Glyceraldehyde and the pancreatic beta-cell.
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Animals↗
Biomedical subjects
A C Elliott
Publications and source records attributed to A C Elliott.
Analysis of calcium oscillations in pancreatic acinar cells.
Since they were first reported in hepatocytes in 1986, agonist-evoked oscillations in intracellular free calcium ([Ca2+]i) have been described in numerous cell types, including pancreatic exocrine and endocrine cells. Pancreatic acinar cells show both 'sinusoidal' [Ca2+]i oscillations (when stimulated with muscarinic agonists) and so-called 'baseline [Ca2+]i spiking' (when stimulated with cholecystokinin, bombesin or analogues of either peptide). These agonist-evoked [Ca2+]i oscillations present novel problems to the investigator, particularly since they show considerable cell-to-cell variability. In this review, we discuss the various approaches available for analysing [Ca2+]i oscillations, particularly with respect to assessing whether drugs or other experimental interventions alter the oscillation pattern.
Animals↗
Interactions between secretin and acetylcholine in the regulation of fluid secretion by isolated rat pancreatic ducts.
1. Interlobular ducts were isolated from the rat pancreas and maintained in short-term tissue culture. Fluid secretion from these isolated ducts was measured using micropuncture techniques, intracellular calcium concentration ([Ca2+]i) by fura-2 microspectrofluorimetry, and cyclic AMP by radioimmunoassay. 2. Applying secretin and ACh simultaneously to ducts caused either a stimulation or an inhibition of fluid secretion depending on the doses employed. 3. The inhibitory effect of secretin and ACh could be relieved by atropine, and by the protein kinase C (PKC) inhibitors staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7). 4. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol 12, 13-dibutyrate (PDBu) inhibited secretin-evoked fluid secretion. 5. ACh and TPA also inhibited fluid secretion stimulated by the adenylate cyclase activator, forskolin. 6. Neither secretin nor the PKC activators and inhibitors had any effect on either the increase in [Ca2+]i evoked by ACh or the increase in intracellular cyclic AMP evoked by secretin and forskolin. 7. We conclude that the inhibitory effect of combined doses of secretin and ACh on ductal fluid secretion is probably mediated by PKC at a point in the secretory mechanism distal to the generation of intracellular messengers.
Acetylcholine↗
Imaging of intracellular calcium stores in individual permeabilized pancreatic acinar cells. Apparent homogeneous cellular distribution of inositol 1,4,5-trisphosphate-sensitive stores in permeabilized pancreatic acinar cells.
Several lines of evidence suggest that the existence of a heterogeneous population of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ stores underlies the polarized agonist-induced rise in cytosolic Ca2+ concentration ([Ca2+]i) in pancreatic acinar cells (Kasai, H., Li, Y. X., and Miyashita, Y. (1993) Cell 74, 669-677; Thorn, P., Lawrie, A. M., Smith, P. M., Gallacher, D. V., and Petersen, O. H. (1993) Cell 74, 661-668). To investigate whether the apical pole of acinar cells contains Ca2+ stores which are relatively more sensitive to Ins(1,4,5)P3 than those in basolateral areas, we studied Ca2+ handling by Ca2+ stores in individual streptolysin O (SLO) permeabilized cells using the low affinity Ca2+ indicator Magfura-2 and an in situ imaging technique. The uptake of Ca2+ by intracellular Ca2+ stores was ATP-dependent. A steady-state level was reached within 10 min, and the free Ca2+ concentration inside loaded Ca2+ stores was estimated to be 70 microM. Ins(1,4,5)P3 induced Ca2+ release in a dose-dependent, "quantal" fashion. The kinetics of this release were similar to those reported for suspensions of permeabilized pancreatic acinar cells. Interestingly, the permeabilized acinar cells showed no intercellular variation in Ins(1,4,5)P3 sensitivity. Although SLO treatment is known to result in a considerable loss of cytosolic factors, permeabilization did not result in a redistribution of zymogen granules, as judged by electron microscope analysis. These results suggest that Ins(1,4,5)P3-sensitive Ca2+ stores are unlikely to be redistributed as a result of SLO treatment. The effects of Ins(1,4,5)P3 were therefore subsequently studied at the subcellular level. Detailed analysis demonstrated that no regional differences in Ins(1,4,5)P3 sensitivity exist in this permeabilized cell system. Therefore, we propose that additional cytosolic factors and/or the involvement of ryanodine receptors underlie the polarized pattern of agonist-induced Ca2+ signaling in intact pancreatic acinar cells.
Adenosine Triphosphate↗
Serotonin elevates intracellular Ca2+ in rat choroid plexus epithelial cells by acting on 5-HT2C receptors.
The effects of serotonin (5-HT) on intracellular calcium activity ([Ca2+]i) in epithelial cells from rat choroid plexuses were examined. Experiments were performed on isolated cells which had been maintained in primary culture. ([Ca2+]i) was measured using micro-spectrofluorimetric techniques and the fluorescent indicator Fura-2. 5-HT was found to increase [Ca2+]i in a dose dependent manner. The [Ca2+]i response was biphasic, with an initial peak of [Ca2+]i (due to release from intracellular stores), followed by an elevated plateau phase (the result of calcium influx). The effect of 1 microM 5-HT was inhibited by mesulergine and mianserin (50 nM), which are antagonists of the 5-HT2C receptor. Spiperone and ketanserin (200 nM), less specific 5-HT2 receptor blockers, caused only a slight reduction in the response to 1 microM 5-HT. The [Ca2+]i response decreased upon repeated challenges with 1 microM 5-HT, probably as a result of receptor desensitisation. Taken together, the data suggest that 5-HT acts at 5-HT2C receptors to increase [Ca2+]i in choroid plexus epithelial cells, both by liberating Ca2+ from intracellular stores and by activating a Ca2+ influx pathway.
Animals↗
Changes in 2',7'-bis(carboxyethyl) 5'(6')-carboxyfluorescein-, fura-2 and autofluorescence in intact rat pancreatic islets in response to nutrients and non-nutrients.
Intracellular pH (pHi) was measured in intact rat islets loaded with the dye 2',7'-bis(carboxyethyl) 5'(6')-carboxyfluorescein. Raising the concentration of glucose from 3 to 13 mM caused a modest, gradual increase in pHi (500:450 fluorescence ratio). The addition of 20 mM lactate caused a gradual decline in pHi which reversed upon withdrawal of lactate. In contrast, the weak acids propionate and acetate (20 mM) induced a rapid, pronounced fall in pHi followed by a gradual recovery. Upon removal of the weak acid, a marked, reversible alkalinization occurred. The addition of 20 mM NH4Cl caused a pronounced intracellular alkalinization, followed by recovery. The subsequent removal of NH4Cl induced a rapid, reversible acidification. The addition of 20 mM KCl did not affect pHi. Epifluorescence at 350 and 380 nm excitation, and the 350:380 fluorescence ratio, an index of cytosolic [Ca2+] ([Ca2+]i), were measured in islets loaded with the calcium indicator fura-2. Approximately 30% of the total fluorescence was estimated to be derived from NAD(P)H autofluorescence. Addition of KCl or acetylcholine to fura-2 loaded islets raised and lowered, respectively, the 350 and 380 signals, thereby causing marked increases in the 350:380 ratio. Neither KCl nor acetylcholine affected cellular NAD(P)H autofluorescence in non-loaded islets. An increase in glucose concentration caused an increase in both the 350 and 380 fluorescence signals and also in the 350:380 ratio. Qualitatively similar, although smaller changes were observed when Ca2+ was omitted from the medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylcholine↗
Molecular recognition of antigenic polysaccharides: a conformational comparison of capsules from Streptococcus pneumoniae serogroup 9.
Aqueous solution conformations of three antigenic bacterial capsular polysaccharides (CPS) from S. pneumoniae serogroup 9 were determined using a combination of NMR data (NOE build-up rates and conformation-dependent chemical shifts), simulated annealing, and molecular dynamics simulations. Each polymer adopts a flexible extended ribbon conformation in solution. Conformations of structural elements shared by each PS are indistinguishable. Differences in conformations are minor and localised at the sites of structural variations; there is no evidence of long-range stabilisation of a secondary structure. It is likely that antigenic specificity of Group 9 PS is dominated by local structural variation rather than by conformational differences.
Bacterial Capsules↗
NMR assignment and conformational analysis of the antigenic capsular polysaccharide from Streptococcus pneumoniae type 9N in aqueous solution.
Complete 1H and 13C NMR assignments, determined by one- and two-dimensional homo- and hetero-nuclear experiments, are reported for the antigenic capsular polysaccharide (CPS) from Streptococcus pneumoniae serotype 9N (S9 in American nomenclature). Distance constraints derived from 1D NOE difference experiments were combined with energy minimisation (simulated annealing) and molecular dynamics (MD) calculations to determine the most favoured conformation of S9 in aqueous solution at 70 degrees C. NOE values simulated for several static conformational models using the NOEMOL program did not correlate well with experimental values, whereas time averaged interproton distances calculated from 500 ps of restrained MD (using the Tropp formalism to account for rapid internal mobility) were in close agreement with experimentally derived distance estimates.
Bacterial Capsules↗
A possible role for glyceraldehyde transport in the stimulation of HIT-T15 insulinoma cells.
D-Glyceraldehyde was transported into HIT-T15 cells at a linear rate for approx. 2 min and appeared to be unsaturable up to a concentration of 50 mM. Evidence was obtained for an electrogenic component of uptake of the triose. The rate of D-glyceraldehyde transport was also reduced in the absence of Na+, suggesting that a component of uptake was Na(+)-linked. Transport of D-glyceraldehyde could be prevented by N-ethylmaleimide but not significantly by p-chloromercuribenzenesulphonic acid, L-glyceraldehyde, nor by a number of inhibitors of known transport systems. However, D-glyceraldehyde transport was inhibited by alpha-cyano-4-hydroxycinnamate, an inhibitor of some anion transport systems. D-Glyceraldehyde caused a marked depolarization of HIT-T15 cells accompanied by a rise in cytosolic [Ca2+] and [Na+] and a gradual intracellular acidification. The glyceraldehyde-induced rise in cytosolic [Na+] and intracellular acidification, but not the depolarization or rise in cytosolic [Ca2+], were reduced by dithiothreitol and 5-aminoguanidine, compounds which form chemical adducts with alpha-ketoaldehydes. Incubation of HIT cells with either D- or L-glyceraldehyde resulted in the formation of large amounts of D-lactate, the end product of methylglyoxal metabolism via the glyoxalase pathway. It is suggested that the depolarizing action of glyceraldehyde is the result, at least in part, of its electrogenic transport, probably via Na(+)-coupled entry into HIT cells involving an unidentified transport system. The intracellular acidification and a component of the increase in cytosolic [Na+] may be largely due to the presence of one or more dicarbonyl contaminants in the glyceraldehyde preparation.
Coumaric Acids↗
The metabolic consequences of an increase in the frequency of stimulation in isolated ferret hearts.
1. The metabolic consequences of an increase in the frequency of stimulation were examined in isolated ferret hearts. Intracellular pH (pHi) and the intracellular concentrations of phosphocreatine ([PCr]i), inorganic phosphate ([Pi]i) and ATP were measured by 31P nuclear magnetic resonance (NMR) spectroscopy. 2. Increasing the stimulus rate from 0.1-0.7 to 2 Hz caused an increase in [Pi]i and a decrease recovery of both [PCr]i and [Pi]i during continued stimulation. There was no change in [ATP]i during stimulation at 2 Hz. Increasing the stimulus rate caused an intracellular acidosis of around 0.1 pH units. 3. Increasing the stimulus rate generally caused an initial increase in developed pressure, followed by a decrease over 1-2 min to a steady level slightly lower than developed pressure at the low (control) stimulus rate. The increase in stimulus rate caused a 4- to 6-fold increase in time-averaged muscle activity. 4. Both oxygen uptake and production of lactate increased on 2 Hz stimulation. Lactate production accounted for less than 5% of ATP production at low or high stimulus rates, suggesting that significant anoxia was not occurring during stimulation. The observed lactate production was, however, sufficient to explain most of the intracellular acidosis observed when the stimulus rate was raised. When glycolysis was prevented by removal of glucose and depletion of glycogen stores, 2 Hz stimulation was accompanied by an intracellular alkalosis rather than an acidosis, suggesting that lactate production by glycolysis was the cause of the intracellular acidosis. 5. Reducing the rate of glycolysis increased the size of changes in [PCr]i and [Pi]i evoked by stimulation at 2 Hz. Furthermore, there was now no partial reversal of the changes in [PCr]i and [Pi]i during 2 Hz stimulation. 6. When oxidative phosphorylation was inhibited by replacing O2 with N2, increasing the rate of stimulation from 0.1-0.7 to 1-2 Hz caused an initial increase followed by a large fall in developed pressure, which declined to a level well below that at the control stimulus rate. The increase in stimulus rate was accompanied by a large fall in [PCr]i, an increase in [Pi]i, and an intracellular acidosis of 0.1-0.3 pH units. The fall in developed pressure was consistent with the known effects of the rise in [Pi]i and the fall in pHi on the contractile apparatus.
Adenosine Triphosphate↗
Activation of protein kinase C does not cause desensitization in rat and rabbit mandibular acinar cells.
We have examined whether activation of protein kinase C by phorbol esters decreases the responsiveness of rat and rabbit mandibular, and rat lacrimal, acinar cells to muscarinic stimulation. Intracellular free calcium concentration ([Ca2+]i) was measured in isolated single acini and cell clusters by fura-2 microspectrofluorimetry. Accumulation of inositol phosphates was measured in acinar cell suspensions. All three cell types showed very similar changes in [Ca2+]i in response to acetylcholine (ACh), although mobilization of Ca2+ required somewhat higher ACh concentrations in rat lacrimal acinar cells than in mandibular acinar cells. There was no evidence for different dose dependencies of the peak and plateau phases of the [Ca2+]i response. The ACh-evoked [Ca2+]i increase in rabbit mandibular acinar cells exhibited desensitization, since it declined in magnitude when cells were stimulated repeatedly with a maximal dose of agonist. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) rapidly and irreversibly decreased the ACh-evoked [Ca2+]i signals in rat lacrimal acinar cells and reduced ACh-stimulated inositol phosphate accumulation. This inhibitory effect of TPA was most marked in cells stimulated with low doses of ACh, implying that TPA treatment shifted the ACh dose response curve to higher ACh concentrations. In contrast to the results obtained with lacrimal acinar cells, TPA had no effect on the [Ca2+]i and inositol phosphate responses to ACh in either rat or rabbit mandibular acinar cells. These results suggest that, although ACh-evoked [Ca2+]i signals, and hence presumably the stimulus-response coupling machinery, are very similar between different acinar cell types, acinar cells show marked differences in their sensitivity to phorbol esters. The insensitivity of mandibular acinar cell [Ca2+]i signals to TPA also suggests that the secretory tachyphylaxis observed in perfused rat and rabbit mandibular salivary glands is unlikely to be a consequence of negative feedback mediated by protein kinase C.
Acetylcholine↗
Stimulation of HIT-T15 insulinoma cells by glyceraldehyde does not require its metabolism.
The addition of the triose D-glyceraldehyde (5-20 mM) to HIT-T15 hamster insulinoma cells caused a rapid, marked depolarisation of the plasma membrane accompanied by a pronounced intracellular acidification, an increase in the cytosolic free calcium concentration [Ca2+]i and enhanced secretion of insulin. D-glyceraldehyde did not reduce the rate of efflux of 86Rb+ from loaded perifused cells. All of the above effects of D-glyceraldehyde were also observed in response to L-glyceraldehyde. The changes in membrane potential and intracellular pH (pHi) caused by D-glyceraldehyde were unaffected by the glycolytic inhibitor iodoacetate, by K(+)-channel blockers (tolbutamide and tetraethylammonium), or by inhibitors of the transport of lactate (alpha-fluorocinnamate), alanine (methylaminoisobutyrate) or glucose (phloretin, phlorrizin). The glyceraldehyde-induced depolarisation and acidification were also observed in the absence of extracellular Ca2+ or Na+. The increase in [Ca2+]i evoked by D-glyceraldehyde was reversed by removal of Ca2+ from the medium. The formation of lactate by HIT-T15 cells was not significantly increased by addition of 10 mM D-glyceraldehyde or L-glyceraldehyde. In contrast, 10 mM glucose caused an approximately fourfold rise in lactate production. The oxidation of D-glyceraldehyde by HIT-T15 cells was also extremely modest compared to glucose oxidation by these cells. These results suggest that the stimulation of HIT-T15 cells by either D-glyceraldehyde of L-glyceraldehyde does not require metabolism of the triose within the cell and may not involve closure of nucleotide-sensitive K+ channels. We propose that the electrogenic transport of glyceraldehyde across the plasma membrane, possibly via H+ cotransport, might lead to depolarisation and hence to Ca2+ entry into the cell.
Animals↗
Regulation of fluid secretion and intracellular messengers in isolated rat pancreatic ducts by acetylcholine.
1. We have studied the effects of acetylcholine (ACh) on fluid secretion and intracellular messengers in interlobular ducts isolated from the rat pancreas and maintained in short-term tissue culture. 2. Ductal fluid secretion was measured using micropuncture techniques. Intracellular free calcium ([Ca2+]i) and cyclic AMP concentrations were measured in single ducts using fura-2 microspectrofluorimetry and radioimmunoassay techniques respectively. Changes in the levels of these intracellular messengers were correlated with fluid secretion. 3. ACh stimulated ductal fluid secretion. The dose required for a half-maximal response was about 0.4 microM and maximal secretion was achieved with 10 microM ACh. These effects of ACh were blocked by atropine and by removal of extracellular Ca2+. 4. ACh was about four orders of magnitude less potent as an activator of ductal fluid transport than the hormone secretin; however, the maximal rates of fluid secretion evoked by these two agonists were similar. 5. ACh caused a dose-dependent rise in duct cell [Ca2+]i, but had no effect on cyclic AMP. In contrast, secretin increased duct cell cyclic AMP, but had no effect on [Ca2+]i. 6. The [Ca2+]i response evoked by ACh resulted from both mobilization of intracellular Ca2+ stores and influx of Ca2+ from the extracellular space. 7. The Ca2+ ionophore, ionomycin, mimicked the effect of ACh on ductal [Ca2+]i and fluid secretion. 8. We conclude that ACh stimulates fluid secretion from rat pancreatic duct cells by activating a 'Ca2+ pathway' which is distinct from the well documented 'cyclic AMP pathway' utilized by secretin.
Acetylcholine↗
The catalytic consequences of experimental evolution. Studies on the subunit structure of the second (ebg) beta-galactosidase of Escherichia coli, and on catalysis by ebgab, an experimental evolvant containing two amino acid substitutions.
1. The ratio of ebgA-gene product of ebgC-gene product in the functional aggregate of ebg beta-galactosidases was determined to be 1:1 by isolation of the enzyme from bacteria grown on uniformly radiolabelled amino acids and separation of the subunits by gel-permeation chromatography under denaturing conditions. 2. This datum, taken together with a recalculation of the previous ultracentrifuge data [Hall (1976) J. Mol. Biol. 107, 71-84], analytical gel-permeation chromatography and electron microscopy, strongly suggests an alpha 4 beta 4 quaternary structure for the enzyme. 3. The second chemical step in the enzyme turnover sequence, hydrolysis of the galactosyl-enzyme intermediate, is markedly slower for ebgab, having both Asp-97----Asn and Trp-977----Cys changes in the large subunit, than for ebga (having only the first change) and ebgb (having only the second), and is so slow as to be rate-determining even for an S-glycoside, beta-D-galactopyranosyl thiopicrate, as is shown by nucleophilic competition with methanol. 4. The selectivity of galactosyl-ebgab between water and methanol on a molar basis is 57, similar to the value for galactosyl-ebgb. 5. The equilibrium constant for the hydrolysis of lactose at 37 degrees C is 152 +/- 19 M, that for hydrolysis of allolactose is approx. 44 M and that for hydrolysis of lactulose is approx. 40 M. 6. A comparison of the free-energy profiles for the hydrolyses of lactose catalysed by the double mutant with those for the wild-type and the single mutants reveals that free-energy changes from the two mutations are not in general independently additive, but that the changes generally are in the direction predicted by the theory of Burbaum, Raines, Albery & Knowles [(1989) Biochemistry 28, 9283-9305] for an enzyme catalysing a thermodynamically irreversible reaction. 7. Michaelis-Menten parameters for the hydrolysis of six beta-D-galactopyranosylpyridinium ions and ten aryl beta-galactosides by ebgab were measured. 8. The derived beta 1g values are the same as those for ebgb (which has only the Trp-977----Cys change) and significantly different from those for ebgo (the wild-type enzyme) and ebga. 9. The alpha- and beta-deuterium secondary isotope effects on the hydrolysis of the galactosyl-enzyme of 1.08 and 1.00 are difficult to reconcile with the pyranose ring in this intermediate being in the 4C1 conformation.
Biological Evolution↗
Subcellular gradients of intracellular free calcium concentration in isolated lacrimal acinar cells.
The spatial distribution of intracellular free calcium concentration ([Ca2+]i) was measured in small clusters of isolated rat lacrimal acinar cells by imaging the fluorescence of the Ca(2+)-sensitive dye fura-2. In the absence of extracellular Ca2+, stimulation with acetylcholine (ACh) caused an increase in [Ca2+]i, due to release of intracellular Ca2+ stores, which was maximal at the luminal pole of the cell. In contrast, the organellar Ca(2+)-ATPase inhibitor 2,5-di(tert-butyl)-hydroquinone caused an increase in [Ca2+]i, which was most marked in the basolateral region of the cell. When the cells were stimulated with ACh in a medium containing Ca2+, the gradients of [Ca2+]i (with [Ca2+]i most elevated at the luminal pole) were maintained for the duration of agonist stimulation. The possible implications of these results concerning the location and identity of intracellular Ca2+ stores, and the location of the sites that underlie agonist-stimulated Ca2+ influx, are considered. In particular, it seems likely that intracellular inositol-1,4,5-trisphosphate (InsP3) binding sites may be concentrated in the luminal region of the cell. It is not clear, however, whether this implies that there is a distinct luminally located InsP3-sensitive organelle.
Acetylcholine↗
Metabolic changes during ischaemia and their role in contractile failure in isolated ferret hearts.
1. The effects of global ischaemia on phosphorus metabolites, intracellular pH (pHi) and developed pressure were measured in isolated whole ferret hearts using 31P nuclear magnetic resonance (NMR) spectroscopy. 2. Brief (10 min) periods of global ischaemia reduced left ventricular developed pressure (LVDP) to undetectable levels. This fall in LVDP was accompanied by a fall in the intracellular concentration of phosphocreatine (PCr) and increases in the concentrations of inorganic phosphate (Pi) and phosphomonoesters. There was no change in the intracellular ATP concentration ([ATP]i). pHi fell approximately linearly at a rate of 0.04 pH units min-1. 3. When ferret hearts were exposed to cyanide (CN-) in the presence of alpha-cyano-4-hydroxycinnamate (CHC), a blocker of lactate efflux, the changes in pHi and [Pi]i which occurred were similar to those observed during global ischaemia. However, developed pressure only fell to around 15% of the control value. 4. Removing the intracellular acidosis (by reducing the CO2 level of the gas with which the perfusate was equilibrated) during exposure to CN- and CHC caused an increase in developed pressure, consistent with the fall in pHi being responsible for a substantial fraction of the fall in developed pressure. 5. Taken together, these results suggest that most, but not all, of the fall in developed pressure during ischaemia can be explained by the effects of the changes in pHi and [Pi]i on the contractile apparatus. 6. Action potential recordings made with a suction electrode during short periods of global ischaemia showed that there was no decrease in action potential duration over the period when developed pressure was falling, eliminating action potential shortening as a possible cause of the fall in developed pressure. 7. In hearts in which the rate of glycolysis had been reduced by glycogen depletion, global ischaemia led to a marked shortening of the action potential. NMR experiments showed that under these conditions [ATP]i decreased by around 50% over the first 10 jin of ischaemia, while the intracellular acidosis which occurred was smaller than that in a control ischaemic period. 8. The time course of the decline of [ATP]i was examined in several hearts during long (45 min and over) ischaemic periods without prior glycogen depletion. After 45 min of ischaemia [ATP]i fell to around two-thirds of the control value, while pHi declined to approximately 6.1. Resting pressure did not increase. On reperfusion pHi recovered rapidly to control levels. [ATP]i, however, did not recover. 9. If ischaemia was prolonged further, [ATP]i eventually became undetectable after 70-90 min.(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Location and quantitation of the sites of O-acetylation on the capsular polysaccharide from Streptococcus pneumoniae type 9V by 1H-n.m.r. spectroscopy: comparison with type 9A.
The 1H-n.m.r. spectra of the Streptococcus pneumoniae type 9V (S68 in American nomenclature) capsular polysaccharide (PS) and its O-deacetylated derivative [which is structurally identical to the S. pneumoniae type 9A (S33) PS] were assigned using COSY, relayed-COSY, and 2D-NOESY experiments. The positions of the OAc groups in the alpha-GlcA, beta-ManNAc, and alpha-Glc residues of the native 9V PS were established using 2D-n.m.r. and chemical shift arguments, and the relative proportions of different O-acetylated species were estimated by integration of well-resolved 1H-n.m.r. signals. The locations of the OAc substituents differ from those previously reported. [formula: see text].
Acetylation↗
The effects of Na+ replacement on intracellular pH and [Ca2+] in rabbit salivary gland acinar cells.
1. The role of Na(+)-dependent mechanisms in regulating the intracellular pH (pHi) and free calcium concentration ([Ca2+]i) in acinar cells of the rabbit mandibular salivary gland was examined. The fluorescent dyes BCECF and Fura-2 were used to measure pHi and [Ca2+]i respectively in suspensions of isolated acini. 2. Replacement of all the extracellular Na+ with N-methyl-D-glucamine (NMDG) decreased resting pHi from a control value of 7.1-7.2 to 6.8-6.9. Re-addition of Na+ or Li+ caused a recovery of pHi towards control values. This recovery was blocked by 10-50 microM-ethylisopropylamiloride (EIPA), suggesting that it was mediated by Na(+)-H+ exchange. The rate of recovery of pHi when Na+ was re-introduced increased with Na+ concentration with an apparent Km for Na+ of around 30 mM. 3. Replacement of all of the extracellular Na+ with Li+ caused only a small decrease in resting pHi. 4. Stimulation of acini with 1 microM-acetylcholine (ACh) evoked an intracellular acidosis both under control conditions and when acini were bathed in Na(+)-free media. Following the acidosis pHi recovered in acini bathed in either control medium or Na(+)-free (Li+) medium, but not in acini bathed in Na(+)-free (NMDG) medium or in control medium containing EIPA. 5. Stimulation of acini bathed in Na(+)-free, HCO(3-)-free medium with ACh did not cause any change in pHi. 6. Re-addition of Na+ to acini bathed in Na(+)-free, HCO(3-)-free medium evoked the same rate of alkalinization whether or not the acini had been stimulated with ACh, suggesting that receptor stimulation per se did not lead to an activation of acid extrusion. 7. Resting [Ca2+]i was elevated in acini bathed in Na(+)-free (NMDG) medium, but not in acini bathed in Na(+)-free (Li+) medium. 8. ACh evoked a maintained rise in [Ca2+]i in acini bathed in control medium and in Na(+)-free media with either NMDG or Li+ as the Na+ substitute. 9. Experiments in which external Ca2+ was reduced to low levels (by the addition of EGTA) just prior to addition of ACh showed that ACh released intracellular Ca2+ stores under both control and Na(+)-free conditions. 10. In acini bathed in Na(+)-free (NMDG) solution and stimulated with ACh, re-addition of either Na+ or Li+ reduced [Ca2+]i. The reduction of [Ca2+]i on Na+ re-addition was blocked by EIPA. [Ca2+]i could also be reduced under these conditions by alkalinizing the cytosol using the weak base trimethylamine.(ABSTRACT TRUNCATED AT 400 WORDS)
Acetylcholine↗