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PubMed1994

Inositol-1,4,5-trisphosphate [ins(1,4,5)P3] and ins(1,4,5)P3 receptor concentrations in heart tissues.

1. The isolation and culture of neonatal cardiomyocytes causes changes in the metabolism of inositol(1,4,5) trisphosphate (Ins(1,4,5)P3) from primarily dephosphorylation in the intact tissue to a combination of phosphorylation and dephosphorylation in the cultured cells (Woodcock et al. 1992). 2. The content of Ins(1,4,5)P3 was found to be higher in intact heart tissue than in the isolated neonatal cells (10.9 +/- 1.3 and 0.5 +/- 0.1 pmol/mg tissue, mean +/- s.e.m., n = 4, P < 0.002, respectively). 3. Despite this difference, Ins(1,4,5)P3 receptors in intact tissue and in isolated cells were not different in terms of affinity (8.0 +/- 1.7 and 10.9 +/- 1.6 nmol/L, n = 3, respectively) or concentration (143.3 +/- 20.5 and 91.2 +/- 16.0 fmol/mg protein, n = 3, respectively). 4. Thus, while there appears to be a relationship between the tissue content of Ins(1,4,5)P3 and its metabolism, no relationship to the properties of Ins(1,4,5)P3 receptors could be demonstrated.

Animals↗
PubMed1991

Accumulation and metabolism of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in muscarinic-receptor-stimulated SH-SY5Y neuroblastoma cells.

Stimulation of M3 muscarinic receptors expressed by SH-SY5Y cells induced a dose- and time-related increase in the mass of Ins(1,4,5)P3 (basal 38.3 +/- 5.8 pmol/mg of protein) and Ins(1,3,4,5)P4 (basal 6.1 +/- 1.2 pmol/mg of protein). Comparison of radioreceptor mass assays with [3H]inositol labelling showed higher-fold stimulations with the former protocol. The later accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 mass was dependent upon extracellular Ca2+.

Animals↗
PubMed2003

Solution conformations of mycothiol bimane, 1-D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins and 1-D-GlcNAc-alpha-(1 --> 1)-L-myo-Ins.

Mycothiol is an abundant small molecular weight thiol found only in actinomycetes, which include mycobacteria. Mycothiol biosynthetic and detoxification enzymes are novel and unique to actinomycetes, thereby representing potential antimycobacterial targets. To better guide inhibitor design, we have determined by NMR the solution conformations of mycothiol bimane (MSmB) and the pseudodisaccharide 1-D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins (D-GI), molecules that represent the natural substrates for the mycothiol-dependent detoxification enzyme mycothiol-S-conjugate amidase (MCA) and the mycothiol biosynthetic enzyme D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins deacetylase (AcGI deacetylase), respectively. Comparison of the mean structure of MSmB and the energy-minimized structures of two competitive spiroisoxazoline-containing MCA inhibitors shows striking similarities between these molecules in the region of the scissile amide bond of MSmB and provides structural evidence that those inhibitors are substrate mimics. Owing to our earlier finding that AcGI deacetylase will not deacetylate the unnatural isomer 1-d-GlcNAc-alpha-(1 --> 1)-L-myo-Ins (L-GI), the solution conformation of L-GI was also determined. The interglycosidic bond angles for all three compounds are comparable. When considered together with the observation that a simplified cyclohexyl thioglycoside mycothiol analogue is a good substrate for MCA, it appears that the stereochemistry of the inositol ring is critical for deacetylase function, superceding the importance of the full complement of hydroxyl groups on the "nonreducing" ring.

Actinomyces↗
PubMed1997

A convenient synthesis of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and L-myo-inositol 1,4,5-trisphosphate (Ins(3,5,6)P3).

An efficient synthesis of an optically active inositol derivative that is a precursor to D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3, (-)) is described. Crystallization of the diastereomers of (+/-)-1-O-[(+)-menthoxycarbonyl]-6-O-benzyl-2,3:4,5-di-O-isopropyl idene-myo- inositol diastereomers from methanol gives only one diastereomer. Alkaline hydrolysis gives the useful inositol derivative (-)-6-O-benzyl-2,3:4,5-di-O-isopropylidene-myo-inositol. Likewise, crystallization of the diastereomers of (+/-)-3-O-[(-)-menthoxycarbonyl]-4-O-benzyl-1,2:5,6-di-O-isopropyl idene-myo- inositol from methanol gave a pure compound which could be hydrolyzed to give (+)-4-O-benzyl-1,2:5,6-di-O-isopropylidene-myo-inositol, a precursor to D-myo-inositol 3,5,6-trisphosphate (Ins(3,5,6)P3,(+)). The ease with which these enantiomerically pure inositol derivatives were isolated may facilitate the synthesis of more complex inositol phosphate derivatives such as D-myo-inositol 1,3,4,5-tetrakisphosphate.

Inositol 1,4,5-Trisphosphate↗
PubMed1999

Regulation of cerebellar Ins(1,4,5)P3 receptor by interaction between Ins(1,4,5)P3 and Ca2+.

We have characterized in detail the Ca(2+)-dependent inhibition of [(3)H]Ins(1,4,5)P(3) ([(3)H]InsP(3)) binding to sheep cerebellar microsomes, over a short duration (3 s), with the use of a perfusion protocol. This procedure prevented artifacts previously identified in studies of this Ca(2+) effect. In a cytosol-like medium at pH 7.1 and 20 degrees C, a maximal inhibition of approx. 50% was measured. Both inhibition and its reversal were complete within 3 s. Ca(2+) decreased the affinity of the receptor for InsP(3) by approx. 50% (K(d) 146+/-24 nM at pCa 9 and 321+/-56 nM at pCa 5.3), without changing the total number of binding sites. Conversely, increasing the [(3)H]InsP(3) concentration from 30 to 400 nM tripled the IC(50) for Ca(2+) and decreased the maximal inhibition by 63%. This is similar to a partial competitive inhibition between InsP(3) binding and inhibitory Ca(2+) binding and is consistent with InsP(3) and Ca(2+) converting InsP(3) receptor into two different states with different affinities for these ligands. Mn(2+) and Sr(2+) also inhibited [(3)H]InsP(3) binding but were respectively only 1/10 and 1/200 as effective as Ca(2+). No inhibition was observed with Ba(2+). This selectivity is the same as that previously reported for the inhibitory Ca(2+) site of InsP(3)-induced Ca(2+) flux, suggesting that the same site is used by Ca(2+) to convert cerebellar InsP(3) receptor to a low-affinity state and to inhibit its channel activity. Our results also suggest a mechanism by which InsP(3) counteracts this Ca(2+)-dependent inhibition.

Amino Acid Sequence↗
PubMed1999

Recombinant p42IP4, a brain-specific 42-kDa high-affinity Ins(1,3,4,5)P4 receptor protein, specifically interacts with lipid membranes containing Ptd-Ins(3,4,5)P3.

We have recently cloned the cDNA of p42IP4, a membrane-associated and cytosolic inositol (1,3,4,5)tetrakisphosphate receptor protein [Stricker, R., Hülser, E., Fischer, J., Jarchau, T., Walter, U., Lottspeich, F. & Reiser, G. (1997) FEBS Lett. 405, 229-236.] p42IP4 is a protein of 374 amino acids with Mr of 42 kDa. The p42IP4 protein has a zinc finger motif at its N-terminus, followed by two pleckstrin homology domains. To characterize further the biochemical and functional properties of p42IP4, it was expressed as a glutathione-S-transferase fusion protein in Sf9 cells using a recombinant baculovirus vector. The protein was affinity adsorbed on glutathione beads, cleaved from glutathione-S-transferase with the protease factor-Xa and purified on heparin agarose. The recombinant purified protein is active because it shows binding affinities similar to those of the native p42IP4, purified from pig cerebellum or rat brain (Ki for inositol(1,3,4,5)P4 of 4.1 nm and 2.2 nm, respectively). Moreover the ligand specificity of the recombinant protein for various inositol polyphosphates is similar to that of the native protein purified from brain. Importantly, we show here that p42IP4 binds phosphatidylinositol(3,4,5)P3 specifically, as the recombinant protein can associate with lipid membranes (vesicles) containing phosphatidylinositol(3,4,5)P3; this binding occurs in a concentration-dependent manner and is blocked by inositol(1,3,4,5)P4. This specific association and the possibility that endogenous p42IP4 can be converted from a membrane-associated state to a soluble state support the hypothesis that p42IP4 might be redistributed between cellular compartments upon hormonal stimulation.

Animals↗
PubMed1998

Inositol hexakisphosphate in Schizosaccharomyces pombe: synthesis from Ins(1,4,5)P3 and osmotic regulation.

Schizosaccharomyces pombe extracts synthesize InsP6 (myo-inositol hexaphosphate) from Ins(1,4,5)P3 plus ATP. An S. pombe soluble fraction converts Ins(1,4,5)P3 into Ins(1,4,5,6)P4 and Ins(1,3,4, 5)P4, in a constant ratio of approximately 5:1, and thence to Ins(1, 3,4,5,6)P5 and InsP6. We have purified a soluble Mg2+-dependent kinase of molecular mass approximately 41 kDa that makes Ins(1,4,5, 6)P4 and Ins(1,3,4,5)P4 in the same ratio and also converts Ins(1,4, 5,6)P4 or Ins(1,3,4,5)P4 into Ins(1,3,4,5,6)P5 and InsP6. Of InsP3 isomers other than Ins(1,4,5)P3, only the non-biological molecule Ins(1,4,6)P3 potently 'competed' with all steps in conversion of Ins(1,4,5)P3 into InsP6. Examination of molecular graphics representations allowed us to draw tentative conclusions about the environment needed for an hydroxyl group to be phosphorylated by this kinase and to predict successfully that the purified kinase would phosphorylate the 5-hydroxyl of Ins(1,4,6)P3. S. pombe that have been cultured with [3H]inositol contains a variety of 3H-labelled inositol polyphosphates, with Ins(1,4,5)P3 and InsP6 the most prominent, and the InsP6 concentration quickly increases in hyper-osmotically stressed S. pombe. This yeast therefore contains InsP6 and Ins(1,4,5)P3 as normal constituents, makes more InsP6 when hyper-osmotically stressed and contains a versatile inositol polyphosphate kinase that synthesizes InsP6 from Ins(1,4,5)P3.

Homeostasis↗
PubMed1997

Rapid kinetic measurements of 45Ca2+ mobilization reveal that Ins(2,4,5)P3 is a partial agonist at hepatic InsP3 receptors.

Ins(2,4,5)P3, a metabolically stable analogue of Ins(1,4,5)P3, is widely used in analyses of Ca2+ signalling pathways, but its utility depends upon it faithfully mimicking the effects of the natural messenger, Ins(1,4,5)P3, at InsP3 receptors. To compare the kinetics of InsP3-evoked 45Ca2+ mobilization, Ins(1,4,5)P3- and Ins(2,4,5)P3-stimulated 45Ca2+ release from the intracellular stores of permeabilized rat hepatocytes was measured using rapid superfusion. Both Ins(1,4,5)P3 and Ins(2,4,5)P3 caused concentration-dependent increases in the rate of 45Ca2+ efflux, which accelerated towards a peak and then abruptly switched to a bi-exponentially decaying release rate. However, the peak rate of 45Ca2+ mobilization evoked by maximal concentrations of Ins(2,4,5)P3 was only 65+/-3% (n = 3) of that evoked by Ins(1,4,5)P3. Furthermore, Ins(2,4,5)P3 inhibited the peak rate of 45Ca2+ efflux evoked by Ins(1,4,5)P3. These results indicate that Ins(2,4,5)P3 is a partial agonist at hepatic Ins(1,4,5)P3 receptors. Additionally, responses to Ins(2,4,5)P3 were less positively cooperative [Hill coefficient (h) = 1.9+/-0.3] than were those to Ins(1,4,5)P3 (h = 3.0+/-0.2) and the kinetics of termination of 45Ca2+ mobilization were slower. The lesser efficacy of Ins(2,4,5)P3 may account for the lower cooperativity in the responses it evokes, the slower inactivation of InsP3 receptors and the characteristic patterns of Ca2+ spiking it evokes in intact cells.

Animals↗
PubMed1997

Conformational changes in plant Ins(1,4,5)P3 receptor on interaction with different myo-inositol trisphosphates and its effect on Ca2+ release from microsomal fraction and liposomes.

The interaction of the only reported plant inositol trisphosphate receptor with different myo-inositol trisphosphates (InsP3 species), namely Ins(1,4,5)P3, Ins(1,3,4)P3, Ins(1,5,6)P3, and Ins(2,4,5)P3, were studied to assess the extent of Ca2+ mobilization from microsomes/vacuoles as well as liposomes in vitro. Ins(1,4,5)P3 and Ins(2,4,5)P3 bind with the receptor with comparable affinities, as evidenced from their dissociation constants (Kd approx. 100 nM at 5 degrees C), whereas the interaction between Ins(1,3,4)P3/Ins(1,5,6)P3 and the receptor was not detected even with these ligands at 5 microM. Ins(1,3,4)P3/Ins(1,5,6)P3 isomers also do not elicit Ca2+ release from liposomes or microsomes/ vacuoles. The ability of any InsP3 to bind the receptor for Ins(1,4,5)P3 is a prime requirement for Ca2+ release. However, the comparison of binding affinities at a single temperature does not help to correlate it directly with the extent of Ca2+ release from the intracellular stores because the concentration of Ca2+ released by Ins(1,4,5)P3 as estimated over a period of 20 s is 3500 +/- 200 nM/mg of protein and is about 4-fold higher than that by Ins(2,4,5)P3 under identical conditions. To understand the role of the receptor conformation in Ca2+ release by different isomers, we have probed the conformational change of the receptor when the different isomers bind to it. Accessibility of the tryptophan residues in the free and Ins(1,4,5)P3/Ins(2,4,5)P3-bound receptor was monitored by a neutral fluorescence quencher, acrylamide. The resulting Stern-Volmer-type quenching plots of the internal fluorescence indicate a change in the conformation of the receptor on binding to Ins(1,4,5)P3 and Ins(2,4,5)P3. It is also detected when far-UV CD spectra (205-250 nm) of the free and ligand [Ins(1,4,5)P3/Ins(2,4,5)P3]-bound receptor are compared. The results from CD spectroscopic studies further indicate that the conformational changes induced by the two isomers are different in nature. When thermodynamic parameters, such as enthalpy (delta H), entropy (delta S) and free energy (delta G), for the formation of the two InsP3-receptor complexes are compared, a major difference in the extent of changes in enthalpy and entropy is noted. All these findings taken together support the proposition that it is the overall interaction leading to the requisite conformational change in the receptor that determines the potency of the InsP3 isomers in their abilities of Ca2+ mobilization from the intracellular stores or reconstituted liposomes.

Acrylamide↗
PubMed1989

Soluble and particulate Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase in bovine brain.

Ins(1,4,5)P3 5-phosphatase catalyses the dephosphorylation of Ins(1,4,5)P3 in the 5 position. At 1 microM Ins(1,4,5)P3, 10-15% of total activity of a bovine brain homogenate was measured in the soluble fraction, whereas 85-90% was in the particulate fraction. Particulate activity could be solubilized by cholate or, to a lower extent, by 2 M KCl. Two soluble enzymes (type I and type II) could be fractionated by DEAE-Sephacel chromatography. Soluble activities have been further purified by blue-Sepharose, Sephacryl S-200 and phosphocellulose chromatography. Specific activities reached 10-30 mumol.min-1 mg protein-1 for type I and were 10-20 times lower for type II. Type I and type II Ins(1,4,5)P3 5-phosphatase displayed different Km values and molecular masses, as estimated by gel filtration. Type I dephosphorylated both Ins(1,4,5)P3 and Ins(1,3,4,5)P4; in contrast, type II specifically dephosphorylated Ins(1,4,5)P3 but not Ins(1,3,4,5)P4. Type I Ins(1,4,5)P3 5-phosphatase eluted as a single peak of activity with an apparent molecular mass of 51 kDa when gel filtration was performed in the presence of cholate. This molecular mass is identical to the molecular mass estimated for the particulate Ins(1,4,5)P3 5-phosphatase that was solubilized by cholate. Km values for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 obtained with type I Ins(1,4,5)P3 5-phosphatase were 11 microM and 1 microM, respectively. Similar values were obtained with particulate Ins(1,4,5)P3 5-phosphatase. In conclusion, the catalytic domains of type I and particulate Ins(1,4,5)P3 5-phosphatase activity may be very similar, if not identical, but different from type II phosphatase.

Animals↗
PubMed1993

Rapid stimulation of Ins (1,4,5)P3 production in rat aorta by NE: correlation with contractile state.

Rapid stimulation of Ins(1,4,5)P3 production in rat aorta by NE: correlation with contractile state. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H126-H132, 1993.--The isomeric composition of inositol phosphates generated in response to norepinephrine (NE) stimulation and the relationship of inositol phosphate production to release of intracellular Ca2+ as measured by contraction were characterized in rat aorta prelabeled with [3H]inositol. NE stimulated a rapid and transient increase in labeled D-myo-inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] levels. A maximal increase in labeled Ins(1,4,5)P3 occurred within 15 s of stimulation followed by a decline to control levels at 5 min. D-Myo-inositol 1,3,4-trisphosphate [Ins-(1,3,4)P3] and D-myo-inositol 1-monophosphate [Ins(1)P] levels also increased rapidly in response to NE. In contrast to the transient production of Ins(1,4,5)P3, Ins(1,3,4)P3 and Ins(1)P production was maintained in the presence of NE. Half-maximal stimulation of Ins(1,4,5)P3 production and Ca2+ release occurred at 0.3 microM NE, and maximal effects were obtained with 10 microM NE. The concentration-response curve and time course for production of Ins(1,4,5)P3 correlated with the neurotransmitter-induced Ca2+ release from intracellular stores, indicating that the level of Ins(1,4,5)P3 regulated the Ca(2+)-release mechanism. In the continued presence of NE, the intracellular pools did not completely refill with Ca2+ despite the return of Ins-(1,4,5)P3 levels to basal at 5 min. These results demonstrate that NE stimulates a rapid increase in Ins(1,4,5)P3 that correlates with contraction in Ca(2+)-free buffer. The reuptake of Ca2+ into intracellular stores is regulated by a mechanism that may not involve Ins(1,4,5)P3.

Animals↗
PubMed1995

Quantitative comparisons of muscarinic and bradykinin receptor-mediated Ins (1,4,5)P3 accumulation and Ca2+ signalling in human neuroblastoma cells.

1. Muscarinic and bradykinin receptor-mediated Ins(1,4,5)P3 accumulation, Ca2+ mobilization and Ca2+ entry have been examined in human SH-SY5Y neuroblastoma cells. This has allowed both direct comparison of signalling events by two receptor types potentially linked to the same transduction pathway and an investigation of the interactions between the components of this pathway. 2. Stimulation of muscarinic receptors with carbachol produced biphasic accumulations of Ins(1,4,5)P3 consisting of a rapid peak followed by a lower sustained phase. Both phases were dose-dependent but the potency of elevation at peak was significantly less than that of the sustained phase. Bradykinin also dose-dependently stimulated Ins(1,4,5)P3 accumulation but responses were smaller and not sustained. 3. Lowering of [Ca2+]e reduced basal Ins(1,4,5)P3 levels. Peak Ins(1,4,5)P3 elevation in response to carbachol and bradykinin were lowered by an amount approximating this reduction over the entire dose-response curves. Sustained Ins(1,4,5)P3 elevation in response to carbachol showed a more marked absolute reduction. Agonist potencies were unaffected by lowering [Ca2+]e. Thus, a consistent but small amount of PLC activity during rapid activation appears to be sensitive to lowered [Ca2+]e, whilst activity during sustained stimulation is greatly facilitated by external Ca2+, probably through Ca2+ entry. 4. The temporal- and dose-dependency of carbachol-mediated Ins(1,4,5)P3 accumulations were unaffected by loading cells with fura-2, thus allowing direct comparison of Ins(1,4,5)P3 and [Ca2+]i changes monitored by fura-2. 5. Changes in [Ca2+]i by both agonists revealed temporal patterns that were similar to Ins(1,4,5)P3 accumulations. Only carbachol stimulated a marked sustained [Ca2+]i signal and this was fully dependent on external Ca2+. 6. All agonist-mediated [Ca2+]i elevations occurred with significantly greater potency than that of the respective Ins(1,4,5)P3 accumulations. Further examination of peak elevations in response to carbachol indicated that this was independent of Ca2+ entry. Thus, a major site for amplification of the potency of rapid agonist-mediated responses lies at the level of the Ins(1,4,5)P3 receptor. 7. The transient nature of Ins(1,4,5)P3 and [Ca2+]i peaks followed by either lower but sustained levels with carbachol or a return to basal levels with bradykinin suggests rapid but partial desensitization of the muscarinic receptor and complete desensitization of the bradykinin receptor. This indicates receptor-specific desensitization. Further analysis of this was provided by detecting accumulations of [3H]-inositol phosphates ([3H]-InsPs) in Li(+)-blocked, myo-[3H]-inositol labelled cells. Carbachol produced a rapid accumulation over the first minute, followed by a slower linear accumulation for at least 29 min. At this point accumulations were dose-related with a potency similar to that of sustained Ins(1,4,5)P3 accumulation.However, bradykinin produced a minor accumulation of [3H]-InsPs, maximal by 1 min. Thus,analysis of PLC activation by measurement of [3H]-InsPs over relatively long time frames will indicate the ability of agonists for predominantly sustained PLC activation, potentially driven by a partially desensitized receptor, as opposed to rapid activation by a fully sensitized receptor.8. These data provide quantitative comparisons between and within components of the receptor mediated phosphoinositide and Ca2+ signalling pathway, provide mechanistic insights into regulation of these components and characterize a model system in which heterologous interaction between two receptors linked to the same transduction pathway may be examined.

Bradykinin↗
PubMed2005

Facilitatory effect of Ins(1,4,5)P3 on store-operated Ca2+-permeable cation channels in rabbit portal vein myocytes.

In rabbit portal vein smooth muscle cells, store-operated Ca2+-permeable cation channels (SOCs) display multi-modal gating mechanisms. SOCs are activated by depletion of intracellular Ca2+ stores but also may be stimulated in a store-independent manner by noradrenaline acting on alpha-adrenoceptors and by diacylglycerol (DAG) via protein kinase C (PKC). In the present study we have investigated whether inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) modulates SOC activity in freshly dispersed rabbit portal vein myocytes with patch pipette recording techniques. Inclusion of 1 mum Ins(1,4,5)P3 in the patch pipette solution increased whole-cell currents evoked by the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) by about 3-fold at -80 mV. In the cell-attached configuration the cell-permeable Ca2+ chelator BAPTA-AM stimulated SOC activity and after excision of an isolated inside-out patch bath application of 1 mum Ins(1,4,5)P3 increased open channel probability (NP(o)) by approximately 3-fold. Ins(1,4,5)P3 also produced a similar increase in NP(o) of SOCs stimulated by the phorbol ester, phorbol 12,13-dibutyrate (PDBu) in inside-out patches and these channel currents had a unitary conductance of about 2 pS. The equilibrium constant of Ins(1,4,5)P3 on increasing PDBu-evoked SOC activity was about 0.4 mum. The facilitatory effect of Ins(1,4,5)P3 was also manifest as markedly increasing the rate of activation of SOCs. The synergistic effect of Ins(1,4,5)P3 was mimicked by the metabolically stable analogue 3-fluoro-Ins(1,4,5)P3 and Ins(1,4)P2, a metabolite of Ins(1,4,5)P3, but was not inhibited by the classical Ins(1,4,5)P3 receptor antagonist heparin. Finally Ins(1,4,5)P3 also increased NP(o) of SOCs activated by a PKC catalytic subunit. It is concluded that Ins(1,4,5)P3 facilitates SOC opening via a heparin-insensitive mechanism at, or close to, the channel protein.

Animals↗
PubMed1997

Ins(1,4,5)P3 receptors in cerebral arteries: changes with development and high-altitude hypoxia.

We and others have shown that adrenergic-mediated contractile responses in cerebral vessels in vitro differ with vessel segment, with developmental age, and with high-altitude, long-term hypoxia. This is associated with significant differences in alpha 1-adrenergic receptor density and norepinephrine (NE)-induced response of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. To test the hypothesis that vessel-specific, developmental, and hypoxic-associated contractility changes are mediated, in part, by changes in Ins(1,4,5)P3-receptor [Ins(1,4,5)P3-R] density or affinity, we performed the following study. In common carotid (Com), circle of Willis, and main branch anterior, middle, and posterior cerebral arteries (MBC) from normoxic fetal (approximately 140 days), newborn (3-5 days), and adult sheep and fetal and adult sheep acclimatized to high altitude, we quantified Ins(1,4,5)P3-R with [3H]Ins(1,4,5)P3. In normoxic Com, Ins(1,4,5)P3-R density values (fmol/mg protein) in fetus, newborn, and adult were 8 +/- 53, 150 +/- 18, and 357 +/- 21, respectively (P < 0.05). In normoxic MBC cerebral arteries, the receptor density values in the three age groups were 115 +/- 15, 105 +/- 9, 99 +/- 5 fmol/mg protein, respectively. For fetal and adult Com, high-altitude, long-term hypoxemia was associated with decreases in Ins(1,4,5)P3-R density of 32 (to 58 +/- 5) and 70% (to 109 +/- 12), respectively, from control values (P < 0.01). In MBC cerebral arteries of fetus and adult, hypoxic-associated decreases in Ins(1,4,5)P3-R density from control were 80 (to 23 +/- 3) and 47% (to 53 +/- 7), respectively (P < 0.01). Ins(1,4,5)P3 binding affinity to the receptor averaged 11.8 +/- 0.5 nM and did not vary significantly as a function of vessel type, developmental age, or hypoxia. In Com, but not in MBC, Ins(1,4,5)P3-R density increased dramatically with developmental age. This suggests that differences in Ins(1,4,5)P3-R density values may account, in part, for differences in contractile responses of the two artery types in the several age groups. In response to long-term, high-altitude hypoxia, Ins(1,4,5)P3-R density values in both fetal and adult Com and MBC decreased significantly, as did their NE-induced contraction. This suggests a cellular basis for changes in cerebrovascular contractility in response to long-term hypoxia and that Ins(1,4,5)P3-R may play a role in acclimatization responses to high altitude.

Aging↗
PubMed2004

2-O-(2-Aminoethyl)-myo-inositol 1,4,5-trisphosphate as a novel ligand for conjugation: physicochemical properties and synthesis of a new Ins(1,4,5)P(3) affinity matrix.

2-O-(2-Aminoethyl)-Ins(1,4,5)P(3), (5), a novel derivative of the Ca(2+)-mobilising second messenger d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], was synthesised from myo-inositol. 5 was found to be a potent mobiliser of intracellular Ca(2+), and an Ins(1,4,5)P(3) affinity matrix synthesised from 5 was effective at selectively binding N-terminal fragments of the Ins(1,4,5)P(3) receptor containing the intact Ins(1,4,5)P(3) binding site. The microprotonation scheme for 5 was resolved and the related constants were determined in comparison with Ins(1,4,5)P(3) and another reactive Ins(1,4,5)P(3) analogue 1-O-(2-aminoethyl-1-phospho)-Ins(4,5)P(2), (2a), by potentiometric and NMR titration methods. The (31)P and (1)H NMR titration curves for compound 5 and Ins(1,4,5)P(3) are remarkably close, indicating analogous acid-base properties and intramolecular interactions for the two compounds. The 1-phosphate-modified Ins(1,4,5)P(3) derivative 2a, on the contrary, behaves as a bisphosphorylated rather than a trisphosphorylated inositol. Thus, 5 is a new reactive Ins(1,4,5)P(3) analogue of considerable potential for investigation of the chemical biology of Ins(1,4,5)P(3)-mediated cellular signalling.

Amines↗