeant divalent cations enhance activation of nondesensitizing alpha(7) nicotinic receptors

Neuronal₇ nicotinic acetylcholine receptors (nAChRs) arepermeable to Ca²⁺ and other divalent cations. Wecharacterized the modulation of the pharmacological properties ofnondesensitizing mutant (L²⁴⁷T andS²⁴⁰T/L²⁴⁷T) ₇ nAChRs bypermeant (Ca²⁺, Ba²⁺, and Sr²⁺) andimpermeant (Cd²⁺ and Zn²⁺) divalent cations.₇ receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh,whereas impermeant divalent cations decreased potency and maximalefficacy. The antagonist dihydro--erythroidine (DHE) was a strongpartial agonist of L²⁴⁷T andS²⁴⁰T/L²⁴⁷T ₇ receptors in thepresence of divalent cations but was a weak partial agonist in thepresence of impermeant divalent cations. Mutation of the"intermediate ring" glutamates (E²³⁷A) inL²⁴⁷T ₇ nAChRs eliminated Ca²⁺conductance but did not alter the Ca²⁺-dependent increasein ACh potency, suggesting that site(s) required for modulation are onthe extracellular side of the intermediate ring. The difference betweenpermeant and impermeant divalent cations suggests that sites within thepore are important for modulation by divalent cations.

     

Isolated mouse pancreatic beta-cells show cell-specific temporal response pattern

The length of the silent lag time beforeelevation of the cytosolic free Ca²⁺ concentration([Ca²⁺]_i) differs between individualpancreatic -cells. One important question is whether thesedifferences reflect a random phenomenon or whether the length of lagtime is inherent in the individual -cell. We compared the lag times,initial dips, and initial peak heights for[Ca²⁺]_i from two consecutive glucosestimulations (with either 10 or 20 mM glucose) in individualob/ob mouse -cells with the fura 2 technique in amicrofluorimetric system. There was a strong correlation between thelengths of the lag times in each -cell (10 mM glucose:r = 0.94, P < 0.001; 20 mM glucose:r = 0.96, P < 0.001) as well as between theinitial dips in [Ca²⁺]_i (10 mM glucose:r = 0.93, P < 0.001; 20 mM glucose:r = 0.79, P < 0.001) and between theinitial peak heights (10 mM glucose: r = 0.51, P < 0.01; 20 mM glucose: r = 0.77, P < 0.001). These data provide evidence that theresponse pattern, including both the length of the lag time and thedynamics of the subsequent [Ca²⁺]_i, isspecific for the individual -cell.

     

Synergism between toxin-gamma from Brazilian scorpion Tityus serrulatus and veratridine in chromaffin cells

Toxin- (T)from the Brazilian scorpion Tityusserrulatus venom caused a concentration- andtime-dependent increase in the release of norepinephrine andepinephrine from bovine adrenal medullary chromaffin cells. T was~200-fold more potent than veratridine judged fromEC₅₀ values, although the maximalsecretory efficacy of veratridine was 10-fold greater than that of T(1.2 vs. 12 µg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca²⁺concentration([Ca²⁺]_o),when 30 µM veratridine plus 0.45 µM T were used. T (0.45 µM) doubled the basal uptake of⁴⁵Ca²⁺,whereas veratridine (100 µM) tripled it. Again, a drastic synergism in enhancing Ca²⁺ entry was seenwhen T and veratridine were combined; this was particularlypronounced at 5 mM[Ca²⁺]_o.Veratridine induced oscillations of cytosolicCa²⁺ concentration([Ca²⁺]_i)in single fura 2-loaded cells without elevation of basal levels. Incontrast, T elevated basal[Ca²⁺]_ilevels, causing only small oscillations. When added together, T andveratridine elevated the basal levels of[Ca²⁺]_iwithout causing large oscillations. T shifted the current-voltage (I-V) curve forNa⁺ channel current to the left.The combination of T with veratridine increased the shift of theI-V curve to the left, resulting in agreater recruitment of Na⁺channels at more hyperpolarizing potentials. This led to enhanced andmore rapid accumulation of Na⁺ inthe cell, causing cell depolarization, the opening of voltage-dependent Ca²⁺ channels, andCa²⁺ entry and secretion.

     

Calcium dependence of C-type natriuretic peptide-formed fast K+ channel

The lipid bilayertechnique was used to characterize theCa²⁺ dependence of a fastK⁺ channel formed by a synthetic17-amino acid segment [OaCNP-39-(1-17)] ofa 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchusanatinus) venom (OaV). TheOaCNP-39-(1-17)-formed K⁺ channel was reversiblydependent on1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic)Ca²⁺ concentration([Ca²⁺]_cis).The channel was fully active when[Ca²⁺]_ciswas >10⁴ M andtrans (luminal)Ca²⁺ concentration was 1.0 mM, butnot at low[Ca²⁺]_cis.The open probability of single channels increased from zero at1 × 10⁶ McisCa²⁺ to 0.73 ± 0.17 (n = 22) at10³ McisCa²⁺. Channel openings to themaximum conductance of 38 pS were rapidly and reversibly activated when[Ca²⁺]_cis,but not transCa²⁺ concentration(n = 5), was increased to >5 × 10⁴ M(n = 14). Channel openings to thesubmaximal conductance of 10.5 pS were dominant at5 × 10⁴ MCa²⁺.K⁺ channels did not open whencisMg²⁺ orSr²⁺ concentrations were increasedfrom zero to 10³ M or when[Ca²⁺]_ciswas maintained at 10⁶ M(n = 3 and 2). The Hill coefficientand the inhibition constant were 1 and 0.8 × 10⁴ McisCa²⁺, respectively. Thisdependence of the channel on high[Ca²⁺]_cissuggests that it may become active under1) physiological conditions whereCa²⁺ levels are high, e.g., duringcardiac and skeletal muscle contractions, and2) pathological conditions that leadto a Ca²⁺ overload, e.g., ischemicheart and muscle fatigue. The channel could modify a cascade ofphysiological functions that are dependent on theCa²⁺-activatedK⁺ channels, e.g., vasodilationand salt secretion.

     

Involvement of G protein betagamma-subunits in diverse signaling induced by G(i/o)-coupled receptors: study using the Xenopus oocyte expression system

We studied the functions of -subunits of G_i/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl^– currents in oocytes expressing ₂-adrenoceptor and the protein kinase A-dependent Cl^– channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala², D-Leu⁵]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing ₂-adrenoceptor-CFTR and 5-HT_1A receptor (5-HT_1AR), -opioid receptor, or GABA_B receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT_1AR. The 5-HT-induced enhancement of G_s-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (G_t). The 5-HT-induced enhancement was further augmented by coexpression of the G-activated form of adenylate cyclase (AC) type II but not AC type III. Thus -subunits of G_i/o protein contribute to the enhancement of G_s-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT_1AR or -opioid receptor alone. They elicited Ca²⁺-activated Cl^– currents in oocytes coexpressing these receptors with the G-activated form of phospholipase C (PLC)-2 but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of G_t, suggesting that -subunits of G_i/o protein activate PLC-2 and then cause intracellular Ca²⁺ mobilization. Our results indicate that -subunits of G_i/o protein participate in diverse intracellular signals, enhancement of G_s-coupled receptor-mediated responses, and intracellular Ca²⁺ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology     

Inflammatory cytokines (IL-1alpha , TNF-alpha ) and LPS modulate the Ca2+ signaling pathway in osteoblasts

Locally derived growth factors and cytokines in bone play acrucial role in the regulation of bone remodeling, i.e., bone formationand bone resorption processes. We studied the effect of interleukin(IL)-1, tumor necrosis factor (TNF)-, andEscherichia coli lipopolysaccharide(LPS) on the hormone-activatedCa²⁺ message system in theosteoblastic cell line UMR-106 and in osteoblastic cultures derivedfrom neonatal rat calvariae. In both cell preparations, IL-1,TNF-, and LPS did not alter basal intracellularCa²⁺ concentration([Ca²⁺]_i)but attenuated Ca²⁺ transientsevoked by parathyroid hormone (PTH) andPGE₂ in a dose (1-100 ng/ml)-and time (8-24 h)-dependent fashion. The cytokines modulatedhormonally induced Ca²⁺ influx(estimated by using Mn²⁺ as asurrogate for Ca²⁺) as well asCa²⁺ mobilization fromintracellular stores. The latter was linked to suppressed production ofhormonally induced inositol 1,4,5-trisphosphate. The effect ofcytokines on[Ca²⁺]_iwas abolished by the tyrosine kinase inhibitor herbimycin A (50 ng/ml).The cytokine's effect was, however, independent of nitric oxide (NO)production, since NO donors (sodium nitroprusside) as well as permeablecGMP analogs augment, rather than attenuate, hormonally inducedCa²⁺ transients in osteoblasts.Given the stimulatory role of cytokines on NO production inosteoblasts, the disparate effects of cytokines and NO on theCa²⁺ signaling pathway may servean autocrine/paracrine mechanism for modulating the effect ofcalciotropic hormones on bone metabolism.

     

Autocrine production of prostaglandin F2alpha enhances phenotypic transformation of normal rat kidney fibroblasts

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor- (TGF-). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around –70 to –20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca²⁺ levels and thereby activates Ca²⁺-dependent Cl^– channels. This compound was identified as prostaglandin F₂ (PGF₂) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 ± 2.5 nM (n = 6), compared with 1.5 ± 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF₂ was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF₂) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (25%) suppressed by AL-8810. Our results demonstrate that PGF₂ acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general. membrane potential; intracellular calcium; mass spectrometry; FP receptor     

Involvement of stretch-activated cation channels in hypotonically induced insulin secretion in rat pancreatic beta-cells

In isolated rat pancreatic -cells, hypotonic stimulation elicited an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_c) at 2.8 mM glucose. The hypotonically induced [Ca²⁺]_c elevation was significantly suppressed by nicardipine, a voltage-dependent Ca²⁺ channel blocker, and by Gd³⁺, amiloride, 2-aminoethoxydiphenylborate, and ruthenium red, all cation channel blockers. In contrast, the [Ca²⁺]_c elevation was not inhibited by suramin, a P₂ purinoceptor antagonist. Whole cell patch-clamp analyses showed that hypotonic stimulation induced membrane depolarization of -cells and produced outwardly rectifying cation currents; Gd³⁺ inhibited both responses. Hypotonic stimulation also increased insulin secretion from isolated rat islets, and Gd³⁺ significantly suppressed this secretion. Together, these results suggest that osmotic cell swelling activates cation channels in rat pancreatic -cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca²⁺ channels and thus elevating insulin secretion. calcium ion; swelling; patch-clamp; gadolinium     

Dual effect of nitric oxide on cytosolic Ca2+ concentration and insulin secretion in rat pancreatic beta-cells

Inisolated rat pancreatic -cells, the nitric oxide (NO) donor NOC-7 at1 µM reduced the amplitude of the oscillations of cytosolicCa²⁺ concentration ([Ca²⁺]_c)induced by 11.1 mM glucose, and at 10 µM terminated them. In thepresence of N^G-nitro-L-arginine(L-NNA), however, NOC-7 at 0.5 and 1 µM increased theamplitude of the [Ca²⁺]_c oscillations,although the NO donor at 10 µM still suppressed them. Aqueous NOsolution also had a dual effect on the[Ca²⁺]_c oscillations. The soluble guanylatecyclase inhibitor LY-83583 and the cGMP-dependent protein kinaseinhibitor KT5823 inhibited the stimulatory effect of NO, and8-bromo-cGMP increased the amplitude of the[Ca²⁺]_c oscillations. Patch-clamp analyses inthe perforated configuration showed that 8-bromo-cGMP inhibited wholecell ATP-sensitive K⁺ currents in the isolated ratpancreatic -cells, suggesting that the inhibition by cGMP ofATP-sensitive K⁺ channels is, at least in part, responsiblefor the stimulatory effect of NO on the[Ca²⁺]_c oscillations. In the presence ofL-NNA, the glucose-induced insulin secretion from isolatedislets was facilitated by 0.5 µM NOC-7, whereas it was suppressed by10 µM NOC-7. These results suggest that NO facilitatesglucose-induced [Ca²⁺]_c oscillations of-cells and insulin secretion at low concentrations, which effectsare mediated by cGMP, whereas NO inhibits them in a cGMP-independentmanner at high concentrations.

     

Mitochondrial calcium uptake stimulates nitric oxide production in mitochondria of bovine vascular endothelial cells

Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]_mt). Stimulation of mitochondrial Ca²⁺ uptake by exposure to different cytoplasmic Ca²⁺ concentrations (1, 2, and 5 µM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]_mt was sensitive to the NOS antagonist L-N⁵-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca²⁺ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl--cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca²⁺ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis. nitric oxide; nitric oxide synthase; calcium; endothelium; mitochondria