Prevalence and phenotype consequence of FRAXA and FRAXE alleles in a large, ethnically diverse, special education-needs population.

We conducted a large population-based survey of fragile X (FRAXA) syndrome in ethnically diverse metropolitan Atlanta. The eligible study population consisted of public school children, aged 7-10 years, in special education-needs (SEN) classes. The purpose of the study was to estimate the prevalence among whites and, for the first time, African Americans, among a non-clinically referred population. At present, 5 males with FRAXA syndrome (4 whites and 1 African American), among 1,979 tested males, and no females, among 872 tested females, were identified. All males with FRAXA syndrome were mentally retarded and had been diagnosed previously. The prevalence for FRAXA syndrome was estimated to be 1/3,460 (confidence interval [CI] 1/7,143-1/1,742) for the general white male population and 1/4, 048 (CI 1/16,260-1/1,244) for the general African American male population. We also compared the frequency of intermediate and premutation FRAXA alleles (41-199 repeats) and fragile XE syndrome alleles (31-199 repeats) in the SEN population with that in a control population, to determine if there was a possible phenotype consequence of such high-repeat alleles, as has been reported previously. No difference was observed between our case and control populations, and no difference was observed between populations when the probands were grouped by a rough estimate of IQ based on class placement. These results suggest that there is no phenotype consequence of larger alleles that would cause carriers to be placed in an SEN class.

Differential effects of GTP gamma S on acid and pepsinogen secretion by permeable gastric glands.

Gastric glands isolated from rabbit stomach were permeabilized with Staphylococcus aureus alpha-toxin. Acid secretion by parietal cells, as measured by the accumulation of weak base, was inhibited by incubation with alpha-toxin but could be restored by addition of exogenous ATP (1 mM). The permeable glands were found to retain acid secretory responses to receptor-linked secretagogues, histamine and carbachol, as well as to intracellular mediators, forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate, indicating the presence of intact, functional intracellular coupling mechanisms. Both basal and stimulated acid secretion by the permeable glands were blocked by the Mg2+ chelator, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA; 5 mM), whereas CDTA had no effect on nonpermeabilized glands. These results are interpreted to show that alpha-toxin permeabilizes parietal cells to moderate sized molecules without causing a loss of critical intracellular components. The acid secretory responses to histamine and carbachol persisted in media containing low ( < 50 nM) levels of free Ca2+ buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (0.5 mM), indicating that changes in bulk Ca2+ are not required for these responses. Inclusion of the nonhydrolyzable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM), resulted in inhibition of spontaneous acid secretion, blocked responses to all agents tested, and inhibited stimulated acid secretion. GTP gamma S had no effect on nonpermeabilized glands. No effects on acid secretion by either permeable or nonpermeable glands were observed with GTP, guanosine diphosphate, or guanosine 5'-O-(2-thiodiphosphate). GTP gamma S had no effect on H+ gradient formation by gastric membrane vesicles, showing that it does not inhibit the gastric H(+)-K(+)-adenosinetriphosphatase directly. These results are interpreted to show that GTP gamma S interacts at a postreceptor site to inhibit or reverse a critical step in stimulus-secretion coupling in parietal cells. In contrast to the effect on parietal cells, GTP gamma S was found to stimulate pepsinogen secretion by alpha-toxin-permeabilized chief cells. The differential effects of GTP gamma S on acid and pepsinogen secretions suggest unique roles for GTP binding proteins in these two secretory processes. The use of alpha-toxin-permeabilized gastric glands should prove useful in defining the stimulus-secretion coupling mechanisms involved in both acid and pepsinogen secretions.

Partial agonism by gastrin for a cholecystokinin receptor mediating pepsinogen secretion.

Isolated gastric glands from rabbit were used to characterize the functional cholecystokinin (CCK)-like peptide receptors that mediate pepsinogen secretion. Pepsinogen secretion was stimulated by both CCK octapeptide sulfate (CCK-8) and A-71378, a selective CCK-A-type receptor agonist, with similar mean effective doses (1.0 and 0.8 nM, respectively). Compared with CCK-8, gastrin-17 (G-17-I) showed reduced potency and only partial efficacy for stimulation of pepsinogen secretion while inhibiting the maximal CCK-8-stimulated response. The nonpeptide inhibitors, asperlicin and L-364,718, inhibited pepsinogen secretion with identical pA2 values for antagonism of both CCK and gastrin, indicating that both peptides interact with the same functional receptor. Specific binding of [3H]CCK-8 to isolated chief cell membranes was displaced fully by both CCK and gastrin, indicating full receptor occupancy by both peptides. A novel synthetic peptide analogue, pseudogastrin [(Glu)5-Ala-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2], was used to investigate the structural basis for the lower potency and efficacy of G-17-I. The potency of CCK and gastrin analogues for pepsinogen secretion was found to be dependent on both sulfation of a tyrosine residue and the position of the tyrosine residue relative to the COOH-terminal phenylalanine amide. The efficacy appears to be determined partially by the extended NH2-terminal sequence of G-17-I. The results of the present study are interpreted to show that pepsinogen secretion is mediated by a CCK-A-type receptor and gastrin acts at the same receptor as a partial agonist.

The site of acid secretion in the mammalian parietal cell.

Initiation of acid secretion in the gastric mucosa is accompanied by a morphological transformation in which the acid pump, the H+/K(+)-ATPase, translocates from a cytoplasmic vesicular location to the secretory surface lining the canaliculi. Associated with the morphological changes, activation of K+ and Cl- pathways are necessary to supply K+ to the extracytoplasmic face of the pump. Although the pump in the secretory membrane is known to secrete acid, it is not known whether activation of the KCl pathway occurs in the tubulovesicular membrane prior to the formation of the canaliculus, or when the pump is in the secretory membrane. The cellular site of activation of acid secretion in the rabbit gastric parietal cell was investigated using the covalent binding of [3H]omeprazole as a probe of acid secretion in rabbit gastric glands that were undergoing stimulation in vitro. This compound depends on an acidic environment for activation and covalent binding to the H+/K(+)-ATPase. Electron microscopic autoradiography showed that activation of the enzyme occurred only when it was present in the canalicular membrane and not when it was present in the cytoplasmic tubulovesicular membrane. Hence there is likely to be a physical separation of K+ and/or Cl- pathways from the ATPase in the resting cell, and stimulation of acid secretion is dependent on colocalization of these pathways in the canalicular membrane.

Interaction of cocaine with nasal mucosa.

Canine nasal mucosa was studied in vitro to examine (1) the production of vasoconstriction by cocaine and, (2) the epithelial permeability of cocaine. Cocaine, by itself, failed to induce any contraction of the nasal blood vessels but did enhance contractions resulting from electrical stimulation or addition of norepinephrine. Results indicate that cocaine produces vasoconstriction by blocking the reuptake of endogenous norepinephrine rather than any direct action on vascular smooth muscle. Cocaine was found to be three times more permeable than sucrose, which has a similar molecular weight. The transepithelial permeability of cocaine was independent of direction and did not display competition. Results indicate that cocaine permeates by simple diffusion and that the relatively high permeability is due to a greater lipid solubility. Cocaine was found to accumulate in the nasal mucosa. A significant portion of the accumulation is associated with specific sites that are characteristic of catecholamine uptake sites.

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.

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).

Gastric H+,K(+)-ATPase as a therapeutic target in peptic ulcer disease.

The presence of unbuffered acid appears to be an essential contributory factor in the pathogenesis of peptic ulcer disease. Treatment has concentrated therefore on the reduction of acidity, and the last decade has seen the widespread and effective use of H2 antagonists. They are, at low doses, more successful in improving the natural history of duodenal ulcer disease than of gastric or esophageal ulceration. The H2 receptor plays a central role in activation of parietal cell acid secretion, and antagonists at this receptor block most (but not all) of the acid secretion due to even gastrinergic or muscarinic (vagal) stimulation. In hypergastrinemic states such as Zollinger-Ellison syndrome, or where acid secretion has to be inhibited by more than 20% over a 24-hr period, such as for treatment of esophagitis, NSAID damage, or gastric ulcers, the dose and frequency of administration of the currently available antagonists must be increased to achieve reliable therapy. This has led to a search for an alternative target for acid inhibitory drugs, such as the gastric acid pump, the H+,K(+)-ATPase. This article focuses on the function of this ATPase and suggests that inhibition of this pump will provide a more efficacious means of reduction of acid secretion by the stomach, hence improving and simplifying therapy of acid related diseases.

Permeable cell models in stimulus-secretion coupling.

A number of mechanical and chemical methods have been developed to achieve selective permeabilization of the plasma membrane. These methods have been applied to many cell types and have proven to be highly useful for studying stimulus-secretion coupling mechanisms. Thus far, this approach has contributed significantly to our understanding of phosphoinositide metabolism and the regulation of intracellular calcium. The permeabilization techniques also have contributed important information regarding cAMP-dependent pathways. In addition to studies of stimulus-secretion coupling, permeabilized cell preparations can be employed for investigations of enzyme activity in situ and the properties of intracellular organelles in general. Since cell permeabilization, particularly with chemical agents, is surprisingly easy, these techniques should find wide application for future studies.

Location of the carbohydrates present in the HK-ATPase vesicles isolated from hog gastric mucosa.

The glycosylation of H+K(+)-ATPase vesicles isolated from hog gastric mucosa was investigated by various methods. Following protein separation on sodium dodecyl sulfate reducing gels and transfer to poly(vinyl difluoride) membranes, binding of concanavalin A was confined to the 94-kDa band which corresponds to the catalytic subunit. In contrast, wheat germ agglutinin binding occurred in a region below the 94-kDa subunit, corresponding to the 60-85-kDa region, and also to protein just above the catalytic subunit. Treatment with glycopeptidase F removed most of the concanavalin A staining and also the wheat germ agglutinin staining found below the 94-kDa region, but spared the higher molecular weight wheat germ agglutinin reactive material. During the deglycosylation experiments a protein of 35-kDa was produced. Sequencing analysis of V8 protease generated peptide fragments of the 35-kDa protein show at least 30% homology with the Na+K(+)-ATPase beta-subunits. Labeling of the carbohydrates by galactosyltransferase and [3H]uridine diphosphate-galactose showed that the sites of labeling were extracellular and were confined to the wheat germ agglutinin staining regions. Two molecular weight regions, below the 94-kDa region, of 60 and 85 kDa were identified. Electron microscopy using postembedding staining techniques showed that both concanavalin A and wheat germ agglutinin staining occurred on the extracellular face of the gastric vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional domains of the gastric HK ATPase.

The gastric H+ + K+ ATPase is a member of the phosphorylating class of transport ATPase. Based on sequence homologies and CHO content, there may be a b subunit associated with the catalytic subunit of the H+ + K+ ATPase. Its function, if present, is unknown. The pump catalyzes a stoichiometric exchange of H+ for K+, but is also able to transport Na+ in the forward direction. This suggests that the transport step involves hydronium rather than protons. The initial binding site is likely to contain a histidine residue to account for the high affinity of the cellular site. The extracellular site probably lacks this histidine, so that a low affinity for hydronium allows release into a solution of pH 0.8. Labelling with positively charge, luminally reactive reagents that block ATPase and pump activity has shown that a region containing H5 and H6 and the intervening luminal loop is involved in necessary conformational changes for normal pump activity. The calculated structure of this loop shows the presence of an a helical, b turn, and b strand sector, with negative charges close to the membrane domain. This sector provides a possible site of interaction of drugs with the H+ + K+ ATPase, and may be part of the K+ pathway in the enzyme.

Gastric H+-K+-ATPase in situ: evidence for compartmentalization.

Gastric glands, isolated from rabbit, were permeabilized with digitonin to permit measurement of H+-K+-adenosinetriphosphatase (ATPase) activity and proton transport in situ. Measurement of proton gradient formation using acridine orange fluorescence showed two phases of ATP-driven proton accumulation; one phase occurs spontaneously in KCl medium and one phase requires the K+ ionophore valinomycin. Valinomycin was found to increase H+-K+-ATPase activity, indicating that the second phase is because of increased proton transport rather than a decrease in proton leak rate. The acid-activated, irreversible inhibitor, omeprazole, was used to selectively eliminate the H+-K+-ATPase molecules associated with the spontaneous component of proton transport. After omeprazole treatment a residual, valinomycin-dependent component of proton transport could be demonstrated. These results are interpreted as evidence for two compartments of H+-K+-ATPase, separated by a barrier that prevents K+ diffusion and pH equilibration. The two compartments may be separated also on the basis of anion selectivity. The spontaneously active compartment was found to be functional with various anions, including sulfate and isethionate, whereas the valinomycin-dependent component is highly selective for chloride. The proportion of H+-K+-ATPase that exists in each compartment was quantitated by measuring the fraction of total ATPase activity that could be inhibited by omeprazole in the absence and presence of valinomycin. For glands that were preconditioned with cimetidine, approximately 30% of the inhibitable enzyme was found associated with the spontaneous compartment, and this fraction increased to approximately 70% with histamine preconditioning.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a chloride conductance in secretory membrane of parietal cells.

A fluorescence-quench method using acridine orange as the probe was employed to monitor acid formation in situ by detergent-permeabilized gastric glands. In KCl medium, the addition of ATP to the permeabilized glands resulted in a rapid decrease in fluorescence and addition of valinomycin resulted in a second phase of fluorescence quench. The fluorescence was restored by addition of the H+-K+-ATPase inhibitor, Sch 28080. An ATP-dependent fluorescence quench was observed also in K2SO4 or K+-isethionate medium; however, valinomycin was ineffective in the Cl-free media. The ATP-dependent quench could be reversed or prevented by the electrogenic protonophore, tetrachlorosalicylanilide (TCS), in KCl medium but not in Cl-free media. The results with TCS are interpreted as demonstrating a large Cl- conductance in the secretory membrane, whereas the results with valinomycin indicate that resting membranes lack a K+ conductance. The data suggest that a complex KCl pathway that may demonstrate a Cl- conductance is used to activate acid secretion.

Interaction of anti-ulcer drugs with the gastric proton pump.

In this review consideration is given to anti-ulcer drugs interaction with the gastric H(+)-K+ ATPase. The review has been divided into three sections. First, properties of the gastric proton pump are described in terms of structure, biological activity and ions transport activity, followed by an account of interactions involving antisecretory agents. Emphasis is given to a new class of drugs (substituted benzimidazole) that shows a unique antisecretory action and is safe and effective for short-term treatment of patients with duodenal or gastric ulcers. The final section briefly examines future directions for the production of more selective inhibitors of the gastric proton pump.

Stimulation of acid formation in permeable gastric glands by valinomycin.

Isolated gastric glands made permeable with digitonin treatment were employed to study the ionic requirements of acid formation. Acid formation was monitored by the accumulation of a novel weak base probe, [14C]benzylamine. ATP-dependent acid formation was found to require K+ in a concentration-dependent manner, with an apparent K0.5 = 7 mM. The anion dependence of acid formation gave a selectivity sequence of Cl = I greater than Br greater than NO3 greater than SO4 = isethionate, with isethionate being approximately 50% as effective as Cl. The dependence of acid formation on [Cl] gave an apparent K0.5 = 6 mM. Addition of the K+ ionophore, valinomycin, to resting glands (cimetidine pretreatment) resulted in a two- to threefold increase in ATP-dependent acid formation. In contrast, stimulated (forskolin pretreated) glands showed a greater accumulation of benzylamine with ATP but significantly less valinomycin stimulation. The valinomycin stimulation required both K+ and Cl- and was inhibited by omeprazole and Sch 28080. The results are interpreted to indicate that major events in the transition from a resting to a stimulated state include changes in both K+ and anion permeability of the secretory membrane of parietal cells.

SCH28080 prevents omeprazole inhibition of the gastric H+/K+-ATPase.

The interaction between SCH28080 and omeprazole, two specific inhibitors of gastric H+/K+-ATPase, was investigated using gastric glands and isolated gastric membranes. For gastric glands, inhibition of acid formation by SCH28080 was not reversed by washing whereas inhibition by omeprazole was partially reversed after washing. These features are opposite to what is found with isolated membranes. However, if gastric glands were permeabilized with digitonin after exposure to the inhibitors and recovery measured as ATP-dependent acid formation or H+/K+-ATPase activity, inhibition by SCH28080 was completely reversed while inhibition by omeprazole was non-reversible. Using a procedure of pretreatment with inhibitors followed by permeabilization and assay of recovered activity, it was found that a combined treatment with SCH28080 plus omeprazole prevented the irreversible inhibition by omeprazole, i.e. acid forming capability and ATPase activity were fully recovered. In order to test the possibility that SCH28080 prevented activation of omeprazole by dissipating an acid environment, control experiments were performed with SCN, which gave equivalent dissipation of the acid gradient but did not prevent the irreversible inhibition by omeprazole. These results were confirmed in isolated gastric membranes where residual p-nitrophenylphosphatase activity was assayed following exposure of acid transporting vesicles to omeprazole. Compared to control conditions, omeprazole inhibited 48% of the phosphatase activity whereas simultaneous addition of SCH28080 reduced the inhibition to 14%. The results therefore suggest that SCH28080 selectively blocks irreversible inhibition by omeprazole and thus that these two agents interact at a common region of the luminal aspect of the gastric H+/K+-ATPase.

Gastric H+-K+-ATPase in situ: relation to secretory state.

Isolated gastric glands from rabbit were used to measure the gastric H+-K+-adenosinetriphosphatase (ATPase) and its partial reaction, a K+ p-nitrophenyl phosphatase (pNPPase), in situ. Measurement of the enzyme activities required permeabilization of the cells with digitonin and the use of several ATPase inhibitors to reduce nonspecific activity. The enzyme activities were identified as the H+-K+-ATPase according to the following criteria: dependence on K+, association with parietal cells, insensitivity to ouabain and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and inhibition by the specific inhibitors omeprazole and Sch 28080. K+-stimulated ATPase, but not K+-pNPPase, was enhanced by K+ ionophores, valinomycin and nigericin, with nigericin resulting in the greatest activity. Comparison of tissues that were preincubated to establish resting and stimulated states showed that prestimulation results in an increase in K+-stimulated ATPase activity with no change in the total activity. With the use of a two-step assay procedure, it could be shown that stimulation also results in an increase in the omeprazole-sensitive maximal ATPase activity. These results indicate that the major effects of stimulation are to enhance KCl activation of the enzyme and to increase the number of active enzyme molecules.