Characterization of gastric mucosal membranes. X. Immunological studies of gastric (H+ + K+)-ATPase.

Gastric mucosal homogenates from hog were fractionated by differential and density gradient centrifugation and free-flow electrophoresis. The two major membrane fractions (FI and FII) thus obtained are distinct both enzymically and in terms of transport reactivity. This heterogenicity extends to their antigenic activity. Purified antibodies which were raised against the K+-ATPase-containing H+ transport fraction FI were of two types: inhibitory and non-inhibitory. Inhibitory antibodies reduced the K+-ATPase activity by approximately 80% and the K+-p-nitro-phenylphosphatase activity by approximately 40% in a concentration-dependent manner, while the small Mg++-dependent component of the enzyme activity was unaffected. Antibodies inhibiting the K+-ATPase also inhibited H+ transport. These antibodies did not cross-react with the other major membrane fraction isolated by free-flow electrophoresis, FII, and gave a single band on rocket immunoelectrophoresis. Antibodies against this FII fraction also did not react with the K+-ATPase and were heterogeneous, giving at least four bands with rocket immunoelectrophoresis and inhibiting both the 5'-nucleotidase and Mg++-ATPase of this fraction. Immunofluorescent staining of tissue sections showed that the FI was derived from the parietal cell of gastric tissue and was localized to the supranuclear area of the cell. Staining of isolated rat gastric cell suspensions by FI antibodies confirmed the selectivity of the antibody and showed a polar, plasma membrane localization. FII antibodies also largely stained the parietal cells in tissue sections. In the 16 hog tissues tested, FI antibodies cross-reacted only with gastric fundus, thyroid and weakly with thymus. Immunoelectronmicroscopy showed that FI antibodies reacted strongly with the secretory membrane at the apical cell surface of the parietal cells and at the secretory canaliculi, weakly with the apical surface of the zymogen cell, and not with the basal-lateral surface of the cells. Thus, the protontranslocating ATPase is localized in the parietal cells and in the region postulated to be the site of acid secretion.

An acid transporting enzyme in human gastric mucosa.

Isolation of a microsomal fraction from human gastric mucosa followed by density gradient centrifugation yielded a vesicular membrane preparation free of mitochondrial markers, containing a K+-activated, ouabain-insensitive ATPase with an activity of 20.7 mumol P1 released/mg protein per h. Sodium dodecyl sulfate gel electrophoresis showed that the human gastric membrane vesicles contained a major polypeptide of 110,000 daltons, which accounted for approximately or equal to 30% of the total protein stained and was phosphorylated by [gamma-32P]ATP and dephosphorylated in the presence of K+. Electron microscopy revealed the presence of vesicles with an average size of 0.13 micrometer in diameter. Addition of 0.65 microM ATP to this vesicular preparation resulted in the uptake of 17 nmol H+/mg protein which was dependent on the presence of K+. The gradient was dissipated by a combination of valinomycin and protonophore after consumption of the ATP. Incubation of fixed human fundic sections or human gastric biopsy with monospecific hog gastric membrane antibody followed by fluorescein-conjugated goat anti-rabbit gamma-globulin, showed fluorescent staining in the middle portion of the gastric glands. These data indicate that human stomach contains a H+ transport ATPase with characteristics similar to those established for lower species.

Purification of cells from livers of carcinogen-treated rats by free-flow electrophoresis.

The tumorigenic roles of specific types of cells that emerge during chemical hepatocarcinogenesis in rats could potentially be determined if the specific types of cells could be purified adequately. Type II cells, defined (J. M. Jacobs, T. P. Pretlow, N. Fausto, A. M. Pitts, and T. G. Pretlow, J. Natl. Cancer Inst., 66: 967-973, 1981) as small, slowly sedimenting cells with histochemically demonstrable gamma-glutamyl transpeptidase, have oval nuclei similar in size to those of lymphocytes. Adult male F344 rats were fed a choline-deficient diet containing 0.05% ethionine for 4 weeks. Liver cells were obtained in suspension by in situ perfusion of collagenase. A two-step process, i.e., sedimentation in an isokinetic gradient followed by free-flow electrophoresis, was used to purify type II cells. We obtained preparations of cells with 32.3 +/- 5.0% (S.D.) type II cells, 13.4 +/- 2.0% erythrocytes, 52.9 +/- 3.3% small nucleated cells, and only 1.4 +/- 0.3% hepatocytes. The small nucleated cells were Kupffer cells and cells smaller than Kupffer cells that included many lymphocytes and unidentified cells similar in size to lymphocytes. Because both the electrophoretic mobilities and the rates of sedimentation of type II cells and hepatocytes are different, there is some advantage to be obtained from the sequential use of these techniques that exploit independent differences in the physical properties of these cells.

Papain fragmentation of the gastric (H+ + K+)-ATPase.

Membrane-bound (H+ + K+)-ATPase purified from hog gastric mucosa was exposed to limited papain digestion. Such treatment resulted in a rapid inhibition of the K+-stimulated adenosine triphosphatase and p-nitrophenyl phosphatase activities, with about 90% of these activities lost after 3 min incubation at 37 degrees C with 0.1 units of papain per mg of enzyme protein. Parallel to the inhibition of the enzyme activities, there was a production of a 77 kDa membrane-bound fragment containing the aspartyl phosphate residue of the phospho-intermediate. This fragment accounted for about 45% of the total enzyme protein after the 3 min papain treatment. The digestion barely affected the steady-state level of phosphorylation, allowed the aspartyl phosphate of the 77 kDa fragment to undergo the transition to the E2P form, and did not significantly alter the fraction of ADP-sensitive phosphoenzyme. The presence of KCl, however, depressed the steady-state level of phosphoenzyme formed from [gamma-32P]ATP considerably less than that of the control enzyme. With further exposure to papain the 77 kDa peptide became fragmented into a 28 kDa soluble peptide that retained the phosphorylating site. Binding of fluorescein 5'-isothiocyanate (FITC) to the native enzyme did not affect the sites of papain hydrolysis because the same peptide fragments were obtained. The FITC reaction site was also in the 28 kDa soluble peptide fragment.

Characterization of gastric mucosal membranes. IX. Fractionation and purification of K+-ATPase-containing vesicles by zonal centrifugation and free-flow electrophoresis technique.

Methods are described for purification of a vesicular membrane fraction of hog gastric mucosa using differential centrifugation, density gradient separation on zonal rotors and free-flow electrophoresis. As a result a fraction is obtained enriched 40-fold in terms of K(+)-ATPase and free of any other enzyme marker other than K(+)-activated p-nitrophenyl phosphatase. The 5'-nucleotidase and basal Mg(2+)-ATPase are clearly separated from the latter enzymes. Osmotic shock, Triton X-100 treatment or K+ ionophores increased the K(+)-ATPase activity in isotonic conditions, but K(+)-p-nitrophenyl phosphatase is not affected by these treatments, nor is the ATPase activity in the presence of NH4+. The results suggest that the electrophoretic fraction contains a major population of tight vesicles, whose permeability to K+ is rate limiting for the ATPase activity but not for the p-nitrophenyl phosphatase activity. It is concluded that K+ site for the ATPase is internal whereas the K+ site for the p-nitrophenyl phosphatase is external, hence, the K+ site must be mobile across the membrane.

Proton transport by gastric membrane vesicles.

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H+ uptake into an osmotically sensitive space. The apparent Km for ATP was 1.7-10(-4) M in the absence of a K+ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+ and inhibited by ATPase inhibitors such as N,N'-dicylclohexyl-carbodiimide. Maximal H+ uptake occurs with an outward K+ gradient but the minimal apparent KA is found in the absence of a K+ gradient. The pH optimum for H+ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphroylation of the enzyme, but is considerably less than the pH maximum of the K+ dependent dephosphorylation. In the presence of an inward K+ gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H+ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K+ conductance but a measurable H+ conductance, hence a K+ gradient can produce an H+ gradient in the presence of valinomycin. The uptake and spontaneous leak of H+ are temperature sensitive with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H+ uptake by these vesicles is probably due to a dimeric (H+ + K+)-ATPase and is probably non-electrogenic.

Transport characteristics of frog gastric membranes.

ATP-induced transport by fractions of frog gastric microsomes prepared either by density gradient centrifugation of by free flow electrophoresis were K+ dependent and hence considered due to a K+-activated ATPase. Significant activity of this enzyme was, however, only found in the anodic peak of the free flow electrophoretic separation, which in addition to separating transporting from non-transporting particles, also separated membranes containing a phosphorylatable peptide (Mr=105 000) region as the major peptide on SDS-polyacrylamide gel electrophoresis from those containing a peptide (Mr=44 000) on SDS-polyacrylamide gel electrophoresis. H+ uptake, measured either by acridine orange or 3,3'-diethyloxadicarbocyanine + tetrachlorosalicylanilide absorbance changes was dependent on K+ intravesicularly. Using 86Rb+, active extrusion of the cation followed ATP addition. SCN-, an inhibitor of acid secretion did not affect the latter, but blocked signals due to H+ uptake, in contrast to mammalian preparations.

Combined endoscopic treatment for cholelithiasis associated with choledocholithiasis.

BACKGROUND: The advent of endoscopic techniques changed surgery in many ways. For the management of cholelithiasis, laparoscopic cholecystectomy (LC) is the treatment of choice. This has created a dilemma in the management of choledocholithiasis. Today a number of option exist, including endoscopic sphinterotomy (ES) before LC in patients with suspected common bile duct (CBD) stones, laparoscopic bile duct exploration, open CBD exploration, and postoperative endoscopic retrograde cholangiopancreatography (ERCP). Also, the alternative technique of peroperative ES is emerging. METHODS: We report our experience of routine intraoperative cholangiography followed either by peroperative ERCP in one step or by transcystic drain and postoperative ERCP. In our technique, to facilitate Vater papilla cannulation we inserted a 450-cm transcystic guidewire that was caught by a duodenoscope. Papillotome was then inserted over the guidewire to ensure cannulation of the CBD. RESULTS: Twenty-eight patients were treated successfully in one step and 24 in two steps. The mean operative time was 181 +/- 41 min for patients treated in one step and 131 +/- 30 min for patients treated in two steps. The mean hospital stay was 4.8 +/- 3.3 days for patients treated in one step and 9.6 +/- 4.0 days for patients treated in two steps. Five patients (18%) with positive intraoperative cholangiography for stones for whom peroperative ERCP was not available showed a normal postoperative transcystic cholangiogram and therefore ERCP was canceled. Fourteen of 25 patients treated in one step and none of 17 treated in two steps had raised serum amylase, which resolved spontaneously with no symptoms. No patient developed postoperative pancreatitis. Three (10%) ERCP complications were observed, consisting of mild bleeding of the papilla. All cases were managed by endoscopic adrenaline injection. There was no mortality. CONCLUSION: We believe peroperative ERCP with the technique described should be considered as the treatment of choice for choledocholithiasis associated with cholelithiasis. When single-stage treatment is not possible, a two-step rendezvous technique should be preferred.

Synergistic intrarenal actions of angiotensin on tubular reabsorption and renal hemodynamics.

There is a growing awareness that the direct intrarenal actions of angiotensin II (ANG II) on both tubular and vascular structures contribute to sodium conservation. Even very low concentrations of ANG II (10(-1)) mol/L) stimulate proximal reabsorption rate. Recent studies indicate that this stimulatory action is due to an enhanced activity of the sodium/hydrogen exchanger of the luminal membrane. Elevated ANG II levels in the renal interstitium, effected either through increased delivery of ANG II via the circulation or as a consequence of conversion of angiotensin I (ANG I) generated locally, can also enhance proximal reabsorption rate. One consequence of enhanced proximal reabsorption rate is reduced distal volume delivery, which would be expected to elicit arteriolar vasodilation mediated by the tubuloglomerular feedback (TGF) mechanism. It has been observed, however, that peritubular capillary infusions of either ANG I or ANG II, at doses sufficiently low to be without obvious direct effects on glomerular dynamics, can increase the sensitivity of the TGF mechanism. This enhanced TGF sensitivity serves to minimize or prevent TGF mediated increases in glomerular filtration rate in the face of reduced distal delivery. With greater increases in interstitial ANG II concentration, reductions in glomerular pressure have been observed, demonstrating a powerful action on preglomerular arterioles that predominates over the well known effects on efferent arterioles. At these higher doses, the direct hemodynamic actions of ANG II, plus the effects on the glomerular filtration coefficient, will directly reduce filtered sodium load. Through these synergistic effects on both tubular reabsorptive and hemodynamic function, ANG II can elicit sustained decreases in distal nephron sodium delivery which contribute greatly to its efficacy as a regulator of sodium excretion.

The interaction of K+ with gastric parietal cells and gastric ATPase.

The gastric H+ secretion in isolated cell requires K+ and is ATP dependent. There is also evidence in the cell system for Na+ inhibition of H+ secretion. The isolated gastric ATPase also shows K+ activation and inhibition by K+ or Na+ located on the ATP binding side of the enzyme, which corresponds to the cytoplasmic face of the enzyme. Gastric vesicles are activated in terms of transport activity by internal K+, and this site, inhibited by reagents that modify carboxyl groups, is required for enzyme turnover and transport.

Aspects of parietal cell biology: cells and vesicles.

Many features of these gastric vesicles satisfy the requirements for the gastric H+ pump. For example, we have: (a) K+ requirement, (b) KA for K+ of about 30 mM; (c) identical cation sequence for tissue and vesicles, (d) similar anion sequence, (e) localization at the microvillus of the secretory canaliculus, (f) TI+ inhibiting H+ transport of both systems, and (g) the K+ gradient satisfying the osmotic gradient requirement for HCl-flow out of the parietal cell. Points that require explanation are lack of SCN- effects and regulation of KCl permeability.

Laparoscopic billroth II gastrectomy for completely stricturing duodenal ulcer: technical details.

BACKGROUND: The authors report a series of three patients who underwent laparoscopic gastrectomy for gastric outlet obstruction due to stricturing duodenal ulcer. MATERIALS AND METHODS: In all cases an intracorporeal resection of the antrum and an antecolic end to side gastrojejunostomy (Billroth II) were performed. Technical details are discussed in the paper. RESULTS: Mean operative time was 260 minutes, mean blood loss was 43 millilitres. There were no postoperative complications and all patients were discharged on the fifth postoperative day. A follow up of three years shows that no patient had recurrence and post-gastrectomy syndromes. CONCLUSIONS: Laparoscopic Billroth II gastrectomy is a safe and feasible procedure with benefits such as quick hospital stay, decreased postoperative pain, good cosmesis and reduced morbidity.

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.

Primary resection with and without anastomosis for perforation of acute diverticulitis.

The results of management of perforated large bowel diverticulitis were retrospectively studied over a 7-year period. 38 patients underwent operation, 20 for generalized peritonitis, 12 for local peritonitis, 5 for colovesical fistula and 1 for colovaginal fistula. The mean age of patients was 63 years (range 30-85 years). Depending on the symptoms, the spreading of the peritonitis and associated cardiovascular and pulmonary disease and diabetes mellitus, 4 types of operation were performed: primary left hemicolectomy and anastomosis with and without defunctioning colostomy, Hartmann procedure, suture and drainage with diverting colostomy. The overall mortality was 10.5%: resection and primary anastomosis entailed 3.8% mortality (1 case), while 3 deaths were observed in the 8 patients group having underwent an Hartmann procedure (37.5%). Drainage and/or diverting colostomy performed in 5 patients entailed no hospital mortality, but was followed by a 80% complication rate, requiring reoperation and several hospital admissions. The low mortality and morbidity rates obtained in the group having primary resection and anastomosis encourage wider application of this operation for perforated acute diverticulitis. Even the Hartmann procedure allows removal of the diseased colon but in a great proportion of cases reconstitution of continuity is not performed; nevertheless staged operation entailing major mortality and morbidity, expose these aged patients to remarkable hazard. Prerequisite of safe primary excision and anastomosis is vigorous intraperitoneal lavage and drainage, by the case associated to on table large bowel irrigation if concomitant obstruction is present.

Emergency surgery of complicated colonic diverticulitis.

Colon diverticular disease is an increasingly frequent disorder especially in Western populations characterized by high living standards. In 30-40% of subjects over 60 years of age barium enema detects the presence of diverticula in the sigma. 10-20% of patients affected by colon diverticular disease develop complications such as inflammation or haemorrhage and 20-30% of these patients undergo surgery (60% of patients aged less than 40). Emergency surgery is performed in 50% of cases and it is currently burdened by high mortality rates. We decided to review our cases history to clarify the indication for emergency surgery, the appropriateness to resect the diseased bowel tract and to perform colic anastomosis at the time of emergency resection.

[3 cases of aberrant pancreas]. / A proposito di tre casi di pancreas aberrante.

Three cases of aberrant pancreas in a gastrointestinal site are described. The relevant literature is examined, together with the symptomatology, diagnosis and treatment of this form. Attention is drawn to the frequency and possible occurence of aberrant pancreas in cases of gastrointestinal bleeding.

[Randomized clinical analysis of a series of 306 patients operated on for benign pathology of the terminal choledochus]. / Analisi clinica randomizzata su di una serie di 306 pazienti operati per patologia benigna del coledoco terminale.

Personal experience in the surgical management of the common bile duct is described. Over 100 cases of benign disease of the terminal choledocus are examined in a random assessment of 300 cases. The diagnostic and therapeutic techniques adopted are discussed.

[Malignant neoplasms of the male breast. Our clinical contribution]. / Le neoplasie maligne della mammella maschile. Nostro contributo clinico.

A survey of literature data on male breast cancer is reported: on this basis the authors report their experience of five cases observed between 1980 and 1989 at Ospedale "S. Corona" Pietra Ligure, out of 500 female breast cancers treated in the same period. Epidemiological data and follow-up records are analysed, reporting survival rate similar to that of larger series.

[Preoperative colonic lavage and one-stage excision-anastomosis in obstruction of the left colon]. / Le lavage colique per-opératoire et la résection-anastomose en un temps dans l'obstruction du côlon gauche.

In a retrospective series of 95 patients requiring emergency surgery for distal colonic obstruction, primary bowel resection followed by immediate anastomosis after intraoperative colonic irrigation was performed. Carcinoma was the cause of obstruction in 81 cases (85%); 13 patients had diverticulitis, and 1 had sigmoid volvulus. The technique of on-table lavage was similar to that described by Dudley in 1980: a caecostomy tube was used in 86 patients (90%) and was removed on the tenth postoperative day. 4 patients died, none from complications of anastomotic leakage. There were three anastomotic leakages (3.1%) and 10 radiologic leaks were observed. 3 patients were reoperated. The mean hospital stay was 23 days. The results of this study suggest that intraoperative colonic irrigation is an effective method, enabling the surgeon to perform primary anastomosis with reasonable safety after emergency resection of selected distal colonic lesions.

[Extended lymphadenectomy in carcinoma of the right colon]. / La linfoadenectomia estesa nel carcinoma del colon destro.

The Authors report on a retrospective study concerning 49 patients treated for a modified Dukes' stage B, C or D right colon cancer. A potentially curative right hemicolectomy with extended lymphadenectomy was performed. Locally advanced disease was treated by "en bloc" resection. Two (4.1%) patients died after surgery, in other 2 (4.1%) cases major postoperative complications were registered. Microscopic examination of the surgical specimens showed lymph node metastases in 44.9% of the cases: in this subgroup a metastatic involvement of the main (III level) nodal groups was detected in 22.7% of the patients. The Authors conclude that extended lymph node dissection does not increase postoperative complications and may play a role in the treatment of right colon cancer. A not negligible group of patients may be understaged or receive inadequate surgical treatment when submitted to traditional right colectomy.