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2',3'-O-(N-Methylanthraniloyl)-ITP (MANT-ITP) is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, K(i), 1 nM) yet discovered and surpasses the potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329:1156-1165, 2009). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP. MANT-ITP was a considerably more potent inhibitor of the purified catalytic domains VC1 and IIC2 of mAC than MANT-GTP (K(i), 0.7 versus 18 nM). Moreover, there was considerably more efficient fluorescence resonance energy transfer between Trp1020 of IIC2 and the MANT group of MANT-ITP compared with MANT-GTP, indicating optimal interaction of the MANT group of MANT-ITP with the hydrophobic pocket. The crystal structure of MANT-ITP in complex with the G(s)α- and forskolin-activated catalytic domains VC1:IIC2 compared with the existing MANT-GTP crystal structure revealed only subtle differences in binding mode. The higher affinity of MANT-ITP to mAC compared with MANT-GTP is probably due to fewer stereochemical constraints upon the nucleotide base in the purine binding pocket, allowing a stronger interaction with the hydrophobic regions of IIC2 domain, as assessed by fluorescence spectroscopy. Stronger interaction is also achieved in the phosphate-binding site. The triphosphate group of MANT-ITP exhibits better metal coordination than the triphosphate group of MANT-GTP, as confirmed by molecular dynamics simulations. Collectively, the subtle differences in ligand structure have profound effects on affinity for mAC.

Membranous adenylyl cyclases play a major role in G-protein-coupled receptor signalling and regulate various cellular responses, such as cardiac contraction. Cardiac apoptosis and development of cardiac dysfunction is prevented in mice lacking AC 5, a predominant isoform in the heart. In the search for a potent and selective AC 5 inhibitor, we recently identified 2'(3')-methylanthraniloyl-inosine-5'-triphosphate(MANT-ITP) as the most potent AC 5 inhibitor with a K ( i ) of 13 nM. Therefore, AC inhibition of MANT-ITP was assessed in ventricular cardiomyocytes and compared to three other MANT-nucleotides to evaluate its effect on cardiac signalling. Basal and isoproterenol-induced L-type calcium currents (I (Ca,L)) in murine ventricular cardiomyocytes were recorded by whole-cell patch-clamp technique, using four different MANT-nucleotides. The effects of the MANT-nucleotides on I (Ca,L) were unexpectedly complex. All MANT-nucleotides exhibited an inhibitory effect on basal I (Ca,L). Additionally, several MANT-nucleotides, i.e., MANT-ITPγS, MANT-ATP, and MANT-ITP, caused a strong initial increase in basal I (Ca,L) within the first 2.5 min that appeared to be unrelated to AC 5 inhibition. However, we detected a significant reduction on isoproterenol-induced I (Ca,L) with MANT-ITP, supporting the notion that AC 5 plays an important role in agonist-stimulated activation of I (Ca,L). Collectively, MANT-nucleotides are useful tools for the characterization of recombinant ACs, for fluorescence studies and crystallography, but in intact cardiomyocytes, caution must be exerted since MANT-nucleotides apparently possess additional effects than AC 5 inhibition, limiting their usefulness as tools for intact cell studies.

Background & Aims: Genetic variation of inosine triphosphatase (ITPA) causing an accumulation of inosine triphosphate (ITP) has been shown to protect patients against ribavirin (RBV)-induced anemia during treatment for chronic hepatitis C infection by genome-wide association study (GWAS). However, the biologic mechanism by which this occurs is unknown., Methods: We examined whether ITP can be used by adenosine triphosphatase (ATPase) in human erythrocytes or recombinant human adenylosuccinate synthase (ADSS). RBV-induced adenosine triphosphate (ATP) reduction in erythrocytes was compared with the genetically determined low or normal activity of ITPA, leading respectively to high or normal ITP levels., Results: Although ITP is not used directly by human erythrocyte ATPase, it can be used for ATP biosynthesis via ADSS in place of guanosine triphosphate (GTP). With RBV challenge, erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype. This difference also remains after inhibiting adenosine uptake using nitrobenzylmercaptopurine riboside (NBMPR). Interestingly, the alleviation of ATP reduction by the hemolysis protective ITPA genotype was canceled by the ADSS inhibitor 6-mercaptoethanol (6-MP)., Conclusions: ITP confers protection against RBV-induced ATP reduction by substituting for erythrocyte GTP, which is depleted by RBV, in the biosynthesis of ATP. Because patients with excess ITP appear largely protected against anemia, these results confirm that RBV-induced anemia is due primarily to the effect of the drug on GTP and consequently ATP levels in erythrocytes., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Etomidate minimally influences hemodynamics at a standard induction dose in young healthy patients, but can cause significant systemic hypotension at higher doses for induction or electroencephalographic burst suppression (i.e., cerebral protection) in patients with advanced age or heart disease, and during cardiopulmonary bypass. However, less is known about its action on systemic resistance arteries., Methods: Using an isometric force recording method and fura-2-fluorometry, we investigated the action of etomidate on vascular reactivity in small mesenteric arteries from young (7-8 wk old, n = 179) and aged (96-98 wk old, n = 10) rats., Results: In the endothelium-intact strips from young rats, etomidate enhanced the contractile response to norepinephrine or KCl (40 mM) at 3 microM but inhibited it at higher concentrations (>or=10 microM). The enhancement was still observed after treatment with N(G)-nitro l-arginine, tetraethylammonium, diclofenac, nordihydroguaiaretic acid, losartan, ketanserin, BQ-123, or BQ-788, but was not observed in aged rats. In the endothelium-denuded strips from young rats, etomidate (>or=10 microM) consistently inhibited the contractile response to norepinephrine or KCl without enhancement at 3 microM. In the fura-2-loaded, endothelium-denuded strips from young rats, etomidate inhibited norepinephrine- or KCl-induced increases in both intracellular Ca(2+) concentration ([Ca(2+)]i) and force. Etomidate still inhibited the norepinephrine-induced increase in [Ca(2+)]i after depletion of the intracellular Ca(2+) stores by ryanodine, which was sensitive to nifedipine. Etomidate had little effect on norepinephrine- or caffeine-induced Ca(2+) release from the intracellular stores or Ca(2+) uptake into the intracellular stores. During stimulation with norepinephrine or KCl, etomidate had little effect on the [Ca(2+)]i-force relation at low concentrations (

In this study, we found that deoxyinosine triphosphate (dITP) could inhibit polymerase chain reaction (PCR) amplification of various family B-type DNA polymerases, and 0.93% dITP was spontaneously generated from deoxyadenosine triphosphate during PCR amplification. Thus, it was hypothesized that the generated dITP might have negative effect on PCR amplification of family B-type DNA polymerases. To overcome the inhibitory effect of dITP during PCR amplification, a dITP pyrophosphatase (dITPase) from Thermococcus onnurineus NA1 was applied to PCR amplification. Genomic analysis of the hyperthermophilic archaeon T. onnurineus NA1 revealed the presence of a 555-bp open reading frame with 48% similarity to HAM1-like dITPase from Methanocaldococcus jannaschii DSM2661 (NP_247195). The dITPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dITP, not deoxyuridine triphosphate. Addition of the purified protein to PCR reactions using DNA polymerases from T. onnurineus NA1 and Pyrococcus furiosus significantly increased product yield, overcoming the inhibitory effect of dITP. This study shows the first representation that removing dITP using a dITPase enhances the PCR amplification yield of family B-type DNA polymerase.

Aim: The aim of the present study was to explore the mechanism for the Ca2+- dependent inactivation of the canonical transient receptor potential (TRPC) 7 channel expressed in human embryonic kidney 293 cells., Method: The whole-cell patch-clamp technique was used in the study., Results: With Ca2+-free external solution, the perfusion of 100 micromol/L carbachol to, or dialysis of the cell with 100 micromol/L guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), induced large inward currents, respectively. These currents were rapidly inhibited by the addition of 1 mmol/L Ca2+ into the bath, and recovery from this inhibition was only partial after the Ca2+ removal, unless vigorous intracellular Ca2+ buffering with 10 mmol/L 1,2 bis(2- aminophenoxy)ethane-N,N,No,No-tetraacetic acid (BAPTA) (plus 4 mmol/L Ca2+) was employed. In contrast, the current induced by a membrane-permeable analog of diacylglycerol (DAG), 1-oleoyl-2-acetyl-sn-glycerol (OAG; 100 micromol/L) did not undergo the inhibition persisting after Ca2+ removal. Interestingly, the inclusion of inositol 1,4,5 trisphosphate (IP3; 100 micromol/L) in the patch pipette rendered the OAG-induced current susceptible to the persistent Ca2+-mediated inhibition independent of the IP3 receptor in the majority of the tested cells, as evidenced by the inability of heparin and thapsigargin in reversing the effect of IP3., Conclusion: The present results suggest that Ca2+ entry via the activated TRPC7 channel plays a critical role in inactivating the channel where the cooperative actions of DAG and IP3 are essentially involved.

Pretreatment of anaesthetized guinea-pigs with either CHF 4226.01 (8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(p-methoxyphenyl)-1-methylethyl]amino]ethyl] carbostyril hydrochloride), formoterol or budesonide reduced acetaldehyde (AcCHO)-evoked responses in the lungs with a rank order of potency CHF 4226.01 (ED(50) values, from 1.88 to 3.31 pmol) > formoterol (ED(50) values, from 3.03 to 5.51 pmol) >> budesonide (ED(50) values, from 335 to 458 nmol). The duration of action of CHF 4226.01 in antagonizing the airway obstruction elicited by AcCHO was also substantially longer than formoterol (area under the curve) at 10 pmol, 763+/-58 and 480+/-34, respectively; P<0.01). Continuous infusion of a subthreshold dose of AcCHO enhanced the intratracheal pressure (ITP) increases caused by subsequent challenges with substance P (from 9.7+/-0.8 to 27.5+/-1.6 cm H(2)O as a peak, P<0.001). Pretreatment with either CHF 4226.01 or formoterol prevented the sensitizing effect of AcCHO on substance P responses (ED(50) values, 2.85 and 6.11 pmol, respectively; P<0.01). The ED(50) value of budesonide (396 nmol) in preventing AcCHO-evoked ITP increase was reduced when this glucocorticoid was combined with 0.1 pmol CHF 4226.01 (ED(50) 76 nmol; P<0.001). CHF 4226.01/budesonide was two-fold more effective (P<0.01) than the formoterol/budesonide combination. These results suggest that CHF 4226.01/budesonide, by optimizing each other's beneficial potential in the control of pulmonary changes caused by AcCHO in the guinea-pigs, may represent a new fixed combination in asthma.

Previous work has shown up-regulation of a UTP-sensitive P2Y receptor in porcine coronary smooth muscle cells (CSMC) of organ-cultured arteries. However, the molecular identity and functional role of this putative receptor remained undefined. Here we report the cloning of the cDNA for this receptor that encodes an open reading frame for a protein of 373 amino acids with the highest homology to the human P2Y(2) receptor (84%). Heterologous expression of this receptor in 1321N1 cells revealed a novel pharmacology in that UTP and ITP were full agonists and UTP was more potent and efficacious than ATP for increasing intracellular [Ca(2+)] and extracellular signal-regulated kinase phosphorylation. Stimulation of subcultured CSMC with UTP, ITP, or ATP induced a concentration-dependent increase in cellular DNA content, protein synthesis, cell number, and proliferating cell nuclear antigen expression, indicating a mitogenic role for P2Y(2) receptors. This was supported by the finding that the treatment of CSMC with antisense oligonucleotides to the cloned cDNA sequence significantly inhibited UTP- and ATP-induced DNA and protein synthesis. In addition, reverse transcription-polymerase chain reaction analysis showed that P2Y(2) receptor mRNA was dramatically increased in cells of organ-cultured arteries compared with freshly harvested arteries, whereas the P2Y(6) receptor mRNA level was unchanged, and the P2Y(4) receptor mRNA was undetectable. This P2Y(2) subtype-specific up-regulation was confirmed in cells of coronary arteries stented in vivo. In conclusion, we have cloned the porcine P2Y(2) receptor with novel pharmacology and demonstrated that this receptor is up-regulated in CSMC of in vitro organ cultures or in vivo stented coronary arteries to mediate the mitogenic effects of nucleotides.

Unlabelled: The muscarinic M(3) receptor of airway smooth muscle has both an orthosteric binding site and an allosteric binding site. Edrophonium may bind to the allosteric site, resulting in the inhibition of the action of the orthosteric site. Therefore, we examined the effects of edrophonium on neostigmine-induced contractile and phosphatidylinositol responses of rat trachea. Neostigmine (100 micro M in final concentration) was added, and ring tension was examined by the addition of edrophonium. After the completion of the experiment, Krebs-Henseleit (K-H) solution containing both edrophonium and neostigmine was changed three times with fresh K-H solution, and the tension was recorded. Tracheal slices were incubated with [(3)H]myo-inositol and 100 micro M neostigmine in the presence or absence of edrophonium. The [(3)H]inositol monophosphate (IP(1)) was measured. Data were expressed as mean +/- SE. Statistical significance (P < 0.05) was determined with analysis of variance. Neostigmine-induced tension and IP(1) accumulation were attenuated by edrophonium at concentrations of 100 micro M or more. This attenuation was reversed to more than 80% of control levels by washing with fresh K-H solution. The results suggest that edrophonium would bind to the allosteric site, resulting in the inhibition of the action of the orthosteric site of muscarinic M(3) receptors of rat trachea., Implications: We examined the effects of edrophonium on neostigmine-induced contractile and phosphatidylinositol responses of rat trachea. Neostigmine-induced tension and inositol monophosphate accumulation were attenuated by edrophonium. This attenuation was reversed by washing. The results suggest that edrophonium would bind to the allosteric site.

Loading slices of rat barrel cortex with 50 microM BAPTA-AM while recording from pyramidal cells in layer II induces a marked reduction in both the frequency and amplitudes of mEPSCs. These changes are due to a presynaptic action. Blocking the refilling of Ca(2+) stores with 20 microM cyclopiazonic acid (CPA), a SERCA pump inhibitor, in conjunction with neuronal depolarisation to activate Ca(2+) stores, results in a similar reduction of mEPSCs to that observed with BAPTA-AM, indicating that the source for intracellular Ca(2+) is the endoplasmic reticulum. Block or activation of ryanodine receptors by 20 microM ryanodine or 10 mM caffeine, respectively, shows that a significant proportion of mEPSCs are caused by Ca(2+) release from ryanodine stores. Blocking IP(3) receptors with 14 microM 2-aminoethoxydiphenylborane (2APB) also reduces the frequency and amplitude of mEPSCs, indicating the involvement of IP(3) stores in the generation of mEPSCs. Activation of group I metabotropic receptors with 20 microM (RS)-3,5-dihydroxyphenylglycine (DHPG) results in a significant increase in the frequency of mEPSCs, further supporting the role of IP(3) receptors and indicating a role of group I metabotropic receptors in causing transmitter release. Statistical evidence is presented for Ca(2+)-induced Ca(2+) release (CICR) from ryanodine stores after the spontaneous opening of IP(3) stores.

To examine the effect of CTP, GTP, ITP, and UTP on calcium binding of Ca2+-ATPase molecules of the sarcoplasmic reticulum, the calcium dependence of the Ca2+-activated hydrolysis activities of these NTPs of the enzyme molecules was examined by comparison with that of calcium binding of the molecules in the absence of the NTPs at pH 7.40. In the sarcoplasmic reticulum membrane, CTP, GTP, and ITP did not affect the noncooperative (Hill value (n(H)) of approximately 1, apparent calcium affinity (K(0.5)) of 2-6 microm)) and cooperative (n(H) approximately 2, K(0.5) approximately 0.2 microm) calcium binding of the molecules, whereas UTP caused the molecules to highly cooperatively (n(H) approximately 4) bind calcium ions with a lowered K(0.5) of approximately 0.04 microm. When the enzyme molecules were solubilized with detergent, all of these NTPs reversibly degraded the calcium affinity of the molecule (from K(0.5) = 3-5 to >40 microm), although the effect of the NTPs on the negatively cooperative manner (n(H) approximately 0.5) of calcium binding was not experimentally obtained. Taking into account the first part of this study (Nakamura, J., Tajima, G., Sato, C., Furukohri, T., and Konishi, K. (2002) J. Biol. Chem. 277, 24180-24190) showing the improving effect of ATP on calcium binding of the membranous and solubilized molecules, the results show that ATP is the only intrinsic substrate for the enzyme molecule. This NTP regulation is discussed in terms of the oligomeric structure of the molecules.

The dynamics of Ca(2+) release and Ca(2+)-activated Cl(-) currents in two related, but functionally distinct exocrine cells, were studied to gain insight into how the molecular specialization of Ca(2+) signalling machinery are utilized to produce different physiological endpoints: in this case, fluid or exocytotic secretion. Digital imaging and patch-clamp methods were used to monitor the temporal and spatial properties of changes in cytosolic Ca(2+) concentration ([Ca(2+)](c)) and Cl(-) currents following the controlled photolytic release of caged-InsP(3) or caged-Ca(2+). In parotid and pancreatic acinar cells, changes in [Ca(2+)](c) and activation of a Ca(2+)-activated Cl(-) current occurred with close temporal coincidence. In parotid, a rapid global Ca(2+) signal was invariably induced, even with low-level photolytic release of threshold amounts of InsP(3). In pancreas, threshold stimulation generated an apically delimited [Ca(2+)](c) signal, while a stronger stimulus induced a global [Ca(2+)](c) signal which exhibited characteristics of a propagating wave. InsP(3) was more effective in parotid, where [Ca(2+)](c) signals initiated with shorter latency and exhibited a faster time-to-peak than in pancreas. The increased potency of InsP(3) in parotid probably results from a four-fold higher number of InsP(3) receptors as measured by radiolabelled InsP(3) binding and western blot analysis. The Ca(2+) sensitivity of the Cl(-) channels in parotid and pancreas was determined from the [Ca(2+)]-current relationship measured during a dynamic 'Ca(2+) ramp' produced by the continuous, low-level photolysis of caged-Ca(2+). In addition to a greater number of InsP(3) receptors, the Cl(-) current density of parotid acinar cells was more than four-fold greater than that of pancreatic cells. Whereas activation of the current was tightly coupled to increases in Ca(2+) in both cell types, local Ca(2+) clearance was found to contribute substantially to the deactivation of the current in parotid. These data reveal specializations of common modules of Ca(2+)-release machinery and subsequent effector activation that are specifically suited to the distinct functional roles of these two related cell types.

Background: Extracellular adenosine 5'-triphosphate (ATP) increases human eosinophil intracellular Ca(2+) concentration; the mechanism of action is not fully known. ATP, a physiologic regulator, acts through 2 purinergic receptor types: cation channels (P2X) and G protein-coupled receptors (P2Y)., Objective: This study is aimed at identifying the functional purinergic receptors in human eosinophils., Methods: The relative potency of ATP, uridine (UTP), cytidine (CTP), and inosine (ITP) 5'-triphosphates (P2Y agonists); 2-methylthio-ATP (P2Y(1) agonist); and 2 P2X agonists, alpha,beta-methylene-ATP and beta,gamma-methylene-ATP on intracellular Ca(2+) concentration was examined in Ca(2+)-sensitive Fura-2-labeled human eosinophils. For comparison, ATP effects were similarly studied in human neutrophils. P2X/P2Y mRNA expression in cells was examined by reverse transcription and PCR., Results: The nucleotide potency order was UTP > or = ATP > ITP >>> 2-methylthio-ATP > alpha,beta-methylene-ATP = beta,gamma-methylene-ATP = CTP = 0 in eosinophils. Pertussis toxin (500 ng/mL) pretreatment abolished the effect of lower (10(-6) mol/L) but not higher (10(-5) mol/L) concentrations of ATP in eosinophils, whereas it attenuated the effects of 10(-4) mol/L ATP in neutrophils. The phospholipase C inhibitor U73122 (2 micromol/L) partially inhibited the effect of ATP in eosinophils but totally blocked it in neutrophils. Both cells constitutively express mRNA for P2X(1), P2X(4), P2X(5), P2Y(1), and P2Y(2), but not P2X(7), with much weaker expressions of P2X(4) and P2X(5) in neutrophils. Eosinophils cultured with the T(H)1 cytokine, IFN-gamma, expressed mRNA for P2X(7), a receptor linked to apoptosis., Conclusions: These results suggest that the P2 purinergic receptor signal transduction pathways in eosinophils and neutrophils are different and are mediated by more than 1 subtype of functional P2Y receptors.

An arginine-glycine-aspartic acid (RGD)-containing peptide has been reported to generate calcium transients in bovine oocytes similar to those observed at fertilization. The research objective herein was to evaluate the response of bovine oocytes to an RGD peptide after injection with known antagonists of calcium releasing mechanisms in order to determine the initial calcium releasing pathway. Oocytes were injected with either heparin, an inhibitor of inositol 1,4,5-trisphosphate (IP3) induced calcium response, or procaine, which inhibits calcium release through the ryanodine receptor. Oocytes injected with heparin prior to RGD exposure did not display a calcium response. Oocytes injected with procaine prior to RGD exposure did exhibit a calcium response. Electroporation of IP3, caffeine, or exposure to RGD alone elicited a calcium response for each treatment group. Injection of heparin, procaine, vehicle medium (VM), or exposure to a non-RGD-containing peptide alone failed to elicit a calcium response. The data indicates that the RGD peptide is able to induce calcium transients in oocytes inhibited with procaine, but not those inhibited with heparin. These data suggest the pathway whereby the RGD peptide induces the first intracellular calcium transient in bovine oocytes is through IP3-mediated stores.

The aim of our study was to examine the effects of different purine nucleotides [GTP, ITP, and xanthosine 5'-triphosphate (XTP)] on receptor/G protein coupling. As a model system, we used a fusion protein of the beta(2)-adrenergic receptor and the alpha subunit of the G protein G(s). GTP was more potent and efficient than ITP and XTP at inhibiting ternary complex formation and supporting adenylyl cyclase (AC) activation. We also studied the effects of several beta(2)-adrenergic receptor ligands on nucleotide hydrolysis and on AC activity in the presence of GTP, ITP, and XTP. The efficacy of agonists at promoting GTP hydrolysis correlated well with the efficacy of agonists for stimulating AC in the presence of GTP. This was, however, not the case for ITP hydrolysis and AC activity in the presence of ITP. The efficacy of ligands at stimulating AC in the presence of XTP differed considerably from the efficacies of ligands in the presence of GTP and ITP, and there was no evidence for receptor-regulated XTP hydrolysis. Our findings support the concept of multiple ligand-specific receptor conformations and demonstrate the usefulness of purine nucleotides as tools to study conformational states of receptors.

We synthesized 27 GTP analogues with modification or substitution at positions C2, C6, C8 and ribose moiety to investigate their effect on microtubule (Mt) assembly. It was found that C2 and C6 are both functional for the analogues supporting Mt assembly. It was surprising to find that 2-amino- ATP (n2ATP) substantially supports assembly, and that the appearance of the assembled Mts was indistinguishable from those assembled in the standard GTP assembly buffer solution. Furthermore, 2-amino dATP and dGTP are even more potent than GTP in supporting assembly. The substitution of oxo group at C6 with reactive thiol largely reduced the activity of the analogue to support assembly. When free rotation of the glycosidic linkage of GTP was blocked by the introduction of sulfur atom between C8 and C2' of ribose moiety, it resulted in total suppression of assembly. Purine nucleoside triphosphate was found to support assembly better than GTP, and even more efficient was 2-amino purine nucleoside triphosphate. Interestingly, their deoxy-type analogues were totally inhibitory. Although 2-amino 8-hydroxy ATP and other analogues supported assembly much better than did GTP, their diphosphate analogues were totally incapable of supporting assembly. Finally, bulky fluorescent probes were introduced at C3' of ribose moiety (Mant-8-Br-GTP or Mant-GTP) to visualize the fluorescent signal in assembled Mts. Even in this case, the number of most protofilaments was found to be 14, consistent with that found in Mts assembled in GTP standard buffer solution.

Histamine, a putative neuromodulator and neurotransmitter, can depolarize supraoptic neurons and enhance depolarizing afterpotentials that play a key role in determining the excitability of these neurons. This study investigated intracellular signal transduction involved in histamine-induced enhancement of depolarizing afterpotentials utilizing immunohistochemical and electrophysiological methods. Abundant inositol 1,4,5-trisphosphate receptor-related immunostaining was seen in all parts of the supraoptic nucleus, mainly within somata and proximal processes of the magnocellular neurons, but also in astrocytes of the ventral glial lamina. In supraoptic neurons displaying depolarizing afterpotentials, three brief depolarizations evoked a slow inward current. Bath application of histamine (1-2.5 microM) reversibly enhanced this slow inward current in almost all supraoptic neurons tested. Amplitudes and durations of the slow inward current were increased by 68.1% and 22.8%, respectively. Pretreatment of cells with a histamine receptor (subtype 1) antagonist (pyrilamine) or inhibitors of phospholipase C activation (neomycin or U73122) prevented histamine-induced enhancement of the slow inward current. When electrodes containing heparin, an inositol 1,4,5-trisphosphate receptor blocker, were used for recording, histamine had no effect on the slow inward current. Heparin, however, failed to abolish norepinephrine-induced enhancement of the slow inward current. After H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], an inhibitor of protein kinase C, was infused into supraoptic neurons via the electrodes, histamine-induced enhancement of the slow inward current was also blocked. These results indicate the presence of, and functional roles for, inositol 1,4,5-trisphosphate receptor-sensitive Ca2+ stores in supraoptic neurons. Following activation of histamine receptors (subtype 1) and phospholipase C, Ca2+ mobilization from internal stores participates in mediating histamine-induced enhancement of depolarizing afterpotentials.

We have previously reported that injection of ryanodine receptor agonists into mature bovine oocytes induces intracellular calcium release, indicative of the existence of ryanodine receptors. In this experiment, further evidence of the ryanodine receptor localization, and developmental regulation in bovine oocytes is presented. The possible physiological significance is also suggested. Using a rabbit antibody against the rabbit cardiac muscle ryanodine receptor, the ryanodine receptor was observed uniformly localized in the periphery of mature bovine oocytes, while a weak and discontinuous signal was observed in the germinal vesicle intact stage of bovine oocytes. As oocytes progress to the metaphase I stage, the ryanodine receptor localization became more intense and continuous, yet not comparable to that observed in the metaphase II oocytes. These modifications correlate with the intracellular calcium responsiveness to ryanodine. A 200-microM injection of ryanodine induces a low intracellular calcium transient in germinal vesicle-stage bovine oocytes, while peaked intracellular calcium transients are subsequently observed in the metaphase II-stage oocytes. However, no significant changes in the amplitude of intracellular calcium transients induced by 250 nM inositol 1,4,5-trisphosphate and 10 microM ionomycin were observed in oocytes at a comparable stage. Fertilization induced a significant decrease in ryanodine receptor signal; similar changes were also observed in oocytes injected with 200 microM ryanodine or incubated with 10 microM ionomycin. However, no changes in ryanodine signal were observed in oocytes injected with vehicle medium. Furthermore, injection of either ryanodine or inositol 1,4,5-trisphosphate induced subsequent pronuclear formation and cleavage. These data indicate that the ryanodine receptor is closely regulated and associated with early cellular changes following fertilization; stimulation of this receptor results in the activation of bovine oocytes, and it is likely that this receptor may play a role at fertilization.

The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary for pump-leak coupling to the Na+,K+-ATPase pump. Despite this pivotal physiological role, the properties of this conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have investigated the properties of this BLM K+ conductance in dissociated, polarized Ambystoma proximal tubule cells. Nearly all seals made on Ambystoma cells contained inward rectifier K+ channels (gammaslope, in = 24.5 +/- 0.6 pS, gammachord, out = 3.7 +/- 0.4 pS). The rectification is mediated in part by internal Mg2+. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding-unbinding model. The channel conducts Tl+ and K+, but there is no significant conductance for Na+, Rb+, Cs+, Li+, NH4+, or Cl-. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0. 2 mM ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K+ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP. We conclude that this K+ channel is the major ATP-sensitive basolateral K+ conductance in the proximal tubule.

Native tropomyosin activated sliding movement in vitro of F-actin with ATP by 30%. Actin cleaved at the 40-50 loop by subtilisin or proteinase K slid on HMM much slower than intact actin, but native tropomyosin strikingly recovered this defective motility of cleaved actin by 2 to 3 times. On the other hand, with ATP analogues of CTP and ITP, sliding movements of cleaved actin and particularly intact actin were inhibited by native tropomyosin, indicating that native tropomyosin augmented specificity of the myosin substrate of NTP. These results suggested that the 40-50 loop in the small domain 2 of actin interacted directly or indirectly with tropomyosin and play a significant role in cross talk between myosin and native tropomyosin.

The uptake and release properties of Ca2+ by several subcellular fractions of the bovine adrenal medulla were investigated. Investigation by the 45Ca2+ tracer method showed that permeabilized cells and the fractions of mitochondria (MT) and microsomes (MC) caused ATP-dependent Ca2+ uptake in a Ca2+ concentration-dependent manner (pCa 8-4), whereas permeabilized cells and the fractions of secretory granules (SG) were able to accumulate a significant amount of Ca2+ even in the absence of ATP, which was completed by the addition of hexokinase and glucose. In these organelle fractions, Ca2+ uptake in the presence of ATP at pCa 7 and pCa 5.8 was well-correlated with the activity of the NADPH cytochrome c reductase (marker enzyme for the endoplasmic reticulum) and cytochrome c oxidase (marker enzyme for mitochondria), respectively. As detected by Fura-2 ratiometry, both inositol 1,4,5-trisphosphate (IP3) and caffeine caused concentration-dependent Ca2+ releases from permeabilized cells and MC, but not from MT and SG. In an ATP-depleted condition, homogenates still took up a significant amount of Ca2+ but was not able to respond to IP3 and caffeine. These results suggest that the endoplasmic reticulum is a major Ca(2+)-storing organelle, which releases Ca2+ in response to IP3 and caffeine in bovine adrenal chromaffin cells.

Superoxide-mediated clastogenesis is characteristic for various chronic inflammatory diseases with autoimmune reactions and probably plays a role in radiation-induced clastogenesis and in the congenital breakage syndromes. It is consistently prevented by exogenous superoxide dismutase (SOD), but not by heat-inactivated SOD, indicating that the anticlastogenic effect is related to the catalytic function of the enzyme. Increased superoxide production by activated monocytes/macrophages is followed by release of more long-lived metabolites, so-called clastogenic factors, which contain lipid peroxidation products, unusual nucleotides of inosine, and cytokines such as tumor necrosis factor alpha. Since these components are not only clastogenic, but can stimulate further superoxide production by monocytes and neutrophils, the genotoxic effects are self-sustaining. It is shown here that anticlastogenic effects of exogenous SOD are preserved despite extensive washing of the cells and removal of all extracellular SOD. Using flow cytometry and confocal laser microscopy, rapid adherence of the fluorescently labeled enzyme to the cell surface could be observed with slow uptake into the cell during the following hours. The degree of labeling was concentration and time dependent. It was most important for monocytes, compared with lymphocytes, neutrophils, and fibroblasts. The cytochrome c assay showed significantly diminished O2- production by monocytes, pretreated with SOD and washed thereafter. The preferential and rapid binding of SOD to monocytes may be of importance not only for the superoxide-mediated genotoxic effects, described above, but also from a therapeutic standpoint. It can explain the observation that beneficial effects of injected SOD lasted for weeks and months despite rapid clearance of the enzyme from the blood stream according to pharmacodynamic studies.

Activation of cells by hormones, growth factors or neurotransmitters leads to an increased production of inositol trisphosphate (InsP3) and, after activation of the InsP3 receptor (InsP3R), to Ca2+ release from intracellular Ca2+ stores. The release of intracellular Ca2+ is characterised by a graded response when submaximal doses of agonists are used. The basic phenomenon, called "quantal Ca2+ release", is that even the maintained presence of a submaximal dose of agonist or of InsP3 for long time periods (up to 20 min) provokes only a partial release of Ca2+. This partial, or quantal, release phenomenon is due to the fact that the initially very rapid InsP3-induced Ca2+ release eventually develops into a much slower release phase. Physiologically, quantal release allows the Ca2+ stores to function as increment detectors and to induce local Ca2+ responses. The basic mechanism for quantal release of Ca2+ is presently not known. Possible mechanisms to explain the quantal behaviour of InsP3- induced Ca2+ release include the presence of InsP3Rs with varying sensitivities for InsP3, heterogeneous InsP3R distribution, intrinsic inactivation of the InsP3Rs, and regulation of the InsP3Rs by Ca2+ store content. This article reviews critically the evidence for the various mechanisms and evaluates their functional importance. A Ca2+-mediated conformational change of the InsP3R is most likely the key feature of the mechanism for quantal Ca2+ release, but the exact mode of operation remains unclear. It should also be pointed out that in intact cells more than one mechanism can be involved.

1. Stimulation of phosphoinositide hydrolysis by human mGlu1 alpha (HmGlu1 alpha) was examined in a non-neuronal cell line (AV12-664) co-expressing both HmGlu1 alpha and a rat glutamate/aspartate transporter (GLAST). 2. Desensitization of HmGlu1 alpha could be elicited by inhibition of the GLAST transporter with the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). Maximal inhibition of HmGlu1 alpha-mediated phosphoinositide hydrolysis was induced upon 24 h pretreatment with trans-PDC. The concentration of glutamate in the extracellular medium also rose significantly in cells pretreated with trans-PDC. Glutamate levels increased upon incubation with trans-PDC in a time-dependent manner, with maximal glutamate levels attained after 24 h incubation with trans-PDC. 3. The time required for desensitization of HmGlu1 alpha by trans-PDC was compared to the time course for desensitization elicited by the direct-acting mGlu receptor agonists, 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) and (R,S)-3,5-dihydroxyphenylglycine (3,5-DHPG). Both direct-acting mGlu receptor agonists elicited desensitization of HmGlu1 alpha more rapidly than did trans-PDC, with maximal inhibition of agonist-induced phosphoinositide hydrolysis upon 12 h pretreatment. Agonist-induced desensitization could be fully reversed upon washout of agonist for 12 h. 4. Both mGlu receptor agonist- and trans-PDC-induced desensitization of HmGlu1 alpha could be blocked by inclusion of (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), an mGlu receptor antagonist, in the pretreatment medium. 5. Agonist-stimulated phosphoinositide hydrolysis by HmGlu1 alpha was found to parallel closely agonist-induced desensitization of HmGlu1 alpha. Thus, the EC50 values for 1S,3R-ACPD- and 3,5-DHPG-stimulated phosphoinositide hydrolysis were similar to the EC50 values for eliciting desensitization of HmGlu1 alpha. 6. These studies demonstrate desensitization of recombinant human mGlu1 alpha receptor in a non-neuronal cell line in which the receptor can be regulated by direct activation or by manipulation of glutamate transporter activity. Desensitization of HmGlu1 alpha was found to be mediated by activation of the receptor since the mGlu receptor antagonist, MCPG, blocked both mGlu receptor agonist- and trans-PDC-induced desensitization of HmGlu1 alpha. Furthermore, agonist-induced desensitization of HmGlu1 alpha was found to parallel receptor-mediated stimulation of phosphoinositide hydrolysis.

The molecular structures of 3'-azido-2',3'-dideoxyribosylthymine 5'-triphosphate (AZTTP), 2',3'-dideoxyribosylinosine 5'-triphosphate (ddlTP), 3'-azido-2',3'-dideoxyribosylthymine 5'-monophosphate (AZTMP) and 2',3'-dideoxyribosyladenine 5'-monophosphate (ddAMP) have been studied by NMR to understand their anti-HIV activity. For ddAMP and ddITP, conformations are almost identical with their nucleoside analogues with sugar ring pucker equilibriating between C3'-endo (approximately 75%) and C2'-endo (approximately 25%). AZTMP and AZTTP on the other hand show significant variations in the conformational behaviour compared with 3'-azido-2',3'-dideoxyribosylthymine (AZT). The sugar rings for these nucleotides have a much larger population of C2'-endo (approximately 75%) conformers, like those observed for natural 2'-deoxynucleosides and nucleotides. The major conformers around C5'-O5', C4'-C5' and the glycosidic bonds are the beta 1, gamma + and anti, respectively.

The contraction of vascular smooth muscle is thought to be regulated by reversible phosphorylation of the 20,000 dalton light chains of myosin, catalyzed by myosin light chain kinase that is dependent on calcium and calmodulin. With phosphorylation, there is a coincident increase in the actin-activated myosin NTPase activity, cross bridge interaction and contractile activity. However, this myosin phosphorylation mechanism may not be the sole factor controlling actin-myosin interaction in vascular smooth muscle. Other mechanisms may function in addition to this myosin-linked regulation. A calcium-insensitive regulation of contraction was observed in helical strips of chemically skinned (Triton X-100) arterial smooth muscle. Millimolar concentrations of inosine triphosphate and uridine triphosphate supported concentration dependent force development in the absence of calcium. Force development was a function of the MgNTP concentration. At high free calcium concentrations, an additional component of force was observed. ITP and UTP, in contrast to ATP, are less effective substrates for the myosin light chain kinase, and their effect on actin-myosin interaction is thus less than that of ATP. They are, however, utilized by the myosin NTPase after treatment by ATP-gamma-S. The efficacy of the substrate for the activated NTPase is greater for UTP than ITP than for ATP.

We have proposed recently that a pertussistoxin-insensitive Ca2+ influx stimulated by Y2-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca2+ concentration ([Ca2+]i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca2+ and independent of internal Ca2+ stores. We describe here the actions of NPY in these same cells, using the activity of Ca(2+)-activated K+ channels as an indicator of [Ca2+]i. The elementary slope conductance of these channels was 110 +/- 3 pS (with an asymmetrical K+ gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca2+, increased the channel open probability. ATP applied in the absence of external Ca2+ caused rises both in channel open probability and [Ca2+]i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca2+ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5'-triphosphate (GTP) by (guanosine 5'-O-(2-thiodiphosphate) (GDP[ beta S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y2-type receptors to Ca2+ influx in CHP-234 cells.

The effect of the biological oxidant H2O2 on purinergic-receptor-stimulated Ca2+ signalling was determined in canine venous endothelial cells. H2O2 increased cytosolic free [Ca2+] ([Ca2+]i), the rate of rise of which was dose-dependently related to H2O2 concentration. The response of [Ca2+]i to H2O2 resulted in part from release of Ca2+ from internal stores. The H2O2-sensitive intracellular Ca2+ pool was characterized in cells suspended in Ca(2+)-free/EGTA buffer and stimulated in sequence with H2O2 and ionomycin or ATP. Under this condition, the rank order of apparent compartment size sensitive to each compound was ionomycin > H2O2 > ATP. Stimulation of cells with H2O2 eliminated any response of [Ca2+]i to subsequent addition of ATP. To test more directly whether H2O2 accesses the inositol trisphosphate-sensitive Ca2+ store, cells were pretreated with thapsigargin, a selective inhibitor of that store's Ca2+ pump. Release of Ca2+ from internal Ca2+ stores by H2O2 declined as the interval after thapsigargin addition increased, a finding that supports the contention that H2O2 accesses the inositol trisphosphate-sensitive Ca2+ store. H2O2-stimulated Ca2+ influx across the cell membrane was sensitive to Ni2+, La3+, and 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole HCl (SKF-96365), a selective inhibitor of the agonist-stimulated Ca(2+)-influx pathway. Ca2+ entry triggered by H2O2 appears to occur via the agonist-sensitive Ca2+ influx pathway. Together, these results suggest that H2O2, which is normally secreted by activated neutrophils and monocytes, may act as an intercellular messenger and stimulate Ca2+ signalling in target endothelial cells.

1. The effect of rolipram (ME3176) on ADP- and IP3-induced repetitive IK(Ca) in rat megakaryocyte was investigated by use of the nystatin perforated patch and conventional whole-cell patch-clamp techniques. 2. The ADP-induced IK(Ca) was depressed by treatment with rolipram in a concentration-dependent manner. The inhibition by rolipram disappeared after treatment with a cyclic nucleotide-dependent protein kinase inhibitor, H-8. The inhibition of IK(Ca) was also observed in the presence of cyclic AMP accumulating agents such as forskolin and isobutylmethylxanthine (IBMX). 3. Rolipram enhanced the inhibitory action of forskolin, suggesting that rolipram facilitates the accumulation of cyclic AMP by blocking its breakdown. Similar results was obtained with adenosine, an endogenous adenylate cyclase activator. 4. Intracellular application of inositol trisphosphate (IP3) induced repetitive IK(Ca) in megakaryocytes. The induced IK(Ca) was also inhibited by rolipram and by other cyclic AMP accumulating agents. 5. It was concluded that megakaryocytes possess rolipram-sensitive phosphodiesterase (PDE), which was not detected in platelets, but plays a distinct modulatory role in megakaryocytes for generating ADP-induced IK(Ca).

1. Using beta-escin and ionomycin-treated skinned smooth muscle strips of the rabbit mesenteric artery, the effects of calyculin A (CL-A, an inhibitor of type 1 and 2A phosphatases) on mechanical activities, phosphorylation of myosin light chain (MLC) and the relationship between the two were studied in Ca(2+)-free solution containing 4 mM EGTA and these effects were compared with those evoked by Ca2+. 2. The threshold concentration of Ca2+ required to increase either tension or MLC-phosphorylation was 0.1 microM and maximum effects were obtained at 10 microM. MLC was mainly monophosphorylated, rather than diphosphorylated, in the presence of Ca2+. ED50 value for Ca2+ was 0.54 microM for either tension or MLC-phosphorylation. The relationship between tension and MLC-phosphorylation is linear in the pCa range 7-5.5. 3. In Ca(2+)-free solution (containing either 20 mM EGTA or 4 mM EGTA with or without 4 mM BAPTA), 3 microM CL-A produced a contraction, the maximum amplitude of which was similar to that evoked by 10 microM Ca2+. CL-A (0.03-3 microM) concentration-dependently increased both tension and MLC-phosphorylation in Ca(2+)-free solution containing 4 mM EGTA. The threshold concentration of CL-A required for the increase in either tension or MLC-phosphorylation was 0.03 microM and maximum effects were obtained at 3 microM. In the presence of CL-A, MLC was not only monophosphorylated but also diphosphorylated. ED50 values for CL-A were 0.39 microM for tension, 0.44 microM for the monophosphorylated form of MLC and 0.54 microM for all phosphorylated (mono + di) forms. The relationship between tension and the monophosphorylated form of MLC was linear over the concentration range studied and was similar to that for Ca2+. 4. H-7 (3 microM, an inhibitor of protein kinase C) inhibited neither the tension nor phosphorylation of MLC induced by 10 microM Ca2+ or 3 microM CL-A. At a high concentration (30 microM), H-7 slightly inhibited both the tension and phosphorylation of MLC induced by either stimulant without a change in the tension-MLC-phosphorylation relationship. KN-62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinase II, did not modify either the tension or the phosphorylation of MLC induced by 10 microM Ca2+ or 3 microM CL-A. CK-II, another inhibitor of Ca(2+)-calmodulin-dependent protein kinase II, did not inhibit the contraction induced by 3 microM CL-A. 5. SM-1 (0.03-0.3 mM) and ML-9 (0.1 and 0.3 mM), inhibitors of MLC-kinase, each lowered the resting level of MLC-phosphorylation in Ca2+-free solution and also inhibited both the tension and MLC-phosphorylation induced by 10 microM Ca2+ or 3 microM CL-A, in a concentration-dependent manner.Neither SM-1 nor ML-9 modified the relationship between tension and either monophosphorylated or all phosphorylated (mono + di) forms of MLC in the presence of Ca2+ or CL-A.6. In a solution containing MgITP (the substrate for myosin ATPase but not for MLC-kinase) with no MgATP, 10 microM Ca2+ failed to produce contraction. Under these conditions, the amplitude of the contraction induced by 3 microM CL-A was greatly diminished in comparison with that induced in the presence of MgATP.7. The present results suggest that in smooth muscle cells of the rabbit mesenteric artery, CL-A in Ca2+-free solution, produces a maximum contraction through an indirect activation of Ca2+-calmodulin independent(constitutively active) MLC-kinase via its inhibitory action on MLC-phosphatases. Based on this evidence, it is hypothesized that, in these cells, a constitutively active MLC-kinase may be present, though its action may be concealed by that of endogenous MLC-phosphatase.

1. Whole-cell patch clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study the biophysical properties of a calcium current activated by depletion of intracellular calcium stores in rat peritoneal mast cells. 2. Calcium influx through an inward calcium release-activated calcium current (ICRAC) was induced by three independent mechanisms that result in store depletion: intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or extracellular application of ionomycin (active depletion), and intracellular infusion of calcium chelators (ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)) to prevent reuptake of leaked-out calcium into the stores (passive depletion). 3. The activation of ICRAC induced by active store depletion has a short delay (4-14 s) following intracellular infusion of InsP3 or extracellular application of ionomycin. It has a monoexponential time course with a time constant of 20-30 s and, depending on the complementary Ca2+ buffer, a mean normalized amplitude (at 0 mV) of 0.6 pA pF-1 (with EGTA) and 1.1 pA pF-1 (with BAPTA). 4. After full activation of ICRAC by InsP3 in the presence of EGTA (10 mM), hyperpolarizing pulses to -100 mV induced an instantaneous inward current that decayed by 64% within 50 ms. This inactivation is probably mediated by [Ca2+]i, since the decrease of inward current in the presence of the fast Ca2+ buffer BAPTA (10 mM) was only 30%. 5. The amplitude of ICRAC was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (KD) of 3.3 mM. Inward currents were nonsaturating up to -200 mV. 6. The selectivity of ICRAC for Ca2+ was assessed by using fura-2 as the dominant intracellular buffer (at a concentration of 2 mM) and relating the absolute changes in the calcium-sensitive fluorescence (390 nm excitation) with the calcium current integral. This relationship was almost identical to the one determined for Ca2+ influx through voltage-activated calcium currents in chromaffin cells, suggesting a similar selectivity. Replacing Na+ and K+ by N-methyl-D-glucamine (with Ca2+ ions as exclusive charge carriers) reduced the amplitude of ICRAC by only 9% further suggesting a high specificity for Ca2+ ions. 7. The current amplitude was not greatly affected by variations of external Mg2+ in the range of 0-12 mM. Even at 12 mM Mg2+ the current amplitude was reduced by only 23%. 8. ICRAC was dose-dependently inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ release was examined in neurons isolated from the mollusc Helix pomatia using Ca2+ indicator fura-2 and fluorescent digital-imaging microscopy technique. Extracellular application of caffeine caused a fast and pronounced augmentation of [Ca2+]i whose amplitude and kinetics differ in the centre of the cell and near its membrane. Mean values of caffeine-induced increase of [Ca2+]i were 0.97 +/- 0.11 microM at the periphery and 0.53 +/- 0.13 microM in the centre. The rates of rise and relaxation of caffeine-evoked [Ca2+]i transients were faster near the membrane. Pressure injection of inositol, 1,4,5-trisphosphate into the same neurons produced an abrupt and significant increase of [Ca2+]i in the centre (mean value of inositol 1,4,5-trisphosphate-induced elevation = 0.55 +/- 0.11 microM) while the response was smaller or even absent near the cellular membrane. Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ transients did not affect each other. The data obtained indicate that in snail neurons these two calcium pools are not overlapping and at least some part of the caffeine-sensitive store is located close to the cellular membrane and that the inositol 1,4,5-trisphosphate-sensitive one is located in the centre of the cell.

Facial motoneurons (FMN) were recorded intracellularly in Sprague-Dawley rats anesthetized with halothane. The animals were vagotomized, paralyzed, and artificially ventilated. The average membrane potential of the cells was 62.6 +/- 1.9 mV and their input impedance ranged from 5 to 30 M omega (9.8 +/- 1.1 M omega, n = 38). The membrane potential of most FMNs varied throughout the central respiratory cycle and four distinct patterns were detected. Type I (post-inspiratory) cells (21/44) showed a two-phase Cl(-)-mediated hyperpolarization during the respiratory cycle, one during central inspiration and the second during late expiration. Type II cells (early inspiratory, n = 10) showed early inspiratory depolarization. Type III (n = 2, stage-2 expiratory) cells displayed late expiratory depolarization and one cell (type IV or throughout inspiratory) exhibited expiratory Cl(-)-mediated hyperpolarization. The remaining 10 cells showed no detectable respiratory modulation. The results reflect the heterogeneity of the central respiratory modulation of FMNs and suggest that these cells receive both excitatory and inhibitory inputs from elements of the central respiratory pattern generating network.

Different pathways of control over the permeability of ionic channels in the receptor domains (RD) of biological membranes and their possible functional role in various receptor systems are discussed. It is suggested that cyclic mononucleotides and inositol-triphosphate may directly act as biochemical modulators of such permeability by blocking the Ca(2+)-binding sites of the gate mechanisms of RD ionic channels by recruiting, in particular, Ca2+ within the cell from both intracellular calcium depots and the intercellular medium. Kinetic schemes of gate mechanisms for all possible types of RD ionic channels have been proposed for a case when, in addition to Ca(2+)-ions, the reaction medium contains another type of ions or molecules capable of blocking only one Ca(2+)-binding site of the gate mechanism. Such kinetic schemes form the basis for qualitative and quantitative analyses of cyclic mononucleotide and inositol-triphosphate influence on the permeability of RD ionic channels.

We have developed a new model system for studying spindle elongation in vitro using the pennate, marine diatom Cylindrotheca fusiformis. C. fusiformis can be grown in bulk to high densities while in log phase growth and synchronized by a simple light/dark regime. Isolated spindles can be attained in quantities sufficient for biochemical analysis and spindle tubulin is approximately 5% of the total protein present. The spindle isolation procedure results in a 10-fold enrichment of diatom tubulin and a calculated 40-fold increase in spindle protein. Isolated spindles or spindles in permeabilized cells can elongate in vitro by the same mechanism and with the same pharmacological sensitivities as described for other anaphase B models (Cande and McDonald, 1986; Masuda et al., 1990). Using this model, in vitro spindle elongation rate profiles were developed for a battery of nucleotide triphosphates and ATP analogs. The relative rates of spindle elongation produced by various nucleotide triphosphates parallel relative rates seen for kinesin-based motility in microtubule gliding assays. Likewise ATP analogs that allow discrimination between myosin-, dynein-, and kinesin-mediated motility produce relative spindle elongation rates characteristic of kinesin motility. Also, isolated spindle fractions are enriched for a kinesin related protein as identified by a peptide antibody against a conserved region of the kinesin superfamily. These data suggest that kinesin-like motility contributes to spindle elongation during anaphase B of mitosis.

Membrane potential changes in endothelial cells in response to caffeine and acetylcholine (ACh) were recorded with microelectrodes from an intact endothelium preparation from the guinea-pig coronary artery. Caffeine induced a transient hyperpolarization of the membrane in a concentration-dependent manner. The hyperpolarization was inhibited by removal of Ca2+ from the bathing medium and by ryanodine (20 microM). It was not affected by 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester hydrochloride (TMB-8, 10 microM) or neomycin (5 mM). ACh induced a sustained hyperpolarization in endothelial cells. At concentrations that caused no significant effects on the caffeine response, TMB-8 and neomycin inhibited hyperpolarization induced by ACh. Ryanodine did not inhibit the response to ACh. The ACh-induced hyperpolarization was also inhibited by caffeine in a concentration-dependent manner. Results from the present study suggest that hyperpolarizations induced by caffeine and ACh are mediated by separate Ca2+ pools.

Preconditioning the brain with sublethal ischemia protects against neuronal damage following subsequent ischemic insult. Using [3H]inositol 1,4,5-triphosphate (IP3), [3H]phorbol 12,13-dibutyrate (PDBu), [3H]cyclic adenosine monophosphate (cAMP) and [3H]rolipram, we performed quantitative autoradiography to determine postischemic alterations in second-messenger systems in the gerbil hippocampus following preconditioning the brain with sublethal ischemia. At 7 days of reperfusion, no alterations were observed in brains subjected to 2 min of forebrain ischemia which produced no neuronal damage. However, 3-min ischemia caused a 75% reduction in [3H]IP3 binding (p < 0.01 vs. control) and 15-25% reductions in [3H]forskolin (p < 0.01 vs. control), [3H]cAMP (p < 0.05 vs. control), and [3H]rolipram (p < 0.01 vs. control) binding in the CA1 subfield coincident with histopathological CA1 pyramidal cell destruction, but no significant alterations in [3H]PDBu binding. Preconditioning the brain with 2 min of ischemia followed by 4 days of reperfusion prevented both histopathological cell death and the reductions in binding following subsequent 3 min of ischemia. Interestingly, [3H]IP3 and [3H]rolipram binding in CA1 showed a transient reduction, by 30% and 20% (both p < 0.01 vs. control), respectively, in the early reperfusion period. This downregulation of the IP3 system may play a role in the protection against cell death.

1. Glomerular epithelial cells (GEC) were cultured from human kidneys and immunologically characterized. 2. The effect of extracellular nucleotides on the cytosolic free calcium activity [Ca2+]i was investigated with the fura-2 microfluorescence method. Extracellular UTP, UDP, UMP, ATP, adenosine 5'-O-(3-thio)-trisphosphate (ATP-gamma-S), inosine-triphosphate (ITP), guanyltriphosphate (GTP), 2-methylthio-ATP, AMP, alpha,beta-methylene-ATP and adenosine led to a rapid, transient, concentration-dependent increase of [Ca2+]i, followed by a plateau above the baseline level. 3. In a calcium-free extracellular solution, the rapid increase of [Ca2+]i was still present, whereas the plateau level was abolished. 4. ATP and UTP (ED50 both: 10(-5) M) stimulated inositol trisphosphate (InsP3) formation in GEC. 5. The order of potency for the purine nucleotides in stimulating InsP3 formation was ATP = ATP-gamma-S greater than ADP greater than 2-methylthio-ATP greater than AMP = a,beta methylene-ATP = adenosine. 6. The increase of InsP3 induced by ATP (10(-5) M) could be inhibited by the P2 receptor blocker suramin (greater than 10(-4) M). Reactive blue 2 exhibited a weak stimulating effect on the InsP3 formation and only a weak inhibitory effect at a concentration of 10(-3) M was observed. 7. Protein kinase C activation by preincubation of GEC with phorbol 12-myristate 13-acetate (PMA, 100 ng ml-1, 15 min) abolished the effect of ATP (10(-5) M) on InsP3 formation. Downregulation of protein kinase C by long term incubation (18 h) with PMA had no significant effect on the phosphoinositol turnover induced by ATP.8. The results indicate that an increase of [Ca2+]i and inositol phosphate breakdown can be mediated via activation of a P2 receptor in human GEC.

There is evidence that one or more metabolites of arachidonic acid can produce positive inotropic effects and may also be implicated in the enhanced alpha 1-adrenoceptor responses in hearts from diabetic rats. We therefore carried out a study to investigate the possibility that arachidonic acid metabolites could be involved in the altered cardiac alpha 1-effect of norepinephrine (in the presence of propranolol) in chronic streptozotocin-diabetic rats. Our results have shown that in the presence of the cyclooxygenase inhibitor indomethacin or the thromboxane synthetase inhibitor imidazole, the norepinephrine-stimulated positive inotropic effect and the formation of inositol 1,4,5-trisphosphate were significantly increased in control hearts but were unaltered in hearts from diabetic rats. The addition of the prostacyclin synthetase inhibitor tranylcypromine reduced the norepinephrine-stimulated positive inotropic effect and inositol 1,4,5-trisphosphate formation only in diabetic hearts and had no effect in the controls. The nature and physiological significance of the enhanced positive inotropic effect and inositol 1,4,5-trisphosphate formation in the control heart with the addition of indomethacin and imidazole are still unclear. The effect of tranylcypromine may indicate the participation of prostacyclin in mediating the enhanced alpha 1-inotropic effect of norepinephrine in the chronic diabetic heart.

In the present paper, P1 and P2 purinergic receptors and their control of signal transduction pathways were investigated in NCB-20 cells. ATP elicited an increase in [Ca2+]i. The purinergic receptor subtype involved was identified by comparing the actions of a range of nucleotides. UTP was the most potent agonist in elevating [Ca2+]i, with an EC50 value of 6.2 +/- 0.5 microM. UTP, ATP (EC50, 17.3 +/- 1.5 microM), adenosine-5'-O-(3-thio)triphosphate (23 +/- 3 microM), and ITP (55 +/- 4 microM) exerted similar maximal effects. Other nucleotides tested, including beta, gamma-methylene-ATP and 2-methylthio-ATP, which are considered prototypic agonists for P2x and P2y receptors, respectively, were ineffective; in general, modifications in the ribose-triphosphate chain and substitution on the 2-position of the purines reduced the efficacy of nucleotides. This pharmacological characterization indicated that a putative P2u receptor mediates the [Ca2+]i elevation elicited by nucleotides in NCB-20 cells. The increase in [Ca2+]i originates from intracellular Ca2+ stores; blockade of Ca2+ entry does not affect the rise in [Ca2+]i. In contrast, pretreatment with the Ca(2+)-ATPase inhibitor thapsigargin or with bradykinin, a hormone that releases Ca2+ from inositol trisphosphate-sensitive stores, does preclude the increase in [Ca2+]i induced by ATP. ATP and UTP also transiently inhibit cAMP accumulation in the intact cell, presumably via a Ca(2+)-mediated mechanism. The finding of a P2u receptor in NCB-20 cells adds to a growing perception that P2 receptors are widely distributed. Besides the P2u receptor, NCB-20 cells express adenosine A2 receptors, coupled to stimulation of cAMP accumulation. The presence of both P1 and P2 purinergic receptors permits a sequential modulation of distinct second messenger levels associated with a common stimulus, ATP.

The latent period before depolarization of Limulus ventral photoreceptors by light flashes was compared with that following brief, intracellular, pressure-injection of d-myo-inositol 1,4,5 trisphosphate. At temperatures between 18 degrees C and 22 degrees C and with an extracellular calcium concentration of 10 mM, the responses of 4 cells to light and to injections of 100 microM inositol trisphosphate displayed average latencies of 71 and 56 ms, respectively. The latencies of responses to InsP3 included an estimated 20 ms dead-time inherent in the injection method. Reducing the temperature lengthened the latency of the response to light (Q10 approximately 3.2 between 7 and 22 degrees C) more than that to inositol trisphosphate (Q10 approximately 2.3). Bathing the photoreceptors in seawater containing no added calcium and 1 mM of the calcium chelator EGTA greatly increased the latency of the light response at all temperatures, but did not increase the latency of the response to inositol trisphosphate. We conclude that the response to inositol trisphosphate lacks the calcium- and temperature-sensitive latent period which characterizes the response to light. If inositol trisphosphate acts, via the release of stored calcium, to stimulate an intermediate in the visual cascade, then that intermediate would appear to be downstream from the latency-generating mechanism.

1. The interaction between inositol 1,4,5-trisphosphate (InsP3) and guanosine 5'-O-(3-thio triphosphate) (GTP gamma S) releasable calcium (Ca2+) pools was examined using 45Ca effluxes in permeabilized cultured airway smooth muscle cells from rabbit trachea. 2. Addition of InsP3 or GTP gamma S caused a concentration-dependent release of intracellular Ca2+. The release of Ca2+ by InsP3 was much greater than with GTP gamma S. Pretreatment with maximally effective InsP3 (10 microM) abolished the GTP gamma S-induced Ca2+ release, whereas pretreatment with 100 microM GTP gamma S reduced the InsP3-induced Ca2+ release by 25%. 3. Ryanodine (100 microM), also gave a large release of intracellular Ca2+. After pretreatment with 100 microM ryanodine, GTP gamma S did not induce Ca2+ release, and InsP3-induced Ca2+ release was reduced by 76%. 4. Caffeine (50 mM), produced a slow release of intracellular Ca2+. Pre-exposure to 50 mM caffeine had no effect on the GTP gamma S-induced Ca2+ release but reduced the InsP3 releasable Ca2+ by 58%. 5. Pretreatment with ryanodine abolished the caffeine-induced Ca2+ release, and addition of caffeine before ryanodine reduced the ryanodine-induced Ca2+ release by 64.4%. 6. These results suggest that there are at least three pools of Ca2+ present within airway smooth muscle cells. The largest pool is released by InsP3 or ryanodine, another is released either by a high concentration of InsP3 or on application of GTP gamma S, and the third by InsP3 alone. Ca2+ may be able to move from the GTP gamma S-sensitive pool into the InsP3- and ryanodine-sensitive pool when this becomes depleted. In contrast, the opposite movement of Ca2 + cannot occur.

Changes in intracellular free calcium ([Ca++]i) play an important role in a variety of biochemical reactions that lead to cellular responses such as proliferation and differentiation. The response of [Ca++]i to extracellular nucleotides (ATP, UTP, ITP, and AMP-PNP) was determined in individual canine keratinocytes using the fluorescent probe fura-2 and digital video fluorescence imaging microscopy. In the presence of 1.8 mM extracellular Ca++, 100 and 500 microM ATP caused a rapid (less than 9 sec) three- to twelvefold rise in [Ca++]i above resting levels of 50-150 nM followed by occasional fluctuations. Small responses were elicited with doses as low as 0.1 microM ATP. The response of cells stimulated with 500 microM ATP in Ca(++)-free medium was characterized by 1.5 to 3 times rapid initial peak followed by a decrease of [Ca++]i below resting levels. Loss of response occurred in the majority of keratinocytes preincubated for 30 min in Ca(++)-free medium. UTP was as effective as ATP in stimulating rises in [Ca++]i in keratinocytes. Smaller elevations in [Ca++]i up to four- to fivefold resting levels were noted with 100 microM AMP-PNP or 500 microM ITP. Desensitization of cells was demonstrated when a second stimulation followed the primary ATP or UTP treatment. These results are suggestive of the presence of purinergic receptors in the cytoplasmic membrane of canine keratinocytes. Experiments using the calcium channel blocker lanthanum suggest that ATP-induced initial rises and sustained levels of [Ca++]i are dependent on the release of Ca++ from intracellular stores. These intracellular Ca++ stores appear to be rapidly depleted after removal of extracellular calcium ([Ca++]e), thereby abolishing ATP-induced [Ca++]i increases.

The degradation of insulin in isolated liver endosomes and the relationships of this process with ATP-dependent endosomal acidification have been studied. Incubation of endosomal fractions containing 125I-insulin in isotonic KCl at 30 degrees C resulted in a rapid loss of insulin integrity as judged from trichloroacetic acid precipitability, Sephadex G-50 chromatography, immunoreactivity and receptor binding ability, with a maximum at pH 5-6 (t1/2: 10, 10, 6 and 6 min, respectively). On a log/log plot, the amount of acid-soluble products generated was linearly related to the amount of insulin associated with endosomes (slope, 0.80). Upon incubation, virtually all acid-soluble products diffused out of endosomes as judged from their solubility in aqueous poly(ethyleneglycol). In permeabilized endosomes, intact insulin was also released in part extraluminally, but only when degradation was inhibited did this release increase with lowering pH. ATP shifted the pH for maximal insulin degradation to about 7.5-8.5 and caused endosomal acidification as judged from the uptake of acridine orange and the fluorescence of internalized fluorescein-labeled dextran and galactosylated bovine serum albumin (delta pH about 0.8-0.9). GTP, ITP and UTP exerted comparable effects but with lower potencies. The ability of ATP to alter the pH dependence of insulin degradation was maximal in the presence of Cl-, other anions being less effective (Br- greater than gluconate = SO4(2-) greater than NO3- = sucrose = mannitol) and/or inhibitory (NO3-). Na+, K+ and Li+ supported more effectively ATP-dependent insulin degradation than did choline. Divalent cations were required for the ATP effect (Mg2+ = Mn2+ greater than Co2+ greater than Ni2+ = Zn2 greater than Ca2+). Little or no effects of ATP occurred in the presence of proton ionophores such as monensin and carbonyl cyanide chlorophenylhydrazone, and inhibitors of the proton ATPase such as N-ethylmaleimide. The abilities of nucleotides, ions and inhibitors to support or inhibit ATP-dependent insulin degradation were well correlated with their abilities to affect ATP-dependent acidification. The acidotropic agents chloroquine and quinacrine caused a leftward shift in the pH dependence of insulin degradation and a decrease in maximal degradation; in the presence of ATP, chloroquine almost completely inhibited degradation at pH 5-9. It is concluded that ATP-dependent acidification, in part by enhancing the dissociation of the insulin-receptor complex, is required for optimum degradation of insulin within liver endosomes.

The effect of GTP analogues on catecholamine secretion and [3H]arachidonic acid release from digitonin-permeabilized adrenal chromaffin cells was examined. Several GTP analogues stimulated Ca2(+)-independent exocytosis, with the order of efficacy being XTP greater than ITP greater than guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The stimulatory effect of the GTP analogues appeared to be due to activation of a conventional GTP-binding protein, as it was inhibited by guanosine 5'-[beta-thio]diphosphate (GDP[S]). In contrast, Ca2(+)-dependent exocytosis was only partially inhibited by high doses of GDP[S]. GTP did not stimulate Ca2(+)-independent exocytosis, but instead was found to inhibit secretion caused by micromolar Ca2+. Arachidonic acid (100 microM) also stimulated Ca2(+)-independent catecholamine secretion. Determination of the effect of GTP analogues on release of free [3H]arachidonic acid into the medium showed that it was stimulated by GTP[S] but inhibited by GTP, p[NH]ppG, ITP and XTP. The inhibition of [3H]arachidonic acid release by XTP was not prevented by GDP[S]. These results demonstrate that activation of a GTP-binding protein by certain GTP analogues can induce Ca2(+)-independent secretion in adrenal chromaffin cells and that the effect of GTP analogues on Ca2(+)-independent secretion can be dissociated from generation of arachidonic acid.

The interaction of the fluorescent ADP analogue lin-benzo-ADP (containing a linearly extended version of adenine, in which a benzene ring is inserted between pyrimidine and imidazole ring) with nucleotide-depleted mitochondrial F1 was investigated. It was found that lin-benzo-ADP is able to occupy all six nucleotide-binding sites present on the enzyme. Two sites exhibit a very high affinity for the analogue (dissociation constant, Kd, less than 10 nM) and bind it rapidly (association rate constant, k+1, about 1.10(6) M-1 S-1). A third site shows a lower affinity for the analogue (Kd = 1-2 microM) and is occupied relatively fast (k+1 approximately 10(4) M-1 S-1). Binding of lin-benzo-ADP to these three sites is prevented not only in the presence of excess ADP and ATP, but also by IDP and ITP, thus indicating that these sites are the catalytic ones. As it will be discussed, this conclusion is further corroborated by the finding that release of the analogue from the two high affinity sites can be promoted by binding of nucleoside di- and triphosphates to the third site. The remaining three sites were found to bind lin-benzo-ADP with identical affinity (Kd = 1-2 microM) and with a rather low association rate (k+1 = 300-600 M-1 S-1). Binding of the analogue to them is only prevented by ADP and ATP, but not by IDP and ITP, which confirms that these sites are the noncatalytic ones. The analogue could be displaced by excess ADP also from these sites; however, in contrast to the catalytic sites, no promotive effect was observed here. The obvious changes in the nucleotide binding behavior of the non-catalytic sites after depletion of endogenous nucleotides will be discussed.

The roles of intracellular free Ca2+ and protein kinase C in the tonic contraction induced by prostaglandin were studied. Prostaglandin F2 alpha induced tonic contraction of rat thoracic aorta in both control and Ca2(+)-free solution. Close correlations were observed between the contractile response of aortic strips and the changes in intracellular free Ca2+ concentration in vascular smooth muscle cells assessed with the fluorescent Ca2+ indicator fura 2, both in control and Ca2(+)-free solutions. Prostaglandin F2 alpha also enhanced the production of inositol 1,4,5-trisphosphate in vascular smooth muscle cells before the rise of the intracellular free Ca2+ concentration. Moreover, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, inhibited the tonic contractions induced by PGF2 alpha and 12-O-tetradecanoyl phorbol-13-acetate, a direct activator of protein kinase C, at similar concentrations. These results suggest that both intracellular free Ca2+ and protein kinase C participate in prostaglandin F2 alpha-induced tonic contraction.

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p-fluorosulfonylbenzoylethenoadenosine (FSB epsilon A) with pseudo-first order kinetics. The dependence of the rate of inactivation on the concentration of FSB epsilon A revealed an apparent Kd of 0.25 mM. ATP and ADP, and to a lesser extent, ITP and IDP provide partial protection against inactivation by the reagent. Isolation and sequence analysis of major radioactive fragments in peptic or cyanogen bromide digests of MF1 inactivated with [3H]FSB epsilon A indicate that modification of Tyr-alpha 244 is associated with the loss of activity observed. Assessment of the amount of Tyr-alpha 244 derivatized with [3H]FSB epsilon A at specific points during inactivation of the ATPase indicates that maximal inactivation is achieved on modification of this residue in slightly greater than one copy of the alpha subunit. The following characteristics of inactivation of MF1 by FSB epsilon A have also been determined. (a) The rate of inactivation of ITPase activity by FSB epsilon A is 1.4 times greater than that observed for inactivation of ATPase activity under identical conditions. (b) After maximally inactivating the capacity of MF1 to hydrolyze saturating ATP with FSB epsilon A, the modified enzyme retained its capacity to hydrolyze substoichiometric ATP. (c) Inactivation of the ATPase by FSB epsilon A is accelerated by Pi. In each of the above characteristics, MF1 modified by FSB epsilon A resembles enzyme inactivated with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) more than it does enzyme inactivated with 5'-p-fluorosulfonylbenzoylinosine (FSBI). Furthermore, prior inactivation of MF1 with FSBA completely prevents labeling of Tyr-alpha 244 with [3H]FSB epsilon A, whereas prior inactivation of the enzyme with FSBI does not. Since a single catalytic site is modified when FSBI inactivates MF1 whereas three noncatalytic sites are modified when it is maximally inactivated with FSBA, it is concluded that FSB epsilon A also modifies noncatalytic sites.