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P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP (pHP-caged ATP) has been investigated for its application as a phototrigger for the rapid activation of electrogenic ion pumps. The yield of ATP after irradiation with a XeCl excimer laser (lambda = 308 nm) was determined at pH 6.0-7.5. For comparison, the photolytic yields of P(3)-[1-(2-nitrophenyl)]ethyl ATP (NPE-caged ATP) and P(3)-[1, 2-diphenyl-2-oxo]ethyl ATP (desyl-caged ATP) were also measured. It was shown that at lambda = 308 nm pHP-caged ATP is superior to the other caged ATP derivatives investigated in terms of yield of ATP after irradiation. Using time-resolved single-wavelength IR spectroscopy, we determined a lower limit of 10(6) s(-1) for the rate constant of release of ATP from pHP-caged ATP at pH 7.0. Like NPE-caged ATP, pHP-caged ATP and desyl-caged ATP bind to the Na(+), K(+)-ATPase and act as competitive inhibitors of ATPase function. Using pHP-caged ATP, we investigated the charge translocation kinetics of the Na(+),K(+)-ATPase at pH 6.2-7.4. The kinetic parameters obtained from the electrical measurements are compared to those obtained with a technique that does not require caged ATP, namely parallel stopped-flow experiments using the voltage-sensitive dye RH421. It is shown that the two techniques yield identical results, provided the inhibitory properties of the caged compound are taken into account. Our results demonstrate that under physiological (pH 7.0) and slightly basic (pH 7.5) or acidic (pH 6. 0) conditions, pHP-caged ATP is a rapid, effective, and biocompatible phototrigger for ATP-driven biological systems.

Replacement of glycine residue 232 with aspartate in the KdpA subunit of the K(+)-translocating KdpFABC complex of Escherichia coli leads to a transport complex that has reduced affinity for K(+) and has lost the ability to discriminate Rb(+) ions (, J. Biol. Chem. 270:6678-6685). This glycine residue is the first in a highly conserved GGG motif that was aligned with the GYG sequence of the selectivity filter (P- or H5-loop) of K(+) channels (, Nature. 371:119-122). Investigations with the purified and reconstituted KdpFABC complex using the potential sensitive fluorescent dye DiSC(3)(5) and the "caged-ATP/planar bilayer method" confirm the altered ion specificity observed in uptake measurements with whole cells. In the absence of cations a transient current was observed in the planar bilayer measurements, a phenomenon that was previously observed with the wild-type enzyme and with another kdpA mutant (A:Q116R) and most likely represents the movement of a protein-fixed charge during a conformational transition. After addition of K(+) or Rb(+), a stationary current could be observed, representing the continuous pumping activity of the KdpFABC complex. In addition, DiSC(3)(5) and planar bilayer measurements indicate that the A:G232D Kdp-ATPase also transports Na(+), Li(+), and H(+) with a reduced rate. Similarities to mutations in the GYG motif of K(+) channels are discussed.

The reductive part of the catalytic cycle of cytochrome c oxidase from Paracoccus denitrificans was examined by using time-resolved potential measurements on black lipid membranes. Proteoliposomes were adsorbed to the black lipid membranes and Ru(II)(2, 2'-bipyridyl)(3)(2+) was used as photoreductant to measure flash-induced membrane potential generation. Single-electron reduction of the oxidized wild-type cytochrome c oxidase reveals two phases of membrane potential generation (tau(1) approximately 20 micros and tau(2) approximately 175 micros) at pH 7.4. The fast phase is not sensitive to cyanide and is assigned to electron transfer from Cu(A) to heme a. The slower phase is inhibited completely by cyanide and shows a kinetic deuterium isotope effect by a factor of 2-3. Although two enzyme variants mutated in the so-called D pathway of proton transfer (D124N and E278Q) show the same time constants and relative amplitudes as the wild-type enzyme, in the K pathway variant K354M, tau(2) is increased to 900 micros. This result suggests uptake of a proton through the K pathway during the transition from the oxidized to the one-electron reduced state. After the second laser flash under anaerobic conditions, a third electrogenic phase with a time constant of approximately 1 ms appears. The amplitude of this phase grows with increasing flash number. We explain this growth by injection of a second electron into the single-electron reduced enzyme. On multiple flashes, both D pathway mutants behave differently compared with the wild type and two additional slow phases of tau(3) approximately 2 ms and tau(4) approximately 15 ms are observed. These results suggest that the D pathway is involved in proton transfer coupled to the uptake of the second electron.

The effects of lyotropic anions, particularly perchlorate, on the kinetics of partial reactions of the Na+,K+-ATPase from pig kidney were investigated by two different kinetic techniques: stopped flow in combination with the fluorescent label RH421 and a stationary electrical relaxation technique. It was found that 130 mM NaClO4 caused an increase in the Kd values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (+/- 0.6) microM and 143 (+/- 17) microM in 130 mM NaCl solution to values of 42 (+/- 3) microM and 660 (+/- 100) microM in 130 mM NaClO4 (pH 7.4, 24 degrees C). The half-saturating concentration of the Na+-binding sites on the E1 conformation was found to decrease from 8-10 mM in NaCl to 2.5-3.5 mM in NaClO4 solution. The rate of equilibration of the reaction, E1P(Na+)3 left arrow over right arrow E2P + 3Na+, decreased from 393 (+/- 51) s-1 in NaCl solution to 114 (+/- 15) s-1 in NaClO4. This decrease is attributed predominantly to an inhibition of the E1P(Na+)3 --> E2P(Na+)3 transition. The effects can be explained in terms of electrostatic interactions due to perchlorate binding within the membrane and/or protein matrix of the Na+,K+-ATPase membrane fragments and alteration of the local electric field strength experienced by the protein. The kinetic results obtained support the conclusion that the conformational transition E1P(Na+)3 --> E2P(Na+)3 is a major charge translocating step of the pump cycle.

Charge transport by the K+ transporting Kdp-ATPase from Escherichia coli was investigated using planar lipid membranes to which liposomes reconstituted with the enzyme were adsorbed. To study reactions in the absence of K+, given some contamination of solutions with K+, we used a mutant of Kdp whose affinity for K+ was 6 mM instead of the wild-type whose affinity is 2 microM. Upon rapid release of ATP from caged ATP, a transient current occurred in the absence of K+. In the presence of K+, a stationary current was seen. On the basis of their structural similarity, we propose a kinetic model for the Kdp-ATPase analogous to that of the Na+K+-ATPase. In this model, the first, K+-independent step is electrogenic and corresponds to the outward transport of a negative charge. The second, K+-translocating step is probably also electrogenic and corresponds to transport of positive charge to the intracellular side of the protein.

The kdpFABC operon of Escherichia coli consists of the four structural genes kdpF, kdpA, kdpB, and kdpC. Expression of the kdpF gene was demonstrated using minicells of E. coli. In addition, it was shown that the KdpF subunit remains associated with the purified complex. Although KdpF is not essential in vivo, the purified complex lacking KdpF exhibits hardly any K(+)-stimulated ATPase activity. This clearly demonstrates that the KdpF subunit is stabilizing the transport complex. Charge translocation by the purified Kdp-ATPase was measured with the potential-sensitive dye DiSC3(5) using proteoliposomes. Upon addition of ATP a fluorescence quench was observed indicating the buildup of a negative potential inside the proteoliposomes. Using the Kdp-ATPase derived from a mutant strain, in which the K(m) value for K+ (1,2 mM) was almost identical to that of Rb+ (1.4 mM), the same fluorescence quench was observed when K+ or Rb+ were present in the lumen of the proteoliposomes. These data clearly indicate that the Kdp-ATPase transports K+ in an electrogenic manner. In order to identify the binding site(s) for the inhibitor concanamycin A within the Kdp complex, concanamycin A was synthesized. Using this compound labeling of KdpA and KdpB, but not of KdpC, could be shown with the purified complex. When everted vesicles were used only KdpB could be labeled.

The lipid bilayer and the giant patch technique were used to study the Na+,K(+)-ATPase. In excised patches from ventricular myocytes, Na+,K(+)-pump currents show a saturable ATP dependence with a Km of approximately 150 microM at 24 degrees C. Partial reactions in the transport cycle were investigated by generating ATP concentration jumps through photolytic release of ATP from caged ATP at pH 7.4 and 6.3. Transient outward currents were obtained at pH 6.3 with a fast rising phase followed by a slow decay to a stationary current. Experiments with purified pig kidney Na+,K(+)-ATPase attached to a planar lipid bilayer resulted in similar pump current signals and the same outcome. It was concluded that the fast rate constant of approximately 200 s-1 at 24 degrees C reflects a step rate limiting the electrogenic Na+ release.

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau1 approximately 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 --> E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and

Charge translocation by the Kdp-ATPase of Escherichia coli was measured by adsorption of proteoliposomes to a planar lipid membrane. The proteoliposomes were prepared by reconstitution of purified Kdp-ATPase into liposomes prepared from E. coli lipids. The protein was activated by a ATP concentration jump produced by photolysis of a protected derivative of ATP, caged ATP. Charge translocation was measured with a time resolution of 15-40 ms. Stationary currents demonstrated the continuous pumping activity of the enzyme. Control measurements with the potential-sensitive dye DiSC3(5) showed a negative potential inside the proteoliposomes after activation with ATP. The measured electrical signals as well as the dye measurements correspond to the transport of positive charge to the intracellular face of the protein. The electrical signal was increased when K+ was inside the proteoliposomes (K0.5 approximately 50 microM) and was inhibited by vanadate. These experiments demonstrate the electrogeneity of the Kdp-ATPase in a purified reconstituted system.

The incidence of both systolic and diastolic hypertension is increased in elderly patients, therefore antihypertensive drugs are commonly used in this population. In addition to changes in blood pressure, the aging process also causes numerous changes in other physiological parameters, resulting in altered pharmacokinetic and pharmacodynamic responses to the drugs. The dosage regimens for thiazide diuretics and amiloride must be individually titrated in the elderly patient, since the elimination of these agents decreases concurrently with decreased renal function, as indicated by compromised creatinine clearance. The initial doses of the calcium antagonists should be decreased in elderly patients, since representative compounds from all 3 chemically heterogeneous classes have been shown to have decreased clearance in these patients which appears to be primarily due to the status of hepatic function in the patient. However, with verapamil, the dosage should be further decreased in association with compromised renal function. The dosage of the angiotensin converting enzyme (ACE) inhibitors should be adjusted according to renal function rather than age. Lisinopril, which is primarily eliminated unchanged, is usually given in lower doses in the elderly, and doses of both captopril and enalapril may need to be reduced, depending on renal function. While there is no need to adjust the dosage regimen for the alpha-adrenoceptor blocking drugs (prazosin, terazosin), caution should be used with the beta-adrenergic blockers, particularly the hydrophilic agents, since they are renally eliminated. Labetalol may be a suitable alternative beta-blocker for the elderly patient, since its pharmacodynamic properties of decreased systemic vascular resistance without changes in heart rate or stroke volume are preferential for the elderly patient, and its pharmacokinetics are relatively unchanged in this population. Drugs that act primarily through the central nervous system, such as clonidine, methyldopa and guanfacine, require smaller doses in the presence of renal dysfunction. In contrast, guanabenz is metabolised primarily by the liver, so it would appear to be useful in elderly patients with renal dysfunction despite the lack of studies in this population. Guanadrel, an adrenergic neuron blocking drug, also requires a dosage reduction in patients with impaired renal function. In addition to the pharmacokinetic changes that occur in the elderly patient, pharmacodynamic changes may also be anticipated due to receptor modifications. Older patients have a decrease in beta-receptor sensitivity, while alpha-receptor sensitivity does not change. When designing the dosage regimen for a senior patient with hypertension, the combination of all these variables must be considered.

Kinetic investigations carried out in a number of laboratories have accumulated evidence favoring modification of the Albers-Post mechanism. The results of the rapid mixing studies involving the eel enzyme indicate that the complex kinetic behavior is confined to the Na(+)-activated reaction pathway (Na-ATPase). The main conceptual problem in interpreting the dephosphorylation experiments involves the intermediate component, which turns over too slowly to account for the overall velocity of Pi production in the presence of Na+ and K+ and exhibits behavior compatible with an ADP-insensitive phosphoenzyme. Attempts to simulate the dephosphorylation reaction using schemes in which the intermediate component represents a precursor to the K(+)-sensitive phosphoenzyme, E2P, were unsuccessful in reproducing both the pre-steady-state and steady-state time dependence. When Na+ and K+ were both present during phosphorylation, the time course of dephosphorylation showed no evidence of an intermediate decay component, implying that K+ either prevents its formation or accelerates its turnover. Complex kinetic behavior was also observed in the phosphorylation reaction under conditions where the reaction was initiated by the simultaneous addition of ATP, Na+, and Mg2+. Preincubation with Na+ eliminated the biexponential pattern of accumulation so that only the fast phase was seen. The proportion of EP in the slow phase of phosphorylation was approximately equal to the fraction of EP in the intermediate phase of dephosphorylation (roughly one-third of the sites), suggesting that the two may be related to the same catalytic activity. To try to explain these observations using recent modifications to the Albers-Post mechanism is difficult without invoking additional complex effects of the transported ions. We propose that a series model for phosphorylation is inadequate and that further modification of the mechanism is required. The alternative to a consecutive mechanism is a parallel pathway scheme: [sequence: see text] In this model the enzyme exists in two distinct forms which are distributed in the upper and lower pathways in a ratio of 2:1. In the lower pathway the rates of phosphorylation and E2P hydrolysis are controlled by the kinetics of ligand binding because of a structural constraint (ion channel?) imposed by the transport protein. When phosphorylation is carried out in the presence of Na+ alone, E2P and E2P' accumulates rapidly and give rise to the fast and intermediate components of dephosphorylation, respectively. Preincubation with Na+ and K+ eliminates the functional differences between these pathways by removing the kinetic dependence of ligand binding, resulting in behavior that conforms to the Albers-Post mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Multifactorial studies were performed in child and adolescent swimmers in the early period of training and in a submaximum state of training in the year's racing period. Blood coagulation, viscosity, acidity, protein metabolism, muscle enzymes, ions, haematologic, cardiac and endocrine parameters have been investigated. In the early period of training, physical exercise resulted in fibrinolysis, and a decrease in the fibrinogen and prothrombin levels and the platelet count. The same exercise in fully-trained organisms failed to produce such changes. Data concerning acidity, protein metabolism, muscle enzymes, ions, haematologic, cardiac and endocrine parameters will be published in subsequent papers.

Fifty-five patients suffering from acute cerebral ischaemia were treated with pentosan polysulphate (SP54) intravenously and subsequently orally. The control group consisting of 35 patients received the infusion solution and vasodilator drugs. The statistically significant changes appearing following infusion of SP54 include the following parameters: haematocrit, blood viscosity and cholesterol concentrations. In individual cases the euglobulin lysis time was shortened within two hours after the beginning of the infusion. As demonstrated by the statistically reliable results in patients treated with SP54 the neurological symptoms improved significantly within the first two weeks after the onset of the disease compared to the control groups. Based on case reports, recommendations are given for the indication and contraindication of SP54 treatment in cerebral ischaemia.

14 cases of transluminal angioplasty and selective clot lysis are reported. One patient removed the catheter before successful lysis and one lost his leg after an almost complete lysis due to advanced necrosis. Treatment was successful in the other 12 cases with complete or partial thrombolysis. Remaining clots were either smashed by catheter and/or successfully treated by oral fibrinolytic therapy. The injected dose of streptokinase was 5,000 U/h after an initial dose of 20,000 U in one hand injection. The duration of infusions was 6-72 hours. A total dose of 60,000-520,000 U streptokinase was adopted. Intraarterial infusion therapy was followed by long-term low-grade fibrinolytic treatment with oral penthosan polysulphate. Four bleedings at the puncture site, one epistaxis and a bleeding at the site of a recently extracted tooth were seen. Only one infusion was stopped due to moderate bleeding and without any drawback.

A randomized, prospective, crossing-over clinico-pharmacological study was conducted on the tolerability by humans, of TISACID (Al-Mg-hydroxy-carbonate), a new antacid of up-to-date composition produced in Hungary. Even a relatively high dose of the preparation is tolerated by the human organism. During a 6-week continuous treatment neither subjective nor objective side-effects were observed. The tablet is immediately decomposed in the gastric juice, a considerable portion of it will permanently stick to the mucosa of the stomach and duodenum. Depending on the dose, it rapidly and permanently reduces the acidity of the gastric content and increases serum gastrin concentration only moderately and for a short time. Administered together with cimetidine, it promotes healing of duodenal ulcer and the cessation of complaints. It does not increase the aluminium and magnesium concentrations of the plasma not even on prolonged administration, and clinical symptoms and laboratory changes characterizing the phosphate depletion syndrome do not develop either. Based on the results, authors consider Tisacid a beneficial preparation as regards both effectivity and tolerability.

The concern over rising health care costs has created an environment that is favorable for the development of efficient health care programs. Pharmacists have been instrumental in developing and implementing programs to reduce drug costs, but many of these lack adequate documentation in the literature. One such area is the impact of disseminating drug bulletins to prescribers. This study was undertaken to assess quantitatively the impact of drug bulletins on physician prescribing habits and to measure the resultant changes in drug cost. This was a retrospective examination of Prime Health's acquisition of certain drugs described in five separate drug bulletins before and after issuance of the bulletins. We were able to demonstrate statistically significant changes in the proportions of drugs acquired after dissemination of each bulletin. In some cases, these changes persisted for the duration of the study (one year after issuance of the bulletin); in other cases, they reverted to pre-bulletin proportions. These changes resulted in mean drug cost reductions of 30 percent per calendar quarter for all of the post-bulletin period. Our findings suggest that the issuance of certain types of drug bulletins to prescribers is associated with a significant change in their prescribing habits, with resultant cost savings.

Acid-base parameters of adolescent swimmers (capacitive sport) and of adult weight-lifters (athletoid sport) were investigated before and after training in the submaximally trained phase of a year-round training period. In swimmers, training induced incompensated metabolic acidosis, which persisted at 10 min after the training. This acidosis showed no correlation to the blood lactate level. In weight-lifters, there was only a slight, compensated posttraining acidosis, which tended to decrease 10 min after the training. The weight-lifters had extremely high posttraining ammonia levels. It might be supposed that ammonia-genesis has a role in the compensation of exercise-induced acidosis in weight-lifters. The differences in acid-base status of swimmers and weight-lifters might be related to the different breathing mechanism needed for the two different sports.

Temperature jump experiments were carried out on purple membranes oriented and fixed in polyacrylamide gel. With green background illumination a relaxation of the photocurrent after an infrared laser pulse could be observed. To simulate the temperature jump signals different models of the bacteriorhodopsin photocycle were tested. The parameters of these models were obtained by measuring absorbance changes and photocurrent after excitation with a 575-nm laser flash.A model with a temperature-dependent branching before the M state turned out to be satisfying. Other models, especially those with a late branching or without branching, could not reproduce the temperature jump measurements.

The etiology, pathogenesis, diagnosis, and treatment of reflux esophagitis are reviewed. Reflux esophagitis is the subjective or objective response to gastroesophageal reflux (GER), which is defined as the entrance of gastroduodenal contents into the esophagus not associated with vomiting or belching. The pathogenesis of reflux esophagitis may involve a number of mechanisms, including changes in lower esophageal sphincter pressure, gastric volume, composition of the refluxate, esophageal acid clearance, and esophageal tissue resistance. The most common symptom of reflux esophagitis is heartburn. Regurgitation of fluid into the mouth, usually after bending or during the night, is an unequivocal symptom of GER. Treatment can be divided into three phases. Phase 1 involves the avoidance of certain foods and habits, elevation of the bed head, antacid, and alginic acid-antacid therapy. Phase 2 involves drug therapy with agents not yet approved by the FDA for this indication: bethanechol chloride, cimetidine, and metoclopramide hydrochloride. Bethanechol chloride 25 mg is generally given four times daily. Cimetidine is given in doses of 300-400 mg after meals and at bedtime. Metoclopramide hydrochloride is administered in doses of 10 mg before meals and at bedtime. Phase 3 is antireflux surgery. Clinical experience has shown that phase 1 therapy is successful for about 75% of all patients. Of the 25% that do not respond to phase 1 therapy, about 90% will respond to phase 2 therapy, leaving only 5-10% of all patients with this disorder who will require phase 3 treatment. Current data favor cimetidine and bethanechol over metoclopramide. The least proof of efficacy and the most frequent adverse side effects are seen with metoclopramide. Cimetidine and bethanechol appear to have similar efficacy and relatively infrequent side effects. Evidence confirming the superiority of cimetidine over bethanechol is lacking. Further research is needed to determine the optimal pharmacologic combinations and treatment regimens.