Generation Of Ins Research Articles

A1 adenosine receptor-mediated Ins(1,4,5)P3 generation in allergic rabbit airway smooth muscle.

The signal transduction pathway for A1 adenosine receptor in airway smooth muscle from allergic rabbits was studied by investigating the effect of the selective A1 adenosine-receptor agonist N6-cyclopentyladenosine (CPA) on tissue levels of inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3] measured by protein binding assay. CPA caused a rapid, transient, and concentration-dependent elevation of Ins(1,4,5)P3 in airways from allergic rabbits. The agonist also produced a concentration-dependent contraction of the airway preparations from these animals. Both the Ins(1,4,5)P3 and contractile responses generated by CPA were attenuated by the phospholipase C (PLC) inhibitor U-73122, indicating the coupling of these responses to PLC. The CPA-induced Ins(1,4,5)P3 production observed in the allergic rabbit tissues was also inhibited by the adenosine-receptor antagonist 8-( p-sulfophenyl)-theophylline, suggesting that the effect was mediated by A1 adenosine receptors. On the other hand, the A2 adenosine-receptor agonist CGS-21680 was ineffective in altering the tissue concentration of Ins(1,4,5)P3, indicating that A2 adenosine receptors may not be involved in the activation of PLC in the allergic rabbit airway smooth muscle. In this preparation, the Gi-Go inhibitor pertussis toxin (PTX) attenuated the CPA-induced Ins(1,4,5)P3 accumulation, providing evidence that the generation of Ins(1,4,5)P3 by A1 adenosine-receptor stimulation is coupled to a PTX-sensitive G protein(s). The results suggest that activation of A1 adenosine receptors in allergic rabbit airway smooth muscle causes the production of Ins(1,4,5)P3 via a PTX-sensitive G protein-coupled PLC, and this signaling mechanism may be involved, at least in part, in the generation of contractile responses. It is hypothesized that this process may contribute to adenosine-induced bronchoconstriction in allergic asthma.

Activation of the 43 kDa Inositol Polyphosphate 5-Phosphatase by 14-3-3ζ

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyzes and thereby inactivates the second messenger molecules inositol 1,4,5-trisphosphate -Ins(1,4,5)P3- and inositol 1,3,4,5-tetrakisphosphate in a signal terminating reaction. Recent studies have shown that the platelet protein pleckstrin forms a complex with the 43 kDa 5-phosphatase and activates Ins(1,4,5)P3 hydrolysis 2-fold [Auethavekiat, V., Abrams, C. S., & Majerus, P. W. (1997) J. Biol. Chem. 272, 1786-1790]. We now show that another platelet protein, 14-3-3zeta, forms a complex with the 43 kDa 5-phosphatase and thereby activates the hydrolysis of Ins(1,4,5)P3. Both pleckstrin and 14-3-3zeta contain one or more pleckstrin-homology domains, both are present in platelet cytosol, and both dimerize and form complexes with other signalling proteins. Purified platelet pleckstrin and 14-3-3zeta enhanced the rate of the hydrolysis of Ins(1,4,5)P3 by the 43 kDa 5-phosphatase 1.9- and 3.8-fold, respectively, but did not activate the 75 kDa 5-phosphatase. We have demonstrated that the mechanism of 5-phosphatase activation by 14-3-3zeta results from specific complex formation between the 43 kDa 5-phosphatase and 14-3-3zeta. Recombinant 43 kDa 5-phosphatase bound to recombinant glutathione S-transferase (GST)/14-3-3zeta fusion protein, but not GST alone, immobilized on glutathione-Sepharose. A potential 14-3-3 binding motif was located in the 43 kDa, but not the 75 kDa, 5-phosphatase. The motif "363RSESEE" is present in close proximity to the proposed catalytic domain of the 43 kDa 5-phosphatase. A synthetic peptide corresponding to the putative 14-3-3 binding motif demonstrated specific, saturable binding to purified 125I-14-3-3, with a Kd of 92 nM. In addition, platelet cytosolic 5-phosphatase bound to recombinant 14-3-3zeta immobilized on glutathione-Sepharose. Thus, 14-3-3zeta serves in human platelets to activate the 43 kDa 5-phosphatase and may thereby function to prevent generation of Ins(1,4,5)P3 -mediated calcium release in unstimulated platelets.

Cis-FFA do not alter membrane depolarization but block Ca 2+ influx and GH secretion in KCl-stimulated somatotroph cells. : Suggestion for a direct cis-FFA perturbation of the Ca 2+ channel opening

It has been reported that cis-unsaturated free fatty acids ( cis-FFA) block intracellular Ca 2+ rise in EGFR T17 and GH 3 cells by perturbing the generation of Ins(1,4,5)P 3. In the present work, it was found that cis-FFA did not alter potassium-induced cell depolarization in GH 3 cells, while blocking Ca 2+ rise and GH secretion. Interestingly enough, saturated or trans-unsaturated FFA exert the opposite actions, i.e., they block cell depolarization without altering Ca 2+ rise and hormone secretion. As depolarization activates GH 3 cells via direct opening of Ca 2+ channels with no generation of intracellular mediators, these results suggest that cis-FFA act by a direct perturbation of the Ca 2+ channel opening.

Ethanol perturbs receptor-operated cytosolic free calcium concentration signals in cultured rat hepatocytes

BACKGROUND & AIMS: Studies of the acute effects of ethanol on hepatocellular free calcium concentration, [Ca2+]i, and on receptor- operated [Ca2+]i signals have produced conflicting results. The effects of ethanol on basal and receptor-operated [Ca2+]i signals in rat hepatocytes cultured on two different extracellular matrices were examined.METHODS: [Ca2+]i was determined by digitized fluorescence microscopy and inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) by high- performance liquid chromatography (HPLC).RESULTS: Ethanol induced an increase in [Ca2+]i in hepatocytes cultured on collagen I but not on the more physiological substratum, matrigel. Compared with hepatocytes cultured on matrigel, cells on collagen I exerted less responsiveness and lower amplitude of [Ca2+]i signals to vasopressin or phenylephrine. The effects of ethanol on receptor-operated [Ca2+]i signals were examined in hepatocytes cultured on matrigel. Incubation of hepatocytes with physiologically attainable concentrations of ethanol (20-30 mmol/L) for 30 minutes to 48 hours perturbed epidermal growth factor, phenylephrine, and lower concentration (< or = 1 nmol/L) of vasopressin- induced [Ca2+]i signaling by reducing the amplitude and changing the pattern of [Ca2+]i signals. Alcohol induced-impairment of [Ca2+]i signaling was associated with decreased production of Ins(1,4,5)P3.CONCLUSIONS: Alcohol does not itself evoke an increase in [Ca2+]i in hepatocytes cultured on matrigel but perturbs receptor-operated [Ca2+]i signaling. This is associated with and could be caused by impaired generation of Ins(1,4,5)P3.(Gastroenterology 1997 Aug;113(2):641-8)

Role of phospholipase C and D signalling pathways in vasopressin-dependent myogenic differentiation.

Arg8-vasopressin (AVP) is a potent inducer of myogenic differentiation stimulating the expression of myogenic regulatory factors. To understand the mechanism of its effect on myogenesis, we investigated the early signals induced by AVP in myogenic target cells. In the rat skeletal muscle cell line L6, AVP selectively stimulates phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) breakdown, through the activation of phospholipases C and D (PLC, PLD), as shown by the generation of Ins(1,4,5)P3 and phosphatidylethanol (PtdEtOH), respectively. AVP induces the biphasic increase of sn-1,2-diacylglycerol (DAG) consisting in a rapid peak followed by a sustained phase, and the monophasic generation of phosphatidic acid (PA). Propranolol (a PA phosphatase inhibitor) and Zn2+ (a PLD inhibitor), abolish the sustained phase of DAG generation. Our data indicate that PtdIns-PLC activity is mainly responsible for the rapid phase of AVP-dependent DAG generation, whereas the sustained phase is dependent upon PtdCho-PLD activity and PA dephosphorylation, ruling out any significant role of DAG kinase. Modifications of PA level correlate with parallel changes of PLC activity, indicating a possible cross-talk between the two signal transduction pathways in the intact cell. PLD activation is elicited at AVP concentrations two orders of magnitude lower than those required for PLC activation. The differentiation of L6 myoblasts into multinucleated fibers is stimulated significantly by AVP at concentrations at which PLD, but not PLC, is activated. These data provide the first evidence for an important role of PLD in the mechanism of AVP-induced muscle differentiation.

Role and source of ATP for activation of nonselective cation channels by AlF complex in guinea pig chromaffin cells.

Intracellular dialysis with the solution containing the G protein activator, AlF complex, induced an inward nonselective cation current (INS) at -55 mV in chromaffin cells. Amplitudes of INS induced by dialysis with ATP-free AlF solutions progressively diminished as cells were pretreated with cyanide, a mitochondrial inhibitor. After a 10-min pretreatment, generation of INS by the AlF complex depended on exogenous ATP delivered from pipette solution. The relationship between amplitudes of INS and concentrations of MgATP was well expressed by a rectangular hyperbola with an EC50 of 0.265 mM. This result suggests that the cyanide treatment almost depleted ATP near the plasma membrane. On the other hand, a similar cyanide treatment of adrenal medullary preparations did not induce a marked decrease in cellular ATP content. GTP, ITP, or UTP could not substitute for ATP in generation of INS by the AIF complex. Similarly, the substitution of ATP with non- or poorly hydrolyzable ATP analogues did not aid in generating INS. Bath application of the kinase inhibitor, H-7 (100 microM), suppressed AlF-induced INS in a manner depending on intracellular Mg2+. We conclude that ATP is a prerequisite for generation of INS as a phosphoryl donor and that mitochondria is the main source of ATP.

Chronic ethanol administration to rats decreases receptor-operated mobilization of intracellular ionic calcium in cultured hepatocytes and inhibits 1,4,5-inositol trisphosphate production: relevance to impaired liver regeneration.

We tested the hypothesis that ethanol impairs liver regeneration by abrogating receptor-mediated elevation of cytosolic free calcium ([Ca2+]i). In rats fed for 16 weeks with ethanol, hepatocellular proliferation induced by partial hepatectomy was greatly impaired. Similarly, EGF-induced DNA synthesis was reduced in cultured hepatocytes from ethanol-fed rats. There was no change in the number or affinity of EGF receptors on hepatocytes from ethanol-fed rats. Despite this, EGF-mediated production of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) was lower in hepatocytes from ethanol-fed rats, and the EGF-induced [Ca2+]i transient appeared to be abrogated. When vasopressin or phenylephrine were used as cell surface receptor ligands, hepatocytes cultured from ethanol-fed rats exhibited major reductions in Ins(1,4,5)P3 synthesis. This was associated with greatly truncated [Ca2+]i transients. These changes were not due to an effect on the Ins(1,4,5)P3 receptor on the endoplasmic reticulum or to a decrease in the size of the Ins(1,4,5)P3-mobilizable intracellular Ca+2 store. Further, mobilization of the same Ca2+ store by 2,5-di-tert-butylhydroquinone or thapsigargin restored the ability of hepatocytes from ethanol-fed rats to proliferate when exposed to EGF. It is concluded that chronic ethanol consumption inhibits liver regeneration by a mechanism that is, at least partly, the result of impaired receptor-operated [Ca2+]i signaling due to reduced generation of Ins(1,4,5)P3.

ADP indirectly supports activation of non-selective cation channels by AIF complex in guinea-pig chromaffin cells

Infusion of the G protein activator, AIF complex, into guinea-pig adrenal chromaffin cells produced an inward non-selective cation current ( I NS) at −55 mV. Under metabolically suppressed conditions, this current was abolished by removal of ATP from the AIF-containing pipette solution. ADP could substitute for ATP in generation of the current. This supporting action of ADP was eliminated by addition to the AIF solution of the ATP-consuming system (hexokinase and 2-deoxyglucose). A similar generation of INS occurred when AMP and phosphocreatine were simultaneously added to AIF solution, but not when either of the agents was added separately. These results suggest that under conditions of whole-cell current recordings, the machinery for ATP production is preserved and that ADP indirectly supports generation of INS by the AIF complex.

Interferon-gamma increases inositol phosphate formation and cellular calcium ion concentration independent of ICAM-1 antigen enhancement in renal tubular cells.

In the present study, we investigated the effect of interferon-gamma (IFN-gamma) on cellular inositol phosphate formation and cellular calcium ion concentration [Ca2+]i in human renal proximal tubular (HRPT) cells. We also examined the possible role of the inositol phosphate-Ca2+ signalling pathway during IFN-gamma-induced intercellular adhesion molecule-1 (ICAM-1) antigen expression. IFN-gamma caused an increase in the formation of inositol 1-monophosphate (Ins 1-P), inositol 1,4-bisphosphate (Ins 1,4-P2), inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4). A rapid time-dependent rise in [Ca2+]i was observed upon IFN-gamma stimulation, with maximal levels reached after 1 min. A lower rise in [Ca2+]i was observed when cells were stimulated in Ca2+-free medium. This correlated with the generation of Ins 1,4,5-P3 by IFN-gamma, a well-known secondary messenger capable of releasing Ca2+ from intracellular stores. The induction of ICAM-1 antigen expression was enhanced by IFN-gamma, 4-bromocalcium ionophore A23187 (Bromo-A23187), and their combinations. However, the calcium antagonist diltiazem and calcium chelator EGTA had no effect on IFN-gamma antigen induction. In conclusion, our data suggest that IFN-gamma stimulation of HRPT cells results in the cleavage of phosphatidylinositol bisphosphate by phospholipase C, generating inositol phosphates, of which Ins 1,4,5-P3 probably releases Ca2+ from intracellular stores. A further increase in [Ca2+]i upon IFN-gamma stimulation results from influx of extracellular Ca2+. IFN-gamma signal transduction in HRPT cells may not be limited to the inositol phosphate-Ca2+ pathway since IFN-gamma-induced ICAM-1 antigen expression was unaffected by calcium antagonist/chelator.

The whoosh and trickle of calcium signalling

The importance of phospholipase C catalysed hydrolysis of phosphatidylinositol-(4,5)bisphosphate (Ptdlns(4,5)P 2) to inositol-(1,4,5)trisphosphate (Ins(1,4,5)P 3) and sn-1,2-diacylglycerol in the signal transduction pathways of eukaryote cells, in response to extracellular stimuli, is now widely recognised. Although nearly 60 naturally occurring inositol phosphates have been identified in mammalian cells, mobilisation of Ca 2+ from the intracellular stores has been most commonly attributed to the generation of Ins(1,4,5)P 3 [1]. However, there is increasing evidence for the presence of ryanodine receptors (RyR) in non-excitable cells and for cADP-ribose (cADPr) as the signalling molecule responsible for Ca 2+ release via the RyR. But what is the purpose for the co-existence of these two intracellular Ca 2+ channels in non-excitable cells and why are they so heterogeneous in their distribution? These questions were explored at the recent International Symposium Calcium Signalling in Inflammatory Cells. Depletion of the intracellular Ca 2+ pools is followed by entry of Ca 2+ into the cell across the plasma membrane, but the mechanism(s) underlying this `capacitative Ca 2+ entry is not well understood. Many potential signalling pathways which may account for capacitative Ca 2+ entry have been proposed although none have been unanimously accepted. New developments in the elucidation of the mechanism responsible for capacitative Ca 2+ entry and how Ca 2+ entry is regulated, together with progress in the characterisation of plasma membrane Ca 2+ entry channels were also discussed at this symposium.

Neuropeptide YY1 ReceptorsMediated Increase in Intracellular Ca2+ Concentration via Phospholipase C-Dependent Pathway in Porcine Aortic Smooth Muscle Cells

In porcine aortic smooth muscle cells, neuropeptide Y (NPY) stimulates mobilization of CA(2+) from intracellular store sites via Y1 receptors. However, it has been debated whether or not Ca(2+) mobilization by Y1 receptors depends on the generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] following activation of phospholipase C. To examine this question, we studied the effect of U73122, an inhibitor of phospholipase C-mediated inositol phosphate accumulation on the NPY-induced rise in cytosolic free Ca(2+) concentration ([Ca(2+)]i) in comparison with that on angiotensin II (AII)-induced [Ca(2+)]i increase, which is dependent on Ins(1,4,5)P3 generation. Digital-imaging microscopy study using the Ca(2+)-sensitive dye fura-2 revealed that application of AII induced a rapid but transient [Ca(2+)]i increase in a single cell, arising from intracellular calcium stores. Application of NPY to the same cell induced a [Ca(2+)]i rise with a pattern similar to that induced by AII. AII increased the formation of Ins(1,4,5)P3 by about 3.0 fold, while the NPY-induced [Ca(2+) formation was very small. U73122 completely inhibited not only Ins(1,4,5)P3 synthesis, but also Ca(2+) mobilization induced by either agonist. The effect of U73122 on the NPY-induced Ca(2+)i increase was about 10-fold more potent that on the AII-induced one. U73343, an inactive analog of U733343, had no influence on any of the AII- and NPY-mediated effects. Herbimycin A completely inhibited the platelet-derived growth factor-induced [Ca(2+]i increase but had no effect on the NPY-induced [Ca(2+)]i increase, indicating that phospholipase C-gamma is not involved in the NPY effect. These results suggest that NPY-induced Ca2+ mobilization from intracellular stores in porcine smooth muscle cells is secondary to the very small generation of Ins(1,4,5)P3 following stimulation of phospholipase C-beta, which may account for the hypersensitivity of the NPY effect to U73122.

Phosphatase is responsible for run down, and probably G protein-mediated inhibition of inwardly rectifying K+ currents in guinea pig chromaffin cells.

The mechanism of G protein-mediated inhibition of an inwardly rectifying K+ current (IIR) in adrenal chromaffin cells was investigated using the whole-cell version of the patch clamp technique. In case of recording with use of ATP-containing patch solution, the IIR was well maintained; otherwise, it ran down within 15 min. This run down was not prevented by replacement with adenylyl-imidodiphosphate, a nonhydrolysable analogue of ATP, but was markedly reduced by the addition to the ATP-free solution of 1 microM calyculin A, a specific inhibitor of serine/threonine phosphatase 1 (PP1) and 2A (PP2A). The addition of alkaline phosphatase to the ATP-containing solution facilitated run down of the current, and application of 100 microM H-7, a general kinase inhibitor, reversibly suppressed IIR. These results taken together suggest that inwardly rectifying K+ channels are under the influence of kinase and phosphatase without external signals. Infusion of nonhydrolysable analogues of GTP, guanosine-5'-O-(3-thiophosphate) (GTP gamma S) or guanylyl-imidodiphosphate, through the pipette produced little inward current at -55 mV, but completely inhibited IIR within approximately 5 or 6 min in all cells tested in the presence of 12 microM Mg2+ inside the cell. In contrast, infusion of aluminum fluoride (AlF) complex, another GTP binding (G) protein activator, consistently produced large inward currents, but did not alter IIR noticeably for 15 min in 17% of the cells tested. In the other cells, the inhibition of IIR developed slowly after long latent periods. This inhibitory potency of AlF was not enhanced by an increase in Mg2+ concentrations. Subtraction of the current-voltage relationship before from that noted during the generation of inward current by AlF complex revealed that the inward current diminished progressively with hyperpolarizations, as is the case with a nonselective cation current (INS) induced by a muscarinic agonist. Thus, AlF complex seems to be potent with the generation of INS, but not with IIR inhibition. The addition of 3 microM calyculin A significantly retarded the IIR inhibition by GTP gamma S, whereas that of 1 microM okadaic acid, another inhibitor of PPI and PP2A, markedly prevented the decline of IIR by AIF complex. Our observations suggest that the low potency of AlF complex in inhibiting IIR may be due to interference with phosphatase activity and that the activation of G protein suppresses IIR, probably by enhancing the apparent activity of phosphatase, which may explain run down of the current.

Mechanism of activation of nonselective cation channels by putative M4 muscarinic receptor in guinea-pig chromaffin cells.

1. Mechanisms involved in the generation of nonselective cation currents (INS) by muscarinic agonists in the chromaffin cell were investigated by the perforated patch method. 2. Bath application of muscarine (0.1-30 microM) produced an inward INS with or without a transient outward current at -40 mV, whereas oxotremorine (0.06-60 microM) induced INS alone. Rectangular hyperbolas with EC50s of 2.01 and 0.21 microM were fitted to muscarine- and oxotremorine-induced INSS, respectively, and the maximal amplitude of the former was about 3.4 times larger than that of the latter. 3. In 36% of the cells exposed to Ca(2+)-free solution, muscarine INS was suppressed, being 53% of control 20 min after the perfusion, and in four cells that were incubated with Ca(2+)-free solution for 2 h or more, the INS averaged 44% of that induced subsequently in normal solution. In contrast, muscarine INS was enhanced by about 30% when A-23187 was added to normal solution. 4. W-7 and W-5, calmodulin-related agents, were almost equally potent in inhibiting muscarine INS, whereas compound 5, a potent inhibitor of calmodulin-dependent kinase II (CaM kinase II), produced no evident inhibition. 5. HA1004, a weak kinase C inhibitor, induced a reversible suppression of muscarine INS with an IC50 of 163 microM, whereas H-8, another kinase inhibitor, produced an even small degree of inhibition. Administration of phorbol 12, 13-dibutyrate did not mimic muscarinic stimulation of NS channels; rather, it led to a progressive inhibition of INS and this inhibition was almost complete within 20 min. An inactive phorbol ester had no such effect. 6. The muscarinic antagonists, pirenzepine and AF-DX 116, shifted the dose-response curve for the muscarine INs to the right in a parallel manner. The KDS for pirenzepine and AF-DX 116 were estimated to be 13 nM (95% confidence interval, 11-16 nM) and 365 nM (283-470 nM), respectively.7. These results suggest that muscarine efficiently produces INS, probably through binding to the M4 subtype, that intracellular Ca2+ has a facilitating, but not an essential role in the generation of INs, and that neither CaM kinase II nor protein kinase C is involved.

Ins [formula omitted] and Ca 2+ signaling in quiescent neonatal cardiac myocytes

Activation of α 1- adrenergic receptors in neonatal cardiac myocytes results in changes in contractile activity and the induction of hypertrophic growth. The biochemical mechanisms responsible for these diverse effects are not yet established, but presumably involve the associated α 1- adrenergic stimulation of phosphatidylinositol (PI) hydrolysis, with concomitant generation of Ins 1,4,5-P 3 and diacylglycerol. This study examined whether α 1- adrenergic generation of Ins 1,4,5-P 3 in intact, quiescent, neonatal cardiac myocytes resulted in a Ca 2+ signal. Stimulation of myocytes with norepinephrine in the presence of propranolol caused accumulation of inositol mono-, bis and trisphosphates. However, α 1- adrenergic stimulation did not alter cytosolic free Ca 2+ levels in 85% of the myocytes examined. Direct generation of Ins 1,4,5-P 3, by photolysis of microinjected caged Ins 1,4,5-P 3, was also unable to alter cytosolic free Ca 2+ levels, despite the presence of Ins 1,4,5-P 3 receptors. Taken together, these data indicated that α 1- adrenergic stimulation did not initiate Ca 2+ signaling because Ins 1,4,5-P 3-induced Ca 2+ mobilization was not operative in quiescent neonatal cardiac myocytes. Normal excitation-contraction Ca 2+ handling mechanisms were present in these cells, as illustrated by depolarization- and caffeine-induced Ca 2+ transients. Analysis of these same myocytes following 48 h in the presence of norepinephrine and propranolol showed a 40% increase in the ration of protein to DNA and a 350% increase in release of atrial naturietic factor, compared to control cells, indicating the normal operation of α 1- adrenergic-induced hypertrophic growth. Therefore, the assumption that Ca 2+-dependent processes will be activated by receptor signaling pathways coupled to enhanced phosphatidylinositol turnover in cardiac cells must be avoided. In addition, the data presented in this study clearly indicated that an increase in cytosolic free Ca 2+ was not necessary for the induction of α 1- adrenergic-mediated cardiac hypertrophy.

Effects of membrane depolarization and changes in intra- and extracellular calcium concentration on phosphoinositide hydrolysis in bovine tracheal smooth muscle

Agonist-stimulated phosphoinositide metabolism plays a central role in pharmacomechanical coupling in airways smooth muscle (ASM). In many other tissues and cells, most noteably excitable cells, membrane depolarization or an increase in intracellular Ca 2+ ([Ca 2+] i) generated by inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3)-induced Ca 2+ release or agonist-mediated Ca 2+influx is able to trigger or augment phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2) hydrolysis and/or initiate PtdIns4P/ PtdIns hydrolysis by direct stimulation of PIC. To assess the importance of these mechanisms in ASM the effects of KCl-induced membrane depolarization, extracellular Ca 2+([Ca 2+] e) chelation, and addition of ionomycin to elevate [Ca 2+] i on basal and agonist-stimulated Ins(1,4,5)P 3 concentration and [ 3H]-InsP x accumulation have been examined. Reducing [Ca 2+] e from 1.8 mM to 6 or 0.8μM caused a progressive inhibition of agonist-stimulated [ 3H]inositol polyphosphate accumulation over 30 min with the histamine-stimulated response being significantly more sensitive to [Ca 2+] e chelation than the response to carbachol. In contrast, the initial accumulation of Ins(1,4,5)P 3 was completely unaffected by such reductions in [Ca 2+] e. Incubation of [ 3H]inositol-prelabelled BTSM slices with buffer containing 80 mM KC1 failed to stimulate [ 3H]InsP x accumulation, causing instead a small inhibition of carbacholstimulated [ 3H]InsP x accumulation with a similar effect seen with respect to Ins(1,4,5)P 3 accumulation. Addition of 5 μM ionomycin to BTSM slices similarily did not stimulate Ins(1,4,5)P 3 generation and only increased [ 3H]InsP x accumulation after prolonged stimulation in the presence of high (mM) [Ca 2+] e. These data indicated that in ASM, membrane depolarization or physiological increases in [Ca 2+] i did not result in either independent activation of PIC or augmentation of initial agoniststimulated PtdIns(4,5)P 2 hydrolysis. However, while the initial agonist-stimulated generation of Ins(1,4,5)P 3 was not dependent on [Ca 2+] e, a normal plasmalemmal Ca 2+ gradient was required to sustain maximal rates of agonist-stimulated PtdIns(4,5)P 2 hydrolysis.

STIMULATION OF PHOSPHATIDYLINOSITOL TURNOVER IN ADULT RAT LEFT ATRIA DOES NOT INVOLVE RELEASE OF INOSITOL (1,4,5) TRI SPHOSPHATE

1. The turnover rate of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) in noradrenaline-stimulated adult rat left atria was calculated from changes in specific activity and was found to equal 110 ct/min per mg tissue. In contrast, the isomers of inositol mono- and bisphosphates accumulated at a rate of 508 ct/min per mg. 2. Neomycin, which inhibits release of Ins(1,4,5)P3, inhibited the accumulation of inositol phosphates in noradrenaline-stimulated isolated neonatal cardiomyocytes but did not inhibit accumulation in left atria. 3. These data demonstrate that most of the inositol phosphates which accumulate in adult rat left atria do not derive from Ins(1,4,5)P3. 4. These data are best explained by a model in which noradrenaline stimulation results mainly in the breakdown of phosphatidylinositol(4)monophosphate (PtIns(4)P1) to inositol(1,4)bisphosphate (Ins(1,4)P2). Thus, heart tissue avoids the generation of Ins(1,4,5)P3.

Modulation of inositol(1,4,5)trisphosphate-sensitive calcium store content during continuous receptor activation and its effects on calcium entry

Changes in intracellular Ca 2+ concentration ([Ca 2+] i) following the activation of muscarinic receptors with carbachol were studied in cells from the exocrine avian nasal gland that had been maintained in culture for 40–48 h. In these cells, the carbachol-induced sustained increase in [Ca 2+] i could be further increased by the subsequent addition of thapsigargin. This increase was due to an additional release of intracellular Ca 2+ and a corresponding further enhancement of Ca 2+ entry. However, thapsigargin-sensitive and Ins(1,4,5)P 3-sensitive stores appeared to be coincident and the initial carbachol stimulus was sufficient to completely empty these stores. It was concluded that the subsequent effect of thapsigargin was due to a partial refilling of the Ins(1,4,5)P 3-sensitive stores despite the continued presence of agonist, an effect that was not the result of any decline in levels of cellular Ins(1,4,5)P 3 or changes in the generation of Ins(1,3,4,5)P 4, which were sustained throughout. Possible explanations for this refilling response include compartmentalization of intracellular Ins(1,4,5)P 3, or a desensitization of the Ins(1,4,5)P 3 receptor/Ca 2+-release channel. Alternatively, the data are also compatible with a recently proposed kinetic separation of Ca 2+ uptake and release sites. An important implication of this particular interpretation of our findings would be an apparent dependence of Ca 2+ entry specifically on the status of the Ca 2+- uptake component of the agonist-sensitive store, rather than the Ca 2+-release component.

Regulation of Ca2+ influx in myeloid cells. Role of plasma membrane potential, inositol phosphates, cytosolic free [Ca2+], and filling state of intracellular Ca2+ stores.

To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.

Calcium-sensitivity of inositol 1,4,5-trisphosphate metabolism in exocrine cells from the avian salt gland.

The generation of inositol phosphates upon muscarinic-receptor activation was studied in [3H]inositol-loaded exocrine cells from the nasal salt glands of the duck Anas platyrhynchos, and the metabolism of different inositol phosphates in vitro was studied in tissue homogenates, with particular reference to the possible interaction of changes in intracellular [Ca2+] ([Ca2+]i) with the metabolic processes. In intact cells, there was a rapid (within 15 s) generation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, followed by an accumulation of their breakdown products, Ins(1,3,4)P3 and inositol bis- and monophosphates. Ca(2+)-sensitivity of the Ins(1,4,5)P3 3-kinase was demonstrated in tissue homogenates, with the rate of phosphorylation increasing 2-fold at free Ca2+ concentrations greater than 1 microM. However, addition of calmodulin or the presence of the calmodulin inhibitor W-7 (up to 100 microM) had no effect. 3-Kinase activity increased proportionally with the initial Ins(1,4,5)P3 concentration up to 1 microM, but a 10-fold higher substrate concentration produced only a doubling in the phosphorylation rate. Ins(1,3,4,5)P4 was dephosphorylated to Ins(1,3,4)P3, which accumulated in the homogenate assays as well as in intact cells. Depending on its concentration, Ins(1,3,4)P3 was phosphorylated [in part to Ins(1,3,4,6)P4] or dephosphorylated. To investigate the Ca(2+)-sensitivity of the 3-kinase in intact cells, excess quin2 was used to buffer the receptor-mediated transient changes in [Ca2+]i in [3H]inositol-loaded cells. These experiments revealed that increasing [Ca2+]i from less than 100 to approx. 400 nM (i.e. within the physiological range) has no effect on the partitioning of Ins(1,4,5)P3 metabolism (phosphorylation versus dephosphorylation) and on the accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. This indicates that activation of the 3-kinase by physiologically relevant Ca2+ concentrations may not play a major role in the generation of Ins(1,3,4,5)P4 signals upon receptor activation in these cells. The latter are mainly achieved by the receptor-mediated increase in Ins(1,4,5)P3 in the cell and its phosphorylation by the 3-kinase in a substrate-concentration-dependent manner.

The role of caffeine-sensitive Ca2+ stores in agonist- and inositol 1,4,5-trisphosphate-induced Ca2+ release from bovine adrenal chromaffin cells

In single bovine adrenal chromaffin cells loaded with fura-2, histamine, angiotensin II (AII) and caffeine elicited large transient increases of intracellular free Ca2+ concentration [( Ca2+]i) in the absence of external Ca2+, with peak amplitudes averaging 726 +/- 138 (n = 14), 710 +/- 102 (n = 21) and 830 +/- 100 nM (n = 30) respectively. A substantial portion of the agonist-induced rise in [Ca2+]i depended on Ca2+ release from caffeine-sensitive stores, as pretreatment with caffeine diminished subsequent agonist responses by 90-95%. Conversely, pretreatment with histamine or AII decreased subsequent caffeine responses by 100% and 90% respectively. The effects of caffeine most likely resulted from activation of a Ca(2+)-induced Ca(2+)-release (CICR) process, whereas histamine and AII initially acted through generation of Ins(1,4,5)P3. The relationship of Ins(1,4,5)P3- and caffeine-sensitive Ca2+ pools was studied by using alpha-toxin-permeabilized chromaffin cells. Evidence was found for three non-mitochondrial, ATP-dependent, Ca2+ pools: one exclusively sensitive to Ins(1,4,5)P3 (pool 1), a second sensitive to both Ins(1,4,5)P3 and caffeine (pool 2), and a third exclusively sensitive to caffeine (pool 3). The existence of pools 1 and 3, and the ability of agonists such as histamine to discharge pool 3 completely, supports a two-pool model in which a caffeine-sensitive CICR mechanism plays a major role in the generation of agonist-induced Ca2+ spikes in bovine chromaffin cells.