Thyrotropin-releasing hormone rapidly activates the phosphodiester hydrolysis of polyphosphoinositides in GH3 pituitary cells. Evidence for the role of a polyphosphoinositide-specific phospholipase C in hormone action

The early actions of thyrotropin-releasing hormone (TRH) have been studied in hormone-responsive clonal GH3 rat pituitary cells. Previous studies had demonstrated that TRH promotes a "phosphatidylinositol response" in which increased incorporation of [32P]orthophosphate into phosphatidylinositol and phosphatidic acid was observed within minutes of hormone addition. The studies described here were designed to establish whether increased labeling of phosphatidylinositol and phosphatidic acid resulted from prior hormone-induced breakdown of an inositol phosphatide. GH3 cells were prelabeled with [32P]orthophosphate or myo-[3H]inositol. Addition of TRH resulted in the rapid disappearance of labeled polyphosphoinositides, whereas levels of phosphatidylinositol and other phospholipids remained unchanged. TRH-promoted polyphosphoinositide breakdown was evident by 5 S and maximal by 15 s of hormone treatment. Concomitant appearance of inositol polyphosphates in [3H]inositol-labeled cells was observed. In addition, TRH rapidly stimulated diacylglycerol accumulation in either [3H]arachidonic- or [3H]oleic acid-labeled cultures. These results indicate that TRH rapidly causes activation of a polyphosphoinositide-hydrolyzing phospholipase C-type enzyme. The short latency of this hormone effect suggests a proximal role for polyphosphoinositide breakdown in the sequence of events by which TRH alters pituitary cell function.     

Thyroliberin stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phosphodiesterase in rat mammotropic pituitary cells. Evidence for an early Ca2+-independent action.

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4'-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4'-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.     

Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes

Accelerated phospholipid catabolism occurs early after the onsetof myocardial ischemia and is likely to be mediated by the activation of one or more phospholipases in ischemic tissue. We hypothesized that hypoxia increases phospholipaseA₂(PLA₂) activity in isolatedventricular myocytes, resulting in increased lysophospholipid andarachidonic acid production, contributing to arrhythmogenesis inischemic heart disease. The majority of ventricular myocyte arachidonicacid was found in plasmalogen phospholipids. Hypoxia increasedmembrane-associated,Ca²⁺-independent,plasmalogen-selective PLA₂activity, resulting in increased arachidonic acid release andlysoplasmenylcholine production. Pretreatment with the specificCa²⁺-independentPLA₂ inhibitor bromoenol lactoneblocked hypoxia-induced increases inPLA₂ activity, arachidonic acidrelease, and lysoplasmenylcholine production. Lysoplasmenylcholineproduced action potential derangements, including shortening of actionpotential duration, and induced early and delayed afterdepolarizationsin normoxic myocytes. The electrophysiological alterations induced bylysoplasmenylcholine would likely contribute to the initiation ofarrhythmogenesis in the ischemic heart.

     

Gonadotropin-releasing hormone receptor binding in bovine anterior pituitary

Synthetic gonadotropin-releasing hormone (GnRH) was monoiodinated at a high specific radioactivity with 125I. The iodinated hormone retained full biological activity as assessed by the release of luteinizing hormone in vitro from bovine anterior pituitary tissue slices. Specific binding of 125I-labeled gonadotropin-releasing hormone of high affinity and low capacity was obtained using dispersed bovine anterior pituitary cells. The binding had sigmoid characteristics, compatible with the presence of more than one binding site. The subcellular fraction responsible for binding was identified with the plasma membranes. However, significant binding also occurred in the secretory granules fraction. The plasma membranes were solubilized with sodium dodecyl sulfate. Using gonadotropin-releasing hormone covalently coupled to a solid phase, a protein was purified by an affinity technique from the solubilized plasma membrane preparation which possessed similar binding propperties as plasma membranes, both intact and solubilized. The protein migrated as a single component on polyacrylamide gel in sodium dodecyl sulfate and the estimated molecular weight was 60 000. The character of the gonadotropin-releasing hormone concentration dependence binding as well as association kinetics were multiphasic and suggested the presence of more than one binding site. When analyzed by the Hill plot, the Hill coefficient of all binding curves was always greater than one which is compatible with positive cooperativity. This was further supported by the dissociation studies where the dissociation rate was inversely proportionate to both the gonadotropin-releasing hormone concentration and the time interval during which the gonadotropin-releasing hormone-gonadotropin-releasing hormone receptor protein complex was formed. Using difference chromatography, aggregation of the purified gonadotropin-releasing hormone receptor protein was demonstrated to occur upon its exposure to gonadotropin-releasing hormone. The formed macromolecular complexes bound preferentially 125I-labeled gonadotropin-releasing hormone. It is concluded that a single receptor protein is responsible for gonadotropin-releasing hormone binding in the bovine anterior pituitary. It is a part of the plasma membranes. Its interaction with gonadotropin-releasing hormone provokes transitions of the protein into different allosteric forms and this may be related to the biological effect of gonadotropin-releasing hormone on gonadotropin secretion.     

Ca2+-independent phospholipase A2 is a novel determinant of store-operated Ca2+ entry

Store-operated cation (SOC) channels and capacitative Ca(2+) entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca(2+) signaling. Here, we present the first evidence that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is a crucial molecular determinant in activation of SOC channels and store-operated Ca(2+) entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA(2) leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca(2+)-release-activated Ca(2+) (CRAC) channels. Transfection of vascular smooth muscle cells (SMC) with antisense, but not sense, oligonucleotides for iPLA(2) impaired thapsigargin (TG)-induced activation of iPLA(2) and TG-induced Ca(2+) and Mn(2+) influx. Identical inhibition of TG-induced Ca(2+) and Mn(2+) influx (but not Ca(2+) release) was observed in SMC, human platelets, and Jurkat T-lymphocytes when functional activity of iPLA(2) was inhibited by its mechanism-based suicidal substrate, bromoenol lactone (BEL). Moreover, irreversible inhibition of iPLA(2) impaired TG-induced activation of single nonselective SOC channels in SMC and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-induced activation of whole-cell CRAC current in rat basophilic leukemia cells. Thus, functional iPLA(2) is required for activation of store-operated channels and capacitative Ca(2+) influx in wide variety of cell types.     

Hypoxic vasorelaxation: Ca2+-dependent and Ca2+-independent mechanisms

The mechanisms of oxygen sensing in vascular smooth muscle have been studied extensively in a variety of tissue types and the results of these studies indicate that the mechanism of hypoxia-induced vasodilation probably involves several mechanisms that combined to assure the appropriate response. After a short discussion of the regulatory mechanisms for smooth muscle contractility, we present the evidence indicating that hypoxic vasorelaxation involves both Ca2+-dependent and Ca2+-independent mechanisms. More recent experiments using proteomic approaches in organ cultures of porcine coronary artery reveal important changes evoked by hypoxia in both Ca2+-dependent and Ca2+-independent pathways.     

Ca2+依赖和Ca2+不依赖的囊泡分泌研究进展

Ca2 是促发囊泡胞吐的关键调节因子.最近的研究表明,分泌囊泡和通道之间的空间距离调节囊泡分泌的过程和性质.Ca2 通道开口附近形成的Ca2 微区和Ca2 钠区和囊泡快速递质释放有非常紧密的联系.SNARE蛋白和钙离子传感器synaptotagmins等在触发分泌中起调控作用.同时另有一类不依赖于Ca2 的囊泡分泌存在.Latrotoxin和mastoparan等可以激活这一类不依赖于Ca2 的信号通路,从而触发囊泡释放.本文主要从ca2 对囊泡胞吐的调控作用着手,综述了Ca2 依赖和Ca2 不依赖的囊泡分泌过程和可能的调控机制.     

Ca2+-dependent and Ca2+-independent somatic release from trigeminal neurons

Besides the nerve endings, the soma of trigeminal neurons also respond to membrane depolarizations with the release of neurotransmitters and neuromodulators in the extracellular space within the ganglion, a process potentially important for the cross-communication between neighboring sensory neurons. In this study, we addressed the dependence of somatic release on Ca²⁺ influx in trigeminal neurons and the involvement of the different types of voltage-gated Ca²⁺ (Cav) channels in the process. Similar to the closely related dorsal root ganglion neurons, we found two kinetically distinct components of somatic release, a faster component stimulated by voltage but independent of the Ca²⁺ influx, and a slower component triggered by Ca²⁺ influx. The Ca²⁺-dependent component was inhibited 80% by ω-conotoxin-MVIIC, an inhibitor of both N- and P/Q-type Cav channels, and 55% by the P/Q-type selective inhibitor ω-agatoxin-IVA. The selective L-type Ca²⁺ channel inhibitor nimodipine was instead without effect. These results suggest a major involvement of N- and P/Q-, but not L-type Cav channels in the somatic release of trigeminal neurons. Thus antinociceptive Cav channel antagonists acting on the N- and P/Q-type channels may exert their function by also modulating the somatic release and cross-communication between sensory neurons.     

Ca2+-dependent and Ca2+-independent mechanisms for phosphatidylinositol hydrolysis and 32P-labeling during cholinergic stimulation of the rat submaxillary gland in vitro

Ca²⁺ was required for carbachol-induced decreases in phosphatidylinositol (PI) and increases in phosphatidic acid (PA) concentrations during incubation of rat submaxillary gland fragments, but was not required for increases in [³²P]P_i incorporation into these phospholipids. Like carbachol, A23187 provoked a Ca²⁺-dependent decrease in PI mass. These results suggest concomitant operation of two separate mechanisms for stimulating PI hydrolysis and ³²P labeling of PA and PI during carbachol action: one mechanism is not dependent on external Ca²⁺ and is manifested by rapid labeling in a relatively small PA-PI pool; the other mechanism is dependent on Ca²⁺ and involves a large PA-PI pool which appears to have a relatively slow renewal (labeling) rate.     

Ca2+-stimulated,Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m  0.25 μM, V_max  24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.     

Microautophagic vacuole invagination requires calmodulin in a Ca2+-independent function

Microautophagy is the uptake of cytosolic compounds by direct invagination of the vacuolar/lysosomal membrane. In Saccharomyces cerevisiae microautophagic uptake of soluble cytosolic proteins occurs via an autophagic tube, a highly specialized vacuolar membrane invagination. Autophagic tubes are topologically equivalent to the invaginations at multivesicular endosomes. At the tip of an autophagic tube, vesicles (autophagic bodies) pinch off into the vacuolar lumen for degradation. In this study we have identified calmodulin (Cmd1p) as necessary for microautophagy. Temperature-sensitive mutants for Cmd1p displayed reduced frequencies of vacuolar tube formation and/or abnormal tube morphologies. Microautophagic vacuole invagination was sensitive to Cmd1p antagonists as well as to antibodies to Cmd1p. cmd1 mutants with substitutions in the Ca2+-binding domains showed full invagination activity, and vacuolar membrane invagination was independent of the free Ca2+ concentration. Thus, rather than acting as a calcium-triggered switch, Cmd1p has a constitutive Ca2+-independent role in the formation of autophagic tubes. Kinetic analysis indicates that calmodulin is required for autophagic tube formation rather than for the final scission of vesicles from the tip of the tube.     

Ca2+-independent contraction of uterine smooth muscle

Rat uterine smooth muscle shows sustained contraction to oxytocin in Ca2+-free medium with EGTA, that is called "Ca-free contraction"(1). Participation of the rise in cytosolic free Ca2+ in this Ca-free contraction was tested. In Ca-free contraction, the cytosolic free Ca2+ level was not changed at all as measured with fura-2. Further, the chelation of cytosolic free Ca2+ with quin-2 did not at all affect Ca-free contraction. These results strongly suggest that Ca-free contraction is not triggered by Ca2+.     

Evidence for a Ca2+-independent hydrolysis of phosphatidylinositol 4,5-bisphosphate in neuron-like cell line NG108-15 cells

The addition of bradykinin to 32Pi-labeled neuroblastoma X glioma hybrid NG108-15 cells caused a substantial loss of radioactivity from phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2). The bradykinin-induced hydrolysis of PI-4,5-P2 was almost equally observed even when extracellular Ca2+ was depleted with EGTA (100 microns). On the other hand, high K+ depolarization of the cells, which allows Ca2+ influx through voltage-dependent Ca2+ channels, failed to induce any significant decrease in the radioactivity of PI-4,5-P2. These data indicate that the bradykinin-stimulated PI-4,5-P2 hydrolysis in NG108-15 cells is independent of extracellular Ca2+ and also that PI-4,5-P2 hydrolysis is not stimulated by an elevation of intracellular Ca2+ concentration.     

PKA induces Ca2+ release and enhances ciliary beat frequency in a Ca2+-dependent and -independent manner

The intent of this work was to evaluate the role of cAMP inregulation of ciliary activity in frog mucociliary epithelium and toexamine the possibility of cross talk between the cAMP- andCa²⁺-dependent pathways in thatregulation. Forskolin and dibutyryl cAMP induced strong transientintracellular Ca²⁺ concentration([Ca²⁺]_i)elevation and strong ciliary beat frequency enhancement with prolongedstabilization at an elevated plateau. The response was not affected byreduction of extracellular Ca²⁺concentration. The elevation in[Ca²⁺]_iwas canceled by pretreatment with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, thapsigargin, and a phospholipase C inhibitor, U-73122. Underthose experimental conditions, forskolin raised the beat frequency to amoderately elevated plateau, whereas the initial strong rise infrequency was completely abolished. All effects were canceled by H-89,a selective protein kinase A (PKA) inhibitor. The results suggest adual role for PKA in ciliary regulation. PKA releasesCa²⁺ from intracellular stores,strongly activating ciliary beating, and, concurrently, producesmoderate prolonged enhancement of the beat frequency by aCa²⁺-independent mechanism.

     

Thyrotropin-releasing hormone activates Na+/H+ exchange in rat pituitary cells.

The effects of thyrotropin-releasing hormone (TRH) and 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytosolic pH (pHi) were studied on GH4C1 pituitary cells loaded with the fluorescent pH indicator bis(carboxyethyl)carboxyfluorescein (BCECF) and the fluorescent Ca2+ indicator quin2. TRH, which was minimally effective at around 10(-9) M, and TPA, 100 nM, produced very small elevations in pHi of about 0.05 pH units from the normal basal resting pHi of GH4C1 cells of around 7.05. The effects were more marked after acid-loading the cells using 1 micrograms of nigericin/ml. Preincubation with amiloride or replacing the extracellular Na+ with choline+ completely blocked the elevations stimulated by TRH or TPA, consistent with an activation of the Na+/H+ antiport mechanism. The effects were completely independent of the cytoplasmic free calcium concentration ([Ca2+]i). The calcium ionophore ionomycin produced an elevation in [Ca2+]i with no concomitant effect on pHi, and amiloride, although completely inhibiting the pH change stimulated by TRH, failed to affect the initial stimulated [Ca2+]i transient. Although the data are consistent with an elevation in pHi by TRH which is caused by stimulation of a protein kinase C and subsequent activation of the antiporter, the rapidity of the onset of the pHi response to TRH could not be mimicked by a combination of TPA and ionomycin. These results, together with previous findings which show that secretion can be mimicked by TPA and ionomycin, suggest that TRH-stimulated Na+/H+ exchange plays no part in the acute stimulation of secretion, but that TRH increases the pH-sensitivity of the antiport system during increased synthesis of prolactin and growth hormone.     

Arachidonic acid activates Ca2+ extrusion in macrophages

Stimulation of macrophages with platelet-activating factor (PAF) elicits an increase of intracellular calcium concentration, Ca2+i, which was monitored here at the single cell level with the calcium-sensitive dye Fura-2. The sustained component of this Ca2+i increase reflects the dynamic balance achieved between enhanced Ca2+ influx and efflux. In macrophages where a steady increase of Ca2+i has been evoked by 50 nM thapsigargin (a molecule known to empty Ca2+ stores and elevate Ca2+i in various cell types), PAF activates Ca2+ efflux, without causing a preceding increase in Ca2+i. This result shows that in this case, Ca2+ extrusion is not merely a consequence of a Ca2+i increase. PAF-evoked Ca2+ extrusion does not result from the activation of the Na+/Ca2+ exchanger. Exogenous arachidonic acid (10-100 microM) elicits Ca2+ efflux in macrophages where Ca2+i has been previously elevated by either PAF or thapsigargin. PAF-induced Ca2+ extrusion is blocked by 4-bromophenacylbromide, an inhibitor of arachidonic acid production by phospholipase A2. Together, these results suggest that arachidonic acid, which is produced in PAF-stimulated macrophages, contributes to the regulation of a Ca2+ extrusion system, which is presumably a Ca2(+)-ATPase.     

Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts

Thus far, determining the relative contribution of Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) and Ca²⁺-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca²⁺, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO₄/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO₄/mol RLC, respectively, within 2.5 min. Ca²⁺ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells. myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation