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Biomedical subjects

G Sachs

Publications and source records attributed to G Sachs.

At least 127 records · Page 7Linked to original sources
PubMed1994

In situ hybridization of mRNA for the gastric H+,K(+)-ATPase in rat oxyntic mucosa.

The H+,K(+)-ATPase member of the phosphorylating ion motive ATPases is composed of two subunits, a large alpha-subunit composed of about 1030 amino acids and a smaller beta-subunit consisting of about 290 amino acids. By biochemical and immunological methods both subunits have been found in high abundance in the gastric parietal cell. In the present study in situ hybridization was used for localizing and comparing concentrations of the mRNA for the two subunits in the gastric epithelium. For this purpose 3H-labelled probes were preferred. Hybridization was detected only in the parietal cells. The older parietal cells in the bottom of the mucosa gave a weaker hybridization signal than the younger parietal cells closer to the surface. The margin of experimental ulcers, where the parietal cells are of low differentiation, showed very weak, if any, hybridization. The differences observed in hybridization densities may reflect differences in mRNA synthesis or stability. It is conceivable that older parietal cells, as well as parietal cells of low differentiation, produce relatively small amounts of H+,K(+)-ATPase.

Animals↗
PubMed1994

Effects of antisecretory agents on parietal cell structure and H/K-ATPase levels in rabbit gastric mucosa in vivo.

The effect of inhibition of acid secretion on parietal cell morphology and the concentration of H,K-ATPase alpha-subunit protein was determined by electron microscopy and western blotting. Omeprazole or famotidine alone or in combination were used. Control animals showed a morphological stimulation index (0 = resting, 1.0 = fully stimulated) of 0.60; omeprazole treatment (1 mg/kg, twice a day) resulted a stimulation index of 0.63, famotidine injection (20 mg/kg twice a day) an index of 0.11, famotidine infusion (0.2 mg/hr) for five days an index of 0.38, and the combination of omeprazole and famotidine injection twice a day gave an index of 0.02. No change in the frequency of degenerating or damaged parietal cells was observed in any of the groups. In control animals, the number of lysosomes was 0.9/cell, with famotidine 1.8 and with omeprazole 5.6/cell. H/K-ATPase levels fell by about 25% with omeprazole and rose by about 23% with famotidine.

Animals↗
PubMed1994

The somatostatin receptor subtype on rat enterochromaffinlike cells.

BACKGROUND/AIMS: Gastric enterochromaffinlike (ECL) cells play an important role in peripheral regulation of acid secretion. This study investigated the somatostatin receptor subtype on ECL cells. METHODS: ECL cells were isolated from rat fundic mucosa to a purity of 90%-95% by combining enzymatic digestion, elutriation, density gradient centrifugation, and culture. RESULTS: Polymerase chain reaction performed with templates from an ECL cell complementary DNA library and primers specific to each of the five known somatostatin receptor subtypes showed that the somatostatin receptor type 2 was significantly enriched in ECL complementary DNA. Single cell videoimaging of highly purified ECL cells in culture showed that only the somatostatin receptor type 2 selective agonist, DC 32-87, inhibited the gastrin-induced calcium signal at 10(-11) mol/L. The type 3 and type 4 selective agonists, DC 25-12 and DC 32-92, and also somatostatin 14 required 100-1000 times higher concentrations (10(-8) mol/L). The somatostatin receptor type 2 analogue also inhibited gastrin-stimulated histamine release with a 50% inhibitory concentration (IC50) value of 2 x 10(-12) mol/L, whereas somatostatin 14 and the type 3 and 4 analogues showed IC50 values of 1 to 5 x 10(-9) mol/L. CONCLUSIONS: The predominant somatostatin receptors on rat gastric ECL cells are of the somatostatin receptor 2 subtype; they inhibit histamine secretion by interfering with the gastrin-induced calcium signal.

Amino Acid Sequence↗
PubMed1994

Psychological moderator variables and metabolic control in recent onset type 1 diabetic patients--a two year longitudinal study.

The relationships between psychosocial adjustment and subsequent glycaemic control were prospectively examined in forty-three adult patients during the first 2 yr after onset of type 1 diabetes mellitus. Decreasing depression was the single psychosocial parameter that changed over time. No correlations were found between the decrease in HbA1c levels and psychological variables at 8- and 16-month follow-ups. Global and specific coping features such as high control attitude, low coping anxiety and low emotional attribution correlated significantly with the decrease in HbA1c levels at the 2-yr follow-up, whereas stressful life events, depression, state-trait anxiety did not correlate. In a regression analysis coping explained 22% variance of the 2 yr decrease in HbA1c levels. We conclude that coping is a better predictor for metabolic control than emotional adaptation and life events. Metabolic control might deteriorate with prolonged stage of the disease being a first sign for psychophysiological coping exhaustion.

Adaptation, Psychological↗
PubMed1994

Differential effects of carbachol on calcium entry and release in CHO cells expressing the m3 muscarinic receptor.

Calcium signalling was examined in CHO-k1 cells that stably express the m3 subtype of the muscarinic receptor. The calcium indicator Fura-2 was retained in these cells only in the presence of probenecid (1 mM), suggesting that Fura-2 efflux was mediated by an organic anion transporter. The addition of carbachol (CCh) to Fura-2 loaded cells in suspension caused a rapid transient increase in intracellular calcium [Ca]i followed by a smaller sustained plateau phase. The transient rise in [Ca]i was dose-dependent with a threshold response of 89 +/- 18 nM above baseline with 10 nM CCh and a maximum stimulation of 734 +/- 46 nM with 10 microM CCh. This phase was accompanied by a similar dose-dependent stimulation of total inositol phosphate production and was assumed to be generated by release from intracellular stores of the endoplasmic reticulum (ER). The sustained increase in [Ca]i was generated by entry from the extracellular bath since it was blocked by pretreatment with La3+ (1 microM) and was absent when bath calcium was chelated with EGTA. This phase was not dependent on CCh dose, and a stimulation of [Ca]i of approximately 90 nM above baseline was observed with CCh concentrations between 50 nM and 10 microM. With this dose range, the rate of Mn2+ quenching of Fura-2 at the Ca-insensitive excitation wavelength of 360 nm was likewise maximally stimulated. At lower CCh concentrations (10-50 nM), it was clear that the activation of Ca entry could not be dissociated from a threshold release of Ca from intracellular stores. The phorbol ester PMA, which uncouples the muscarinic receptor from phospholipase C, reduced the transient rise in [Ca]i by approximately 50% with little or no effect on Ca entry at higher CCh levels (> or = 1 microM). At lower CCh concentrations (< or = 100 nM) however, pretreatment with PMA completely blocked all Ca mobilization and supports the contention that Ca entry is coupled to Ca release from stores or to store depletion. The emptying of inositol trisphosphate-sensitive stores with thapsigargin (10 nM) stimulated Ca entry and also the rate of Mn2+ quenching. Store depletion by incubation in Ca-free media likewise stimulated Mn2+ uptake without a rise in [Ca]i. Our data are therefore consistent with a 'capacitative' coupling model, whereby the activation of the plasma membrane receptor leads to an InsP3-induced change in the degree of filling of the ER Ca pool.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

[A simple bite protector for the laryngeal mask].

A new simple gag is presented securing the width of the tube of the laryngeal mask. The device is a 45 degrees-angled, u-shaped, synthetic-coated aluminium rod. This gag, while being additionally an aid to ensure the proper position of the laryngeal mask, is easy to handle and can be re-used after disinfection. Due to its rounded shape and the soft synthetic coating, accidental damage of the teeth seems to be nearly impossible.

Equipment Design↗
PubMed1994

Continuing development of acid pump inhibitors: site of action of pantoprazole.

Both receptor antagonists and acid pump inhibitors are clinically useful suppressants of acid secretion. The latter class of drugs, the substituted benzimidazoles, inhibit acid secretion more effectively and, therefore, provide superior symptom relief and healing in all acid-related diseases. The H2-receptor antagonists competitively block the action of histamine on the H2-receptors of parietal cells. This histamine is released from enterochromaffin-like cells (ECL cells) due to gastrin, acetylcholine or epinephrine stimulation. In addition, parietal cells have M3-receptors which can function independently of H2-receptors. Hence, there is no single common pathway for parietal cell stimulation. Stimulation of acid secretion by parietal cells requires activation of the acid pump, the gastric H+,K(+)-ATPase. The target site for the benzimidazoles is the activated gastric H+,K(+)-ATPase, and, in particular, the cysteines of the pump that are exposed to the acid space of the secretory canaliculus of the parietal cells. Pantoprazole in its protonated form selectively reacts with cysteines present in both the fifth and sixth membrane segments of the ATPase, explaining its mechanism of inhibiting proton transport by this enzyme.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1994

Studies on a novel series of acyl ester prodrugs of prostaglandin F2 alpha.

A novel series of prostaglandin F2 alpha (PGF2 alpha) prodrugs, with acyl ester groups at the 9, 11, and 15 positions, was prepared in order to design clinically acceptable prostaglandins for treating glaucoma. Studies involving isolated esterases and ocular tissue homogenates indicated that 9-acyl esters cannot provide a prodrug since PGF2 alpha would not be formed as a product. In contrast, 11-mono, 15-mono, and 11, 15-diesters were converted to PGF2 alpha in ocular tissues and could, therefore, be considered as prodrugs of PGF2 alpha. Carboxylesterase (CE) appeared critically important for the hydrolytic conversion of those PGF2 alpha prodrugs where the 11 or 15-OH group was esterified and such prodrugs were not substrates for acetylcholinesterase (ACHE) or butyrylcholinesterase (BuCHE). The enzymatic hydrolysis of PGF2 alpha-1-isopropyl ester was also investigated for comparative purposes. This PGF2 alpha prodrug was a good substrate for CE, but was also hydrolysed by BuCHE, albeit at a much slower rate. The most striking feature of the enzymatic hydrolysis of PGF2 alpha-1-isopropyl ester in ocular tissue homogenates was that it was much faster than for prodrugs esterified at the 11 and/or 15 positions. In terms of ocular hypotensive activity, all prodrugs which showed detectable conversion to nascent PGF2 alpha were potent ocular hypotensives. Although no separation of ocular hypotensive and ocular surface hyperaemic effects was apparent for PGF2 alpha-1-isopropyl ester, a temporal separation of these effects was apparent for the novel PGF2 alpha ester series. This difference may reflect an unfavourably rapid conversion of PGF2 alpha-1-isopropyl ester in ocular surface tissues compared with anterior segment tissues.

3T3 Cells↗
PubMed1994

Ion transport asymmetry and functional coupling in bovine pigmented and nonpigmented ciliary epithelial cells.

The solute and water transport properties of the bovine ciliary epithelium were studied using isolated pigmented (PE) and nonpigmented (NPE) cells. It was shown that these cells were functionally coupled by demonstrating dye diffusion between paired PE and NPE cells after microinjection of lucifer yellow. Electronic cell sizing was used to measure cell volume changes of isolated PE and NPE cells in suspension after anisosmotic perturbations and after transport inhibition under isosmotic conditions. The PE cells showed the presence of a regulatory volume increase when subjected to osmotic shrinkage with NaCl, whereas the NPE cells did not demonstrate a regulatory volume increase under these conditions. In contrast, the NPE cells exhibited a regulatory volume decrease when subjected to osmotic swelling, whereas the PE cells did not recover from swelling. The regulatory volume decrease in NPE cells was inhibited by increased bath K or pretreatment with quinine (1 mM). The presence of a bumetanide-sensitive mechanism capable of moving measurable amounts of solute and water, probably Na-K-2Cl cotransport, was demonstrated in the PE cells but absent in the NPE cells. Bumetanide produced a dose-dependent shrinkage of PE cells at concentrations as low as 1 microM. Isosmotically reducing bath Cl, Na, or K concentration caused a rapid shrinkage of PE cells that was bumetanide inhibitable. The asymmetry of transport properties in PE and NPE cells supports a functional syncytium model of aqueous humor formation (39) across the two layers of the ciliary epithelium wherein ion uptake from the blood is carried out by the PE cells and ion extrusion by the NPE cells. Gap-junction coupling between the cells allows the ions taken up by the PE cells to move into the NPE cells. Extrusion of Na by the Na-K pump across the aqueous facing (basolateral) membranes of the NPE cells, most likely accompanied by Cl, determines the formation of the aqueous humor.

Animals↗
PubMed1994

Effect of histamine on rat gastric H(+)-K(+)-ATPase alpha-subunit expression.

The H(+)-K(+)-adenosinetriphosphatase (ATPase) is expressed in the parietal cell and is responsible for acid secretion by the stomach. Histamine binds to an H2 receptor and activates adenylate cyclase and intracellular calcium concentration ([Ca2+]i) elevation, stimulating acid secretion. It has been shown that omeprazole administered to rats increases serum gastrin and transiently increases the level of mRNA for the alpha-subunit of the pump, but this increase is blocked by the presence of the H2-receptor antagonist, famotidine [A. Tari, G. Yamamoto, K. Sumii, M. Sumii, Y. Takehara, K. Haruma, G. Kajiyama, V. Wu, G. Sachs, and J. H. Walsh. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G752-G758, 1993]. These observations suggest that the release of histamine induced by gastrin is essential for the increase of the expression of mRNA induced by omeprazole. Infusion of histamine at 15 mumol.kg-1.h-1 i.v. for 1 h increased the alpha-subunit mRNA level by 144 +/- 2.4% and induced a stimulated morphological appearance of the parietal cell. These changes were inhibited completely by the competitive H2-receptor antagonist famotidine, which elevated gastric pH and serum gastrin. Famotidine also reduced the level of H(+)-K(+)-ATPase mRNA compared with control animals. No change in the expression of beta-actin mRNA was observed in any group of animals. These data provide direct evidence for histamine stimulation of H(+)-K(+)-ATPase alpha-subunit gene expression by activation of the H2 receptor.

Animals↗
PubMed1994

Gastrin effects on isolated rat enterochromaffin-like cells in primary culture.

The hormone gastrin stimulates acid secretion by releasing histamine from gastric enterochromaffin-like (ECL) cells and induces ECL cell proliferation in vivo. This study uses a > 90% pure ECL cell preparation in culture to compare gastrin effects on histamine release, histidine decarboxylase (HDC) activity, and DNA synthesis. Gastrin and the cholecystokinin octapeptide (CCK-8, nonsulfated) induced histamine release from ECL cells (24-96 h of primary culture) within 5 min of incubation [concentration eliciting 50% of maximal response (EC50), 4 and 2 x 10(-11) M, respectively]. The CCK-B antagonist L-365,260 inhibited this effect [concentration inhibiting 50% of maximal response (IC50), 2 x 10(-8) M], whereas the CCK-A antagonist L-364,718 (10(-8) M) and the tyrosine kinase inhibitor genistein (10(-4) M) had no effect. Histamine release was associated with a biphasic elevation of intracellular Ca2+. Gastrin stimulated HDC activity two- to threefold after 60 min of incubation (EC50, 10(-10) M). Gastrin also increased DNA synthesis in ECL cells, with an EC50 of 1.7 x 10(-12) M as measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). Positive nuclear immunostaining increased two- to threefold in up to 20% of ECL cells after 48-96 h of incubation. This effect was inhibited by L-365,260 (IC50, 5 x 10(-9) M) and by genistein (10(-4) M) but was not altered by L-364,718 (10(-8) M). The antisecretory drugs omeprazole, lansoprazole, and pantoprazole did not affect BrdU incorporation in isolated ECL cells. In conclusion, acute and chronic gastrin effects on the ECL cell are mediated via CCK-B receptors but differ in apparent receptor affinity and signal transduction pathways.

Animals↗
PubMed1994

[Initial experience with endoscopic subfascial dissection of perforating veins in 100 interventions].

Progress in minimal invasive surgery made us look for a new concept to treat our patients with varicosis, which is one of the most frequent operations in our department. We changed our method from an epifascial ligation to an endoscopic subfascial dissection of the perforating veins (ESDP). This method was developed and first described by Hauer in 1985 [2]. Between March 1992 and May 1993 we treated 85 patients by this method, performing ESDP on 100 legs. In most of the cases the operation is combined with a classic Babcock's procedure, although it can be done isolated. Sectioning of the perforating veins needs special instruments and must be performed in partial deprivation of blood supply, which is produced by a sterile Löfqvist roller cuff [5]. The sectioning of the perforating veins through a small incision in the medial part of the lower leg allows a dissection of all perforating veins in the medial and dorsal sections of the lower leg. The subfascial procedure reduces delayed wound healing, especially in patients with trophic skin disorders. The operation in deprivation of blood supply allows smaller and therefore cosmetically better incisions also for the other subcutaneous varices.

Endoscopes↗
PubMed1994

[Phase-specific function of denial in type 1 diabetic patients after disease onset].

In a longitudinal study we examined 43 patients with type 1 diabetes one week after onset as well as 8 and 24 month later in order to analyze the psychological role of denial processes in correlation to metabolic functions. Only depression decreased over the studied period while coping and denial remained stable. However, the adaptive function of denial after onset with low anxiety, good coping and few complaints became maladaptive over the first two years and the correlation of denial with a centripetal kinship behavior loosened. The destructive effect of denial was indicated only by delayed requests for assistance while no correlation could be shown for phase-specific internal restructuring of the psychological function of denial to compliance and metabolic control.

Adaptation, Psychological↗
PubMed1993

The site of action of pantoprazole in the gastric H+/K(+)-ATPase.

Pantoprazole is a pyridinyl-2-methylenesulfinyl-2-benzimidazole derivative. This compound inhibits the vesicular gastric H+/K(+)-ATPase (cytoplasmic side out) under acid transporting conditions by accumulating in the acid space generated by the pump. Pantoprazole is then converted in an acid-catalysed reaction to a cationic sulfenamide and reacts with cysteines available in or from the acidic extracytoplasmic space. This compound binds to the hog gastric H+/K(+)-ATPase with a stoichiometry of 3 nmol per mg protein, resulting in 94% inhibition of ATPase activity. Tryptic cleavage of the intact vesicles which had been reacted with [14C]pantoprazole at a 1 to 4 trypsin to protein ratio removed most of the cytoplasmic domain leaving the pairs of membrane spanning segments and their connecting extracytoplasmic loops intact. The peptides remaining in the membrane were dissolved in SDS and available cysteine residues labelled with fluorescein-5-maleimide. The peptides were separated on Tricine gradient gels, transferred to PVDF membranes and identified by fluorescence and radioactivity. From N-terminal sequence, fluorescence and molecular mass, it is concluded that pantoprazole is able to label both Cys-813 and Cys-822. These cysteines are predicted to be located in the extracytoplasmic loop connecting membrane segments 5 and 6 and in membrane segment 6. The major cytoplasmic tryptic cleavage site at this location moved from position 776 in unmodified enzyme to positions 784 and 792 following pantoprazole labelling, showing that the configuration of this region changed with pantoprazole labelling. A similar result was obtained by reduction of the enzyme with dithiothreitol. Covalent binding of the cationic sulfenamide to this region of the enzyme is able to block the conformation necessary for phosphorylation of the enzyme by ATP, accounting for its inhibitory effect on acid secretion.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1993

Membrane topology and omeprazole labeling of the gastric H+,K(+)-adenosinetriphosphatase.

The gastric H+,K(+)-ATPase is an alpha beta heterodimer with close homology to the Na+,K(+)-ATPase. Digestion of intact cytoplasmic-side-out vesicles at a trypsin to protein ratio of 1/4 removed most of the cytoplasmic protein, leaving membrane-spanning pairs in high yield. These were visualized on gels and poly(vinylidene difluoride) (PVDF) membranes by sodium dodecyl sulfate solubilization of the membrane-embedded segments and labeling of the cysteine residues with fluorescein maleimide prior to electrophoresis. The membrane-spanning residues of the alpha subunit were found between positions 104 and 162 (M1/M2), 291 and 358(M3/M4), 776 and 835 (M5/M6), and 853 and 946 (M7/M8). Although this method did not detect membrane retention of the hydrophobic sequences subsequent to position 946, it provided biochemical evidence for at least eight membrane segments in the catalytic subunit. Intact vesicles containing this enzyme transport acid in the presence of KCl, valinomycin, and MgATP. Omeprazole accumulates in these acidified vesicles and converts to a cationic sulfenamide. This forms disulfides with accessible cysteines. The reaction with this extracytoplasmic thiol reagent inhibits ATPase activity. Full inhibition was obtained with a stoichiometry of 2.2 mol of omeprazole bound/mg of protein. Only the alpha subunit was labeled. The cysteines reacting with omeprazole were defined by proteolytic cleavage of 3H- or 14C-omeprazole-labeled enzyme followed by peptide sequencing of fragments separated on tricine gradient gels and transferred to PVDF membranes. Tryptic digestion at a 1/40 trypsin to protein ratio in the presence of ligands that stabilize the E2P form of the enzyme produced two large fragments, one of 68 kDa stretching from Glu47 to probably Arg666 that contained minor labeling and the other of 333 kDa beginning at Ala671 and extending to probably Arg946 that contained greater than 85% of the label. Digestion of labeled vesicles at 1/75 or 1/4 trypsin to protein ratios gave radioactive patterns consistent with labeling at Cys813 and/or Cys822 and at Cys892 and/or Cys927 and/or Cys938. V8 protease digestion of the solubilized alpha subunit produced a fragment extending from Ser838 to possible Asp900 that was omeprazole-labeled, showing that Cys892 was labeled and Cys927 and Cys938 were not. Hence, omeprazole labels the H+,K(+)-ATPase at cysteines within the M5/M6 and M7/M8 regions of the alpha subunit, accounting for its inhibitory action in vivo and in vitro.

Adenosine Triphosphate↗
PubMed1993

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.

Aminopyrine↗