Biomedical subjects

G Sachs

Publications and source records attributed to G Sachs.

At least 163 records · Page 9Linked to original sources

PubMed1992

A K+ competitive, conformational probe of the H,K-ATPase.

The H,K-ATPase was noncovalently labelled with a fluorescent quinoline derivative, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo [3,2-c]quinoline, (MDPQ). MDPQ competitively inhibited the K+ stimulated ATP hydrolysis with a Ki of 0.22 microM but did not inhibit the MgATP-dependent phosphoenzyme to an extent greater than 10% of control. Inhibitor binding to the H,K-ATPase enhanced MDPQ fluorescence. This fluorescence was quenched by lumenal K+ with a K0.5 of 1.8 mM. MDPQ binding to the H,K-ATPase shifted the fluorescence Ex/Em maxima from 342/478 nm to 342/453 nm. Phosphorylation of the H,K-ATPase by MgATP further enhanced fluorescence with a difference spectra [MgATP-(MgATP+KCl)] emission peak at 446 nm. Trypsin dependent proteolysis of the H,K-ATPase stabilized within the E2K conformation eliminated the phosphoenzyme response, but enhanced the K+ specific dephosphoenzyme response. These observations show that MDPQ is a fluorescent, competitive inhibitor of the H,K-ATPase that interacts with a lumenal cation binding site. Under specific conditions, both the cation and MDPQ binding sites remain intact within trypsin produced cleavage peptides of the H,K-ATPase.

Aminoquinolines↗

PubMed1992

Chemomechanical coupling in the gastric H,K ATPase.

The gastric H,K ATPase is investigated in terms of its secondary structure by analysis of the binding sites of the extracytoplasmic inhibitors and by tryptic cleavage of intact, inside-out gastric vesicles. The inhibitors affect phosphorylation and other partial reactions of the ATPase that depend on cytoplasmic conformational changes. The K competitive imidazopyridine, SCH28080 binds to the first pair of transmembrane segments, M1/M2, probably at phe124 and asp136. Omeprazole which generates a cationic sulfenamide in acid spaces binds to either cys813 or cys822 at one site and cys892 at the other. These cysteines are located at the membrane spanning pairs, M5/M6 and at M7/M8. Tryptic cleavage of intact inside out vesicles followed by labelling with fluorescein-5-maleimide provides direct evidence for 8 membrane spanning segments between positions 104/162 (M1/M2), 291/358(M3/M4), 776/835 (M5/M6),853/946 (M7/M8). Evidence is lacking so far for M9/M10, postulated on the basis of hydrophobicity for the Ca ATPase. Conformational studies suggest that there is interaction between the cytoplasmic loop between M4 and M5 (ATP domain) and the extracytoplasmic domain of the enzyme at the inhibitor binding sites.

Adenosine Triphosphate↗

PubMed1991

Regulation of rat gastric H+/K(+)-ATPase alpha-subunit mRNA by omeprazole.

The H+/K(+)-ATPase is the dimeric enzyme responsible for H+ secretion by the gastric parietal cells. The present study examined the response of rat fundic mRNA levels of H+/K(+)-ATPase alpha-subunit and somatostatin to the inhibition of H+/K(+)-ATPase enzyme activity and gastric pH elevation by oral omeprazole administration. Omeprazole inhibits the alpha-subunit of H+/K(+)-ATPase covalently and stabilizes stimulated morphology of the parietal cell. After a single administration of omeprazole (100 mg/kg), H+/K(+)-ATPase alpha-subunit mRNA levels increased significantly by 57% at 3 h and remained elevated for 6 h, returning to the basal level by 24 h. After multiple administrations of omeprazole (100 mg/kg per day, every 24 h for 3 days), H+/K(+)-ATPase alpha-subunit mRNA levels were already elevated at the time of the last dose, reached maximum at 6 h (95% increase above control), and returned to the pre-treatment level after 36 h. Nuclear run-on assay indicated H+/K(+)-ATPase gene transcription was significantly increased by omeprazole pretreatment in vivo. In contrast, a significant decrease in fundic somatostatin mRNA occurred at 12 h after a single dose, and the inhibition was more pronounced and lasted longer after multiple doses of omeprazole. These data indicate that omeprazole, while effectively inhibiting H+/K(+)-ATPase activity, induces H+/K(+)-ATPase gene expression in the parietal cells. An inverse relationship exists between the regulation of somatostatin gene expression in fundic D-cells and H+/K(+)-ATPase gene expression. The increase in H+/K(+)-ATPase alpha-subunit mRNA could be due to alterations in extracellular gastrin/somatostatin ratios or could be induced by intracellular effects of omeprazole.

Actins↗

PubMed1991

The rat H+/K(+)-ATPase beta subunit gene and recognition of its control region by gastric DNA binding protein.

The rat gastric H+/K(+)-ATPase beta subunit gene was cloned, and its nucleotide sequence was determined. The coding region is separated by 6 introns, whereas the related human Na+/K(+)-ATPase beta subunit gene was shown to have 5 introns (Lane, L.K., Shull, M.M., Whitmer, K.R., and Lingrel, J.B. (1989) Genomics 5, 445-453). The positions of introns 1, 2, and 5 of the two genes were the same. The similarities in intron/exon organizations and primary structures (30-40% identical residues) suggest that the beta subunit genes for H+/K(+)-ATPases were derived from a common ancestor. The upstream region of the rat H+/K(+)-ATPase beta subunit gene contains direct repeat sequences and palindromes, potential binding sites for RNA polymerase II and E4TF1, and CACCC box sequences. Gel retardation assay demonstrated that the stomach, but not other tissues (liver, brain, kidney, spleen, and lung), has a nuclear protein(s) capable of binding to the regions upstream of the potential RNA polymerase II binding sites (TATA box). The nuclear protein(s) are suggested to recognize three tandem GATAGC sequences and may be important for controlled transcription of the H+/K(+)-ATPase beta subunit gene in gastric parietal cells.

Adenosine Triphosphatases↗

PubMed1991

Agonist-sensitive and -insensitive intracellular Ca2+ pools. Separate Ca(2+)-releasing mechanisms revealed by manoalide and benzohydroquinone.

The mechanism of action of a novel compound, 2,5-di-(t-butyl)-1,4-benzohydroquinone (BHQ), used to modulate cell free cytosolic Ca2+ concentration ([Ca2+]i) was studied in AR42J cells and pancreatic acini by using single-cell fluorescence techniques applied to Fura-2-loaded cells. In the presence of extracellular Ca2+ (Ca(2+)out), BHQ induced a biphasic [Ca2+]i increase, an initial and rapid transient followed by a sustained increase. The initial increase was due to Ca2+ release from intracellular stores, being independent of Ca(2+)out. The sustained response was due to Ca2+ entry, being dependent on Ca(2+)out, blocked by La3+ and correlated with an increased rate of Mn2+ entry, all indicative of increased plasma-membrane permeability to Ca2+. Treatment of AR42J cells with BHQ for about 5 min reversibly blocked agonist-dependent Ca2+ release and oscillations, whereas agonist pretreatment decreased, but did not prevent, the effects of BHQ on [Ca2+]i. Accordingly, depletion of the Ins(1,4,5)P3-mobilizable pool in permeabilized AR42J cells by BHQ required 5 min of incubation, although inhibition of the internal Ca2+ pump by BHQ was rapid. These observations suggest that BHQ mobilized an additional intracellular Ca2+ pool that did not respond to changes in Ins(1,4,5)P3. Manoalide, an inhibitor of Ca2+ channels, inhibited agonist-evoked [Ca2+]i oscillation and [Ca2+]i increase in a dose- and time-dependent manner without significant effect on internal Ca2+ pumps and Ca2+ content of the internal stores. Manoalide also inhibited the BHQ-evoked [Ca2+]i increase in the absence and presence of Ca(2+)out. Neither BHQ nor manoalide affected Ins(1,4,5)P3 levels in resting or stimulated cells. Therefore, the effect of BHQ appears to involve unmasking of passive Ca(2+)-permeation pathways in the plasma and intracellular membranes that do not respond to cholecystokinin octapeptide, following its described inhibition of the internal-store Ca2+ pumps responsible for accumulating Ca2+ in these pools.

Animals↗

PubMed1991

The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K-pumps.

Na,K-ATPase and H,K-ATPase are the only members of the P-type ATPases in which a glycosylated beta-subunit is part of the purified active enzyme. In this study, we have followed the synthesis and the posttranslational processing of the beta-subunit of H,K-ATPase (beta HK) in Xenopus oocytes injected with beta HK cRNA and have tested whether it can act as a surrogate for the beta-subunit of Na,K-ATPase (beta NaK) to support the functional expression of Na,K-pumps. In Xenopus oocytes, beta HK is processed from an Endo H-sensitive 51-kDa coreglycosylated form to an Endo H-resistant 71-kDa fully glycosylated form. Similar to beta NaK, beta HK can stabilize and increase the trypsin resistance of alpha-subunits of Na,K-ATPase (alpha NaK). Finally, expression of beta HK together with alpha NaK leads to an increased number of ouabain binding sites at the plasma membrane accompanied by an increased Rb+ uptake and Na,K-pump current. Our data suggest that beta HK, similar to beta NaK, can assemble to alpha NaK, support the structural maturation and the intracellular transport of catalytic alpha NaK, and ultimately form active alpha NaK-beta HK complexes with Na,K-pump transport properties.

Adenosine Triphosphatases↗

PubMed1991

Identification of an extracytoplasmic region of H+,K(+)-ATPase labeled by a K(+)-competitive photoaffinity inhibitor.

The photoaffinity reagent 8-[(4-azidophenyl)-methoxy]-1-tritiomethyl-2, 3-dimethylimidazo-[1,2-alpha]pyridinium iodide ([3H]mDAZIP) has been synthesized and used to photoinactivate and label purified hog gastric H+,K(+)-ATPase. The specific (K(+)-sensitive) components of both photoinactivation and labeling showed dependences on inhibitor concentration consistent with covalent modification at an extracytoplasmic site of reversible K(+)-competitive binding in the dark. The maximum amount of specific labeling (1.2 nmol/mg) was similar to the number of phosphorylation sites measured (1.0 +/- 0.14 nmol/mg). Specific labeling was distributed 76% on the alpha chain, 18% on the beta chain, and 6% on undefined peptides. Various digestions with trypsin, protease V8, and thermolysin were employed to fragment the labeled enzyme. Gasphase sequencing of the radioactive peptides identified the major site of specific labeling to be within a region where only two stretches of amino acids (Leu105 to Ile126 and Leu139 to Phe155, designated H1 and H2, respectively) are predicted to span the membrane. This in turn suggested that the labeling site was located within or close to the proposed loop between them (Gln127 to Asn138). A computer-driven energy minimization protocol yielded a loop structure to which SCH 28080 (the parent structure of [3H]mDAZIP) could be docked. Conversely, modeling of the corresponding region of Na+,K(+)-ATPase (a homologous enzyme with much lower affinity for SCH 28080) yielded no apparent binding site. Similarities in the inhibition of H+,K(+)-ATPase by SCH 28080 and of Na+,K(+)-ATPase by ouabain lead to the hypothesis that, in each case, inhibitor binding to E2-P is associated with an increase in the hydrophobicity of the environment of the loop between H1 and H2.

Adenosine Triphosphatases↗

PubMed1991

A K(+)-competitive fluorescent inhibitor of the H,K-ATPase.

The interactions of a novel fluorescent compound, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-c ]quinoline (MDPQ) with the gastric H,K-ATPase were determined. MDPQ was shown to inhibit the H,K-ATPase and its associated K(+)-phosphatase competitively with K+, with Ki values of 0.22 and 0.65 microM, respectively. It also inhibited H+ transport with an IC50 of 0.29 microM, but at a concentration of 3.5 microM, reduced the steady-state level of phosphoenzyme by only 28%. The fluorescence of the inhibitor increased upon binding to the enzyme. 70% of this increment was quenched by K+, independently of Mg2+. The binding of MgATP to a high affinity site (K0.5(ATP) less than 1 microM) markedly increased the fluorescence due to the formation of an inhibitor-phosphoenzyme complex saturating with a K0.5(MDPQ) of 0.94 microM. The K(+)-dependent fluorescent quench (K0.5(K+) = 1.8 mM) required the ionophore, nigericin, indicating that K+ and MDPQ were competing at an extracytosolic site on the enzyme. Formation also of an enzyme-vanadyl-inhibitor complex was shown by the fact that Mg2+ plus vanadate enhanced MDPQ fluorescence in the absence of MgATP and decreased fluorescence in the presence of MgATP. The minimal stoichiometry of bound MDPQ determined by fluorescence titrations in the presence of MgATP was 1.4 mol/mol phosphoenzyme. The data suggest that this compound can serve as a probe of conformation at an extracytosolic site of the H,K-ATPase.

Adenosine Triphosphatases↗

PubMed1991

Identification of H+/K(+)-ATPase alpha,beta-heterodimers.

Glutaraldehyde treatment of the C12E8 solubilized H+/K(+)-ATPase crosslinks the catalytic subunit with an apparent molecular mass of 94 kDa in SDS polyacrylamide gels into two Coomassie stained particles migrating at approx. 147 and 173 kDa. The subunit composition of these particles was determined from the comparative distribution of FITC fluorescence, wheat germ agglutinin and anti-beta antibody reactivity in control and crosslinked preparations. FITC exclusively labelled the catalytic monomer of the native preparation and its fluorescence was initially distributed into two broad bands centered at approx. 147 and 173 kDa after crosslinking. These fluorescent bands coincided with the Coomassie stained particles. A glycoprotein(s) detected by wheat germ agglutinin reactivity was present in diffuse areas between 65 and 86 kDa and 95 to 134 kDa in the control preparation. This area was also labelled by the anti-beta antibodies. With crosslinking, the distribution of the wheat germ agglutinin reactive protein and anti-beta antibodies coincided with the crosslinked particles labelled by FITC. The presence of both the catalytic monomer and the beta subunit glycoprotein in the crosslinked particles indicated that these proteins were closely associated in the C12E8 solution. This suggests that the minimal structural particle of the H+/K(+)-ATPase is an alpha,beta-heterodimer.

Adenosine Triphosphatases↗

PubMed1991

Diversity of K+ channels in the basolateral membrane of resting Necturus oxyntic cells.

Patch-clamp techniques have been applied to characterize the channels in the basolateral membrane of resting (cimetidine-treated, nonacid secreting) oxyntic cells isolated from the gastric mucosa of Necturus maculosa. In cell-attached patches with pipette solution containing 100 mM KCl, four major classes of K+ channels can be distinguished on the basis of their kinetic behavior and conductance: (1) 40% of the patches contained either voltage-independent (a) or hyperpolarization-activated (b), inward-rectifying channels with short mean open times (16 msec for a, and 8 msec for b). Some channels showed subconductance levels. The maximal inward conductance gmax was 31 +/- 5 pS (n = 13) and the reversal potential Erev was at Vp = -34 +/- 6 mV (n = 9). (2) 10% of the patches contained depolarization-activated and inward-rectifying channels with gmax = 40 +/- 18 pS (n = 3) and Erev was at Vp = -31 +/- 5 mV (n = 3). With hyperpolarization, the channels open in bursts with rapid flickerings within bursts. Addition of carbachol (1 mM) to the bath solution in cell-attached patches increased the open probability Po of these channels. (3) 10% of the patches contained voltage-independent inward-rectifying channels with gmax = 21 +/- 3 pS (n = 4) and Erev was at Vp = -24 +/- 9 mV (n = 4). These channels exhibited very high open probability (Po = 0.9) and long mean open time (1.6 sec) at the resting potential. (4) 20% of the patches contained voltage-independent channels with limiting inward conductance of 26 +/- 2 pS (n = 3) and Erev at Vp = -33 +/- 3 mV (n = 3). The channels opened in bursts consisting of sequential activation of multiple channels with very brief mean open times (10 msec). In addition, channels with conductances less than 6 pS were observed in 20% of the patches. In all nine experiments with K+ in the pipette solution replaced by Na+, unitary currents were outward, and inward currents were observed only for large hyperpolarizing potentials. This indicates that the channels are more selective for K+ over Na+ and Cl-. A variety of K+ channels contributes to the basolateral K+ conductance of resting oxyntic cells.

Animals↗

PubMed1991

Muscarinic responses of gastric parietal cells.

Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol (CCh, 100 microM) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC50 of 13 microM; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC50 of 110 microM; and by the M1/M3 antagonist, diphenyl-acetoxy-4-methylpiperidinemethiodide (4-DAMP), with an IC50 of 35 nM. The three antagonists displayed equivalent IC50 values for the inhibition of carbachol-stimulated production of 14CO2 from radiolabeled glucose, which is a measure of the turnover of the H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 microM La3+. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC50 of 1.7 nM, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations (greater than 30 nM), suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the [Ca]i transient. Displacement of 3H N-methyl scopolamine (NMS) binding to purified parietal cells by CCh showed the presence of two affinities for CCh, but only a single affinity for 4-DAMP and lower affinity for pirenzepine and AFDX 116, providing further evidence for the parietal cell location of the [Ca]i response. Elevation of steady-state [Ca]i levels with either ionomycin or arachidonic acid did not replicate M3 stimulation of acid secretion or glucose oxidation, hence elevation of [Ca]i is necessary but not sufficient for acid secretion.

Aminopyrine↗

PubMed1991

Mammalian phosphorylating ion-motive ATPases.

The pumps discussed in this review are three members of the phosphorylating class of ion transport ATPases. They are the Na(+)-K(+)-, Ca(2+)- and H(+)-K(+)-ATPases. Recent work on their topology, possible transport mechanisms, ion-binding sites and role of the different subunits found for the Na(+)-K(+)- and H(+)-K(+)-ATPases is presented, with a suggestion of a unifying 10-membrane segment model for the catalytic subunit of this class of enzyme.

Amino Acid Sequence↗

PubMed1991

Neurokinin receptor-mediated regulation of [Ca]i and Ca-sensitive ion channels in mammalian colonic muscle.

In conclusion, these findings suggest the following: (1) NK receptor activation results in [Ca2+]i oscillations; (2) the receptor-mediated [Ca2+]i increase is partially due to influx of Ca2+ through L-type Ca2+ channels and partially to release from intracellular stores; (3) the receptor-mediated depolarization results from activation of a Cl- channel at the cell resting potential; (4) NK receptor-mediated release of [Ca2+]i may play a role in Cl- channel activation; (5) there is no evidence for multiple NK receptor types involved in cell activation.

Animals↗

PubMed1991

Second messengers in the gastric gland: a focus on calcium.

The rabbit gastric gland model was used to study the nature of the muscarinic cholinergic and gastrin responses of parietal cells. Carbachol (100 microM) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist pirenzepine with an IC50 of 13 microM; by the M2 antagonist 11,2-(diethylamino)methyl-1-piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4-benzodiazepin-6-one (AF-DX 116) with an IC50 of 110 microM; and by the M3 antagonist diphenylacetoxy-4-methylpiperidinemethiodide (4-DAMP) with an IC50 of 35nM. The three antagonists displayed similar IC50 values for the inhibition of carbachol-stimulated production of 14CO2 from radiolabeled glucose, which is a measure of the turnover of the H(+)-H(+)-ATPase. Intracellular calcium levels wer measured in gastric glands loaded with FURA2. Carbachol was shown both to release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 microM La3+. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or metabolism. However, the rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC50 of 1.7 nM. Image analysis confirmed that the effect of carbachol and of the antagonists on intracellular calcium was occurring in the partial cell. In particular, the high-affinity inhibition of calcium release by 4-DAMP occurs in the parietal cell. Accordingly, it appears that the secretory receptor of the parietal cell is of the M3 type, and acid secretion depends on the entry of calcium rather than on calcium release from intracellular stores. In parallel experiments gastrin (G-17-sulfated) produced a dose-dependent increase in intracellular calcium (EC50, 0.14 +/- 0.013 microM). No stimulation of acid secretion was observed, but pepsinogen secretion was stimulated dose-dependently (EC50 = 1.17 +/- 0.21 microM).

Acetylcholine↗

PubMed1991

[Effect of structured ambulatory training of patients with chronic respiratory tract diseases on the efficiency of long-term care].

In spite of the availability and widespread use of a number of effective medications, the frequency of severe attacks of asthma bronchiale including those with fatal outcome has not decreased. One possible reason for this is the delay in therapeutic intervention of up to 30 minutes resulting either from transport time to a hospital or from the response time of mobile ambulance services. Treatment delays are also frequent co-factors for decreased well-being in non-emergency cases. Prompt initiation of countermeasures, regardless of time or place, can only be guaranteed when the patient himself is capable of carrying them out with the same expertise as his attending physician. Toward this end, a model patient-training program was investigated. The didactic goal of the training was to make the patient an expert in his chronic disease. The medical goal of the training was to improve the quality of a complex long-term therapy and with it, the patient's well-being and quality of life. The content of the training corresponded largely to that contained in a lecture series for medical students or physicians on the same subject. Style and choice of words (avoidance or explanation of medical terminology) was of course adapted to the lay-status of the patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Psychological↗

PubMed1991

[Glycosylated hemoglobin and diabetes--self monitoring (compliance) in depressed and non-depressed type I diabetic patients].

128 type I diabetics were examined in view of connections between psychosocial factors and metabolic control. Indications of a direct interaction between depressivity, incidence of complaints and the level of hyperglycemia were found. The prevalence of manifest depression ranges at a total of 25 percent. The results further show connections between coping with the disease and behavior. High recording frequency, frequent measurements and medical consultations are found in active coping. Changes in relations to close persons and frequent change of job are psychosocial effects of the disease manifesting themselves in poorer metabolic control. In conclusion, high-risk groups of depressive diabetes patients and those under particular stress are defined.

Adaptation, Psychological↗

PubMed1990

Regulation of intracellular Ca2+ oscillation in AR42J cells.

Recordings of [Ca2+]i in single AR42J cells loaded with Fura 2 were used to study regulation of [Ca2+]i oscillation. Continuous stimulation with the cholecystokinin analogue, (t-butyloxycarbonyl-Tyr-(SO3)-norleucine-Gly-Trp-Nle-Asp-2-phenylethyl ester) or carbachol evoked long lasting oscillation in [Ca2+]i. Removal of CCK-JMV-180 after brief stimulation did not abruptly stop the oscillation. Rather, removal of CCK-JMV-180 resulted in time-dependent reduction in amplitude with little change in frequency of oscillation. The patterns of [Ca2+]i oscillation were affected by activation of protein kinase C and protein kinase A. However, down-regulation of protein kinase C activity did not prevent stimulation of [Ca2+]i oscillation. Hence, we conclude that an active protein kinase C pathway is not crucial for [Ca2+]i oscillation in this cell line. Variation in extracellular Ca2+ concentration (Ca2+out) was used to further characterize the oscillation. Reducing Ca2+out to approximately 10 microM resulted in a time dependent inhibition of [Ca2+]i oscillation. Subsequent step increases in Ca2+out up to 2-3 mM resulted in increased amplitude and frequency of oscillation. Further increase in Ca2+out or an increase in plasma membrane permeability to Ca2+, brought about by an increase in pHo, resulted in increased amplitude, decreased frequency, and modified shape of the [Ca2+]i spikes. These observations point to the existence of regulatory mechanisms controlling the duration of Ca2+ release and entry during [Ca2+]i oscillation.

1-Methyl-3-isobutylxanthine↗

PubMed1990

Conformational transitions of the H,K-ATPase studied with sodium ions as surrogates for protons.

Following a recent demonstration that H,K-ATPase can active transport Na+ at a low rate (Polvani, C., Sachs, G., and Blostein, R. (1989) J. Biol. Chem. 264, 17854-17859), we have looked for and found effects of Na+ ions on the conformational state of gastric H,K-ATPase labeled with fluorescein isothiocyanate. Na+ ions reverse the K(+)-induced quench of the fluorescein fluorescence and somewhat enhance fluorescence in the absence of K+ ions. Equilibrium titrations of the cation effects show that Na+ and K+ ions are strictly competitive with apparent dissociation constants of KNa+ = 62 mM (n = 2) and KK+ = 6.6 mM (n = 2). The observations demonstrate that Na+ ions bind to and stabilize the high fluorescence E1 form of the protein while K+ ions stabilize the low fluorescence E2 form. Elevation of pH from 6.4 to 8.0 increased the apparent affinity of the Na+ ions from approximately 62 to 10.2 mM, consistent with competition between protons and Na+. The action of Na+ to stabilize the E1 form was used to measure the rate of the E2K----E1Na transition with a stopped-flow fluorimeter. The rate at pH 6.4 and 20 degrees C is 18.1 s-1. In addition the rate of the reverse conformational transition E1K----E2K has been measured at several K+ concentrations. From the hyperbolic dependence on K+ concentration a maximal rate of 211 +/- 32 s-1 and intrinsic K+ dissociation constant on E1 of 64.6 +/- 3.3 mM have been estimated. The kinetic and equilibrium data are self-consistent and thus support the proposed action of Na+ and K+ ions. Compared with Na,K-ATPase, the H,K-ATPase exhibits a lower affinity for Na+ on E1 and a much faster rate of the E2K----E1Na transition, but a similar affinity for K+ ions on E1 and rate of the transition E1K----E2K. The significance of the similarities and differences in cation specificity and rates of conformational changes of Na,K- and H,K-ATPases is discussed.

Adenosine Triphosphatases↗