The 2-chloroadenosine inhibition of histamine-induced inositol phosphates accumulation in mouse brain and calcium ions.
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2-Chloroadenosine↗
Biomedical subjects
D A Kendall
Publications and source records attributed to D A Kendall.
Chronic lithium treatment enhances inositol polyphosphate formation in mouse brain in vivo.
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Animals↗
Stereochemistry of phospho group transfer catalyzed by a mutant alkaline phosphatase.
The stereochemical course of the phospho group transfer catalyzed by mutant (S102C) alkaline phosphatase from Escherichia coli was investigated by using 31P nuclear magnetic resonance spectroscopy. Transphosphorylation from 4-nitrophenyl (Rp)-[16O, 17O, 18O]phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus. This result is consistent with the view that the hydrolysis of substrates by this mutant enzyme proceeds by way of a covalent phosphoenzyme intermediate in the same manner as the wild-type alkaline phosphatase.
Alkaline Phosphatase↗
A functional decaisoleucine-containing signal sequence. Construction by cassette mutagenesis.
An alkaline phosphatase signal sequence optimized for formation of a hydrophobic alpha-helix functions very efficiently in the transport process. This mutant contained a core region comprised of 9 consecutive leucine residues (Kendall, D. A., Bock, S. C., and Kaiser, E. T. (1986) Nature 321, 706-708). We have now constructed a second mutant containing a decaisoleucine core region. Isoleucine was chosen because it is an isomer of leucine with comparable hydrophobicity but in synthetic peptides isoleucine favors beta-sheet formation. Surprisingly, this mutant precursor was also processed efficiently, and mature alkaline phosphatase was correctly targeted to the Escherichia coli periplasm. Since the effective length of a beta-strand is extended relative to an alpha-helix, conformational differences should be mirrored by the relative effectiveness of shortened polyisoleucine and polyleucine core regions. However, analysis of two additional mutants containing truncated segments of either polyleucine or polyisoleucine did not reveal any differences and both accumulate as precursors. We conclude that these mutants do not adopt critically different structures. This comparative analysis was facilitated by construction of a new plasmid, CASS3. This plasmid contains unique restriction sites flanking the DNA region coding for the signal sequence hydrophobic core segment. Consequently, the wild type core-encoding region can be readily replaced with synthetic oligonucleotides coding for new structural units and multiple amino acid substitutions can be made without the need for step-wise mutagenesis.
Alkaline Phosphatase↗
Use of site-directed mutagenesis to elucidate the role of arginine-166 in the catalytic mechanism of alkaline phosphatase.
The guanidinium group of arginine-166 has been postulated to act as an electrophilic species during phosphorylation of alkaline phosphatase. Its role could be either to stabilize the developing negative charge on the oxygen of the leaving group or the pentacoordinate transition state or to help bind the -PO2-3 group. We have produced via site-directed mutagenesis two Escherichia coli alkaline phosphatase mutants (lysine-166 and glutamine-166) to test whether the guanidinium group plays a critical role in catalysis. Comparative kinetic characterization of the lysine-166 and glutamine-166 mutants indicates that the charge at residue 166 is not required for the hydrolysis of phosphate monoesters. Small decreases in kcat are observed for both the lysine and glutamine mutants, relative to the wild-type enzyme, but the value for the uncharged glutamine mutant is only one-third that of lysine. Thus, the stabilizing effect of the positively charged guanidinium group does not appear to play a major role in the rate-limiting step for substrate hydrolysis. A significant effect on the Km value is seen only for the glutamine mutant.
Alkaline Phosphatase↗
Adenosine inhibition of histamine-stimulated inositol phospholipid hydrolysis in mouse cerebral cortex.
The effects of adenosine on inositol phospholipid hydrolysis in mouse cerebrocortical slices were examined. Despite having no effect alone, adenosine and some structural analogues inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The responses to carbachol, noradrenaline, 5-hydroxytryptamine, and elevated KCl levels were unaffected. The effect of adenosine was on the maximal response to histamine rather than on its EC50. Several adenosine antagonists competitively blocked the inhibition due to adenosine. The results are discussed in relation to the previously reported enhancement of histamine-stimulated hydrolysis of inositol phospholipids in guinea pig brain.
Adenosine↗
Lithium selectively inhibits muscarinic receptor-stimulated inositol tetrakisphosphate accumulation in mouse cerebral cortex slices.
The in vitro effects of Li on agonist- and depolarization-stimulated accumulation of inositol phosphates were determined in mouse cerebral cortex slices. Of the agents examined, only the cholinergic agonist carbachol produced a significant accumulation of inositol tetrakisphosphate (InsP4) in the absence of Li. Lithium at 5 mM enhanced the accumulation of inositol monophosphate (InsP1) and inositol bisphosphate (InsP2) due to all the stimuli used and potentiated inositol trisphosphate (InsP3) accumulation due to histamine and noradrenaline, although at lower Li concentrations, carbachol-stimulated InsP3 accumulation was reduced. Li also enhanced InsP4 accumulation in the presence of noradrenaline, histamine, and elevated KCl level but, in marked contrast, reduced carbachol-stimulated InsP4 accumulation with an IC50 of 100 microM. There was a significant time delay between the initiation of carbachol stimulation and the beginning of the InsP4 inhibition due to Li. The phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate did not mimic the effects of Li. The results suggest that muscarinic receptor-mediated InsP4 production might be one of the targets for the therapeutic action of Li.
Animals↗
Depolarisation-evoked release of acetylcholine can mediate phosphoinositide hydrolysis in slices of rat cerebral cortex.
Depolarisation of [3H]inositol prelabelled slices of cerebral cortex of the rat, with elevated extracellular K+ or the alkaloid veratrine, induced a marked accumulation of [3H]inositol monophosphate in the presence of 5 mM Li+. The effects of these stimuli were concentration-related with maximal responses obtained at 30 mM K+ and 30 microM veratrine. Larger concentrations produced submaximal responses but also markedly suppressed the incorporation of [3H]inositol into phospholipid. The responses to K+ or veratrine were not sensitive to atropine, prazosin, mepyramine, ketanserin or the peptidase inhibitor bacitracin. However, in the presence of the cholinesterase inhibitor physostigmine, the responses to these stimuli were greatly enhanced and this could be blocked by atropine. Both veratrine and K+ markedly stimulate release of endogenous acetylcholine from the slices. Release appears to be linear with time over the 45 min period of continuous stimulation. Reduction of extracellular calcium severely suppressed both the release of acetylcholine and the atropine-sensitive component of the phosphoinositide response to K+. The results suggest that endogenous acetylcholine can stimulate phosphoinositide metabolism by interacting with muscarinic receptors. The atropine-insensitive component, at least in part, represents entry of Ca2+ through voltage-sensitive channels and perhaps a direct effect on phosphoinositide metabolism.
Acetylcholine↗
Studies on the adenosine-receptor mediating the augmentation of histamine-induced inositol phospholipid hydrolysis in guinea-pig cerebral cortex.
Incubation (45 min) of slices of guinea-pig cerebral cortex with adenosine alone had no significant effect on the accumulation of [3H]-inositol phosphates but enhanced the response to histamine H1-receptor stimulation in a concentration-dependent manner. The effect of adenosine on agonist-stimulated inositol phospholipid hydrolysis appeared to be selective for histamine H1-receptor stimulation since it did not augment the phosphoinositide responses to carbachol, noradrenaline, 5-hydroxytryptamine or elevated KCl. The accumulation of [3H]-inositol phosphates induced by histamine increased linearly between 5 and 45 min incubation with agonist. However, following the simultaneous addition of histamine and adenosine, there was a marked delay in the appearance of the augmentation produced by adenosine. The augmentation of [3H]-inositol phosphate accumulation was mimicked by a number of adenosine analogues. The rank order of potency was; cyclopentyladenosine greater than R-phenyl-isopropyladenosine 5'-N-ethylcarboxamidoadenosine greater than 2-chloroadenosine. This is consistent with the order expected for an adenosine A1-receptor effect but the EC50 values were in the micro- rather than nanomolar range. The response to 2-chloroadenosine was antagonized by the xanthine adenosine-antagonists, cyclopropyltheophylline, 8-phenyltheophylline, 3-isobutyl-1-methylxanthine and theophylline, and the non-xanthine alloxazine.
Adenosine↗
Acute and chronic lithium treatments influence agonist and depolarization-stimulated inositol phospholipid hydrolysis in rat cerebral cortex.
The ability of acute and chronic Li treatment to influence agonist and depolarization-induced phosphoinositide metabolism was examined in rat cerebral cortex slices. After acute treatment (6.75 mEq/kg, 18 hr), [3H]inositol phosphates accumulating in the presence of 100 microM carbachol (muscarinic), 31 mM K+, 300 microM histamine (H1) and 300 microM 5-hydroxytryptamine (5-hydroxytryptamine2) were reduced significantly even after preincubation of slices with 2.5 mM myo-inositol. However, the response to noradrenaline (100 microM) (alpha-1) was unaffected. In the absence of a drug-free period, chronic Li (2 weeks) maintained the reduced phosphoinositide response to receptor agonists and K+, and now even noradrenaline responses were reduced significantly. Dose-response curves revealed that reduction in the response to carbachol was due to a fall in maximal response and not in EC50. When rats were withdrawn from chronic treatment for 18 hr, the responses to carbachol were enhanced significantly with respect to untreated controls. Neither acute nor chronic Li treatments altered significantly the overall incorporation of [3H]inositol into phospholipids. Furthermore, Li treatment did not influence the activity of phospholipase C assayed in crude homogenates of cerebral cortex. In conclusion, acute and chronic Li treatments producing less than [1 mM] in cerebral tissue, severely disrupts phosphoinositide metabolism. Although such effects may well be secondary to inhibition of inositol-monophosphatase, they are not reversed by inositol and therefore do not appear to result from depleted phosphoinositides.
Animals↗
Receptors and phosphoinositide metabolism in the central nervous system.
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Adaptation, Physiological↗
Electrophysiological test of an amphiphilic beta-structure in LHRH action.
Micropipettes were used to record electrical activity from single neurons in hypothalamic tissue slices, in the hypothalamic arcuate nucleus (ARC), and in the periventricular and suprachiasmatic preoptic nuclei (POA). Responses were measured following in vitro application of luteinizing hormone releasing hormone (LHRH), and two analogues: LHRH models 1 and 3. Model 1 (pyroGlu-His-Trp-Ser-Phe-Thr-Ile-Lys-Ile-ThrNH2) had amino acid substitutions in residues 5-10 designed to form an amphiphilic beta-strand structure. Model 3 (pyroGlu-His-Trp-Ser-Phe-Gly-Ile-Lys-Pro-SerNH2) was also designed to possess amphiphilic characteristics, but also more closely to resemble the native peptide. Electrical recording results showed that LHRH was able both to excite or inhibit different hypothalamic neurons, and that it was more effective in the preoptic area than in the arcuate nucleus. Responses to LHRH model 3 were strongly correlated with responses to LHRH, in their occurrence and their direction, for both of the brain regions studied. Moreover, LHRH models 1 and 3 also retained the neuromodulatory effects of LHRH on cellular responses to norepinephrine and serotonin. Thus, LHRH analogues, designed to possess an amphiphilic beta-structure, preserved some of the properties of LHRH when tested electrophysiologically in the central nervous system.
Amino Acid Sequence↗
Cyclic GMP formation and inositol phosphate accumulation do not share common origins in rat brain slices.
Cyclic GMP formation and inositol phospholipid hydrolysis were studied in rat brain slices to determine if the two processes have common origins. Muscarinic cholinergic stimulation enhanced [3H]inositol phosphate ([ 3H]IP) accumulation from slices prelabelled with [3H]inositol but did not affect cyclic GMP formation in the cortex, striatum, or cerebellum. An elevated level of extracellular K+ stimulated accumulation of both cyclic GMP and [3H]IP in cortex slices. The former, but not the latter, was reduced by lipoxygenase and phospholipase A2 inhibition. Calcium channel activation enhanced and blockade reduced K+-stimulated [3H]IP formation without affecting the cyclic GMP level, and there were differences in the Ca2+ requirements for the two responses. Thus, there is no support for the concept that guanylate cyclase activation inevitably accompanies inositol phospholipid breakdown, and the evidence presented demonstrates that K+ stimulation promotes cyclic GMP and [3H]IP accumulation by different transducing pathways.
1-Methyl-3-isobutylxanthine↗
Dihydropyridine calcium channel activators and antagonists influence depolarization-evoked inositol phospholipid hydrolysis in brain.
Increased inositol phospholipid hydrolysis induced by elevated extracellular K+ was directly monitored by assaying [3H]inositol phosphate accumulation following prelabelling of cerebral cortical slices with [3H]inositol. Depolarization evoked by K+ increased [3H]inositol phosphate accumulation with a 2-3-fold stimulation observed at 18 mM K+. Higher concentrations of K+ failed to further increase accumulation though a suppression of the incorporation of [3H]inositol into phospholipid at higher K+ could complicate these results. Slices incubated with the dihydropyridine calcium channel activator BAY-K-8644 resulted in a much increased response to 12 mM and 18 mM K+ with substantially smaller enhancement of basal (6 mM) or much higher (30 and 55 mM) K+. The [3H]inositol phosphate response induced by 18 mM K+ + 1 microM BAY-K-8644 was markedly reduced when incubations were performed in the presence of reduced Ca2+. Similarly, preincubation of slices with the dihydropyridine antagonist PN-200-110 suppressed the response to K+ and to K+ + BAY-K-8644. This effect was stereospecific with the (+)-enantiomer being at least 100-fold more potent than the (-)-enantiomer. These data provide primary evidence for functional dihydropyridine-sensitive calcium channels in brain.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Alpha 1-adrenoceptor-mediated inositol phospholipid hydrolysis in rat cerebral cortex: relationship between receptor occupancy and response and effects of denervation.
The characteristics of catecholamine-mediated breakdown of inositol phospholipids in rat cerebral cortex slices have been examined using a direct assay involving prelabeling with [3H]inositol and examining the production of labelled inositol phosphates in the presence of lithium. Noradrenaline produced a marked stimulation of inositol phosphate accumulation and this response could be potently and competitively antagonised by the alpha 1-adrenoceptor antagonist prazosin. The alpha 2-antagonist yohimbine was almost 1000-fold less potent at antagonising noradrenaline inositol phospholipid response. Noradrenaline and adrenaline were full agonists at alpha 1-adrenoceptors but phenylephrine and methoxamine were only partial agonists in their ability to stimulate inositol phospholipid metabolism. There was a significant correlation between the ability of a variety of agonists and antagonists to activate or inhibit [3H]inositol phosphate accumulation and their ability to displace the alpha 1-adrenoceptor selective ligand [3H]prazosin from specific binding sites when assays were performed on rat cerebral cortical slices under identical conditions. The similarity of EC50 values of agonists stimulating inositol phosphate accumulation and their IC50 values in [3H]prazosin binding experiments suggested a close relationship between receptor occupancy and alpha 1-mediated inositol phosphate accumulation. Further experiments were performed to examine this directly by inactivating alpha 1-adrenoceptors with the alkylating antagonist phenoxybenzamine. After washing out unbound antagonist, [3H]prazosin binding was reduced to a very similar proportion to that observed on the maximal noradrenaline-stimulated accumulation of [3H]inositol phosphates in the slices. The EC50 values for noradrenaline-stimulated inositol phosphate accumulation was unaltered and the affinity of [3H]prazosin for the remaining sites was equally unaffected. In rats treated 14 days previously with i.c.v. 6-hydroxydopamine (2 X 250 micrograms) there was a small increase in alpha 1-adrenoceptor binding sites but a parallel shift to the left in the noradrenaline [3H]inositol phosphate accumulation dose-response curve. On the other hand, the partial agonist phenylephrine induced a larger maximal response in denervated animals without a change in the EC50 values. When slices from 6-hydroxydopamine treated animals were preincubated with phenoxybenzamine, the loss in alpha 1-adrenoceptor binding sites was greater than the reduction in the maximal response to noradrenaline. This may indicate the development of a small receptor reserve after denervation.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Amphiphilic cationic peptides mediate cell adhesion to plastic surfaces.
Four amphiphilic peptides, each with net charges of +2 or more at neutrality and molecular weights under 4 kilodaltons, were found to mediate the adhesion of normal rat kidney fibroblasts to polystyrene surfaces. Two of these peptides, a model for calcitonin (peptide 1, MCT) and melittin (peptide 2, MEL), form amphiphilic alpha-helical structures at aqueous/nonpolar interfaces. The other two, a luteinizing hormone-releasing hormone model (peptide 3, LHM) and a platelet factor model (peptide 4, MPF) form beta-strand structures in amphiphilic environments. Although it contains only 10 residues, LHM mediated adhesion to surfaces coated with solutions containing as little as 10 pmoles/ml of peptide. All four of these peptides were capable of forming monolayers at air-buffer interfaces with collapse pressures greater than 20 dynes/cm. None of these four peptides contains the tetrapeptide sequence Arg-Gly-Asp-Ser, which has been associated with fibronectin-mediated cell adhesion. Ten polypeptides that also lacked the sequence Arg-Gly-Asp-Ser but were nonamphiphilic and/or had net charges less than +2 at neutrality were all incapable of mediating cell adhesion (Pierschbacher and Ruoslahti, 1984). The morphologies of NRK cells spread on polystyrene coated with peptide LHM resemble the morphologies on fibronectin-coated surfaces, whereas cells spread on surfaces coated with MCT or MEL exhibit strikingly different morphologies. The adhesiveness of MCT, MEL, LHM, and MPF implies that many amphiphilic cationic peptides could prove useful as well defined adhesive substrata for cell culture and for studies of the mechanism of cell adhesion.
Amino Acid Sequence↗
Pirenzepine indicates heterogeneity of muscarinic receptors linked to cerebral inositol phospholipid metabolism.
Muscarinic receptor-mediated inositol phospholipid metabolism was examined in rat cerebral cortex and medulla-pons slices prelabelled with 3H-inositol. Carbachol stimulated accumulation of 3H-inositol phosphate in both regions with identical EC50 values though the maximal stimulation was considerably greater in cortex. The carbachol response was potently suppressed by atropine with very similar affinities in both regions. However, the M1 antagonist pirenzepine displayed a 10-fold greater affinity for the cortical carbachol response. The data supports the concept of heterogeneity of cerebral muscarinic receptors and suggests that both M1 and M2 sites may be linked to inositol phospholipid metabolism in brain.
Animals↗
5-Hydroxytryptamine-stimulated inositol phospholipid hydrolysis in rat cerebral cortex slices: pharmacological characterization and effects of antidepressants.
The ability of 5-hydroxytryptamine (5-HT) and related agonists to stimulate hydrolysis of inositol phospholipids was examined in rat cerebral cortex slices using a direct assay which involves prelabeling with [3H]inositol and assaying [3H]inositol phosphates in the presence of lithium. 5-HT agonists stimulated [3H]inositol phosphate accumulation in a dose-related but biphasic manner and only the high-affinity component of the dose-response curve was sensitive to antagonists. This response to 5-HT was blocked potently by ketanserin and other putative 5-HT2 antagonists but the overall pattern of apparent drug affinities was inconsistent with that seen at 5-HT2 sites labeled with [3H]ketanserin in cortical membranes. Pretreatment of slices with the alkylating antagonist phenoxybenzamine reduced the inositol phospholipid response to 5-HT to a greater extent than the suppression of [3H]ketanserin binding. Similarly, chronic but not acute treatment of rats with the antidepressants iprindole and imipramine resulted in a greater loss of 5-HT-induced inositol phospholipid hydrolysis than specific [3H]ketanserin binding. However, the effect of antidepressants was agonist-specific in that neither alpha-1 adrenoceptor nor muscarinic receptor stimulation were altered by acute or chronic treatment.
Affinity Labels↗