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Objectives The objective of this clinical trial was to evaluate the long-term effect of endothelin receptor antagonism with bosentan on the morbidity and mortality of patients with severe chronic heart failure. Background Endothelin may play a role in heart failure, but short-term clinical trials with endothelin receptor antagonists have reported disappointing results. Long-term trials are lacking. Methods In 2 identical double-blind trials, we randomly assigned 1,613 patients with New York Heart Association functional class IIIb to IV heart failure and an ejection fraction Results Bosentan did not influence clinical status at 9 months in either trial (p = 0.928 and p = 0.263). In addition, 321 patients in the placebo group and 312 patients in the bosentan group died or were hospitalized for heart failure (hazard ratio [HR]: 1.01; 95% confidence interval [CI]: 0.86 to 1.18; p = 0.90). The bosentan group experienced fluid retention within the first 2 to 4 weeks, as evidenced by increased peripheral edema, weight gain, decreases in hemoglobin, and an increased risk of hospitalization for heart failure, despite intensification of background diuretics. During follow-up, 173 patients died in the placebo group and 160 patients died in the bosentan group (HR: 0.94; 95% CI: 0.75 to 1.16). About 10% of the bosentan group showed meaningful increases in hepatic transaminases, but none had acute or chronic liver failure. Conclusions Bosentan did not improve the clinical course or natural history of patients with severe chronic heart failure and but caused early and important fluid retention.

Polycystin-2, the product of the human PKD2 gene, whose mutations cause autosomal dominant polycystic kidney disease, is a large conductance, Ca(2+)-permeable non-selective cation channel. Polycystin-2 is functionally expressed in the apical membrane of the human syncytiotrophoblast, where it may play a role in the control of fetal electrolyte homeostasis. Little is known, however, about the mechanisms that regulate polycystin-2 channel function. In this study, the role of pH in the regulation of polycystin-2 was assessed by ion channel reconstitution of both apical membranes of human syncytiotrophoblast and the purified FLAG-tagged protein from in vitro transcribed/translated material. A kinetic analysis of single channel currents, including dwell time histograms, confirmed two open and two close states for spontaneous channel behavior and a strong voltage dependence of the open probability of the channel (P(o)). A reduction of cis pH (pH(cis)) decreased P(o) and shifted the voltage dependence of channel function but had no effect on the single channel conductance. An increase in pH(cis), in contrast, increased NP(o) (channel number times P(o)). Elimination of the H(+) chemical gradient did not reverse the low pH(cis) inhibition of polycystin-2. Similar findings confirmed the pH effect on the in vitro translated, FLAG-tagged purified polycystin-2. The data indicate the presence of an H(+) ion regulatory site in the channel protein, which is accessible from the cytoplasmic side of the protein. This protonation site controls polycystin-2 cation-selective channel activity.

Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of unknown function, is a major cause of autosomal dominant polycystic kidney disease (ADPKD), whose manifestation entails the development of fluid-filled cysts in target organs. Here, we demonstrate that polycystin-2 is present in term human syncytiotrophoblast, where it behaves as a nonselective cation channel. Lipid bilayer reconstitution of polycystin-2-positive human syncytiotrophoblast apical membranes displayed a nonselective cation channel with multiple subconductance states, and a high perm-selectivity to Ca 2+ . This channel was inhibited by anti-polycystin-2 antibody, Ca 2+ , La 3+ , Gd 3+ , and the diuretic amiloride. Channel function by polycystin-2 was confirmed by patch-clamping experiments of polycystin-2 heterologously infected Sf9 insect cells. Further, purified insect cell-derived recombinant polycystin-2 and in vitro translated human polycystin-2 had similar ion channel activity. The polycystin-2 channel may be associated with fluid accumulation and/or ion transport regulation in target epithelia, including placenta. Dysregulation of this channel provides a mechanism for the onset and progression of ADPKD.

The rate-limiting step for the maternofetal exchange of low molecular-weight solutes in humans is constituted by transport across a single epithelial layer (syncytiotrophoblast) of the placenta. Other than the well-established presence of a large-conductance, multisubstate Cl- channel, the ionic channels occurring in this syncytial tissue are, for the most part, unknown. We have found that fusion of apical plasma membrane-enriched vesicle fractions with planar lipid bilayers leads, mainly (96% of 353 reconstitutions), to the reconstitution of nonselective cation channels. Here we describe the properties of this novel placental conductance at the single-channel level. The channel has a large (200 pS) and variable conductance, is cation selective (P(Cl)/P(K) approximately or approximately equal 0.024), is reversibly inhibited (presumably blocked) by submillimolar La3+, has very unstable kinetics, and displays a large number (10) of current sublevels with a "promiscuous" connectivity pattern. The occurrence of both "staircaselike" and "all-or-nothing" transitions between the minimum and maximum current levels was intriguing, particularly considering the large number of conductance levels spanned at a time during the concerted current steps. Single-channel data simulated according to a multistate linear reaction scheme, with rate constants that can vary spontaneously in time, reproduce many aspects of the recorded subconductance behavior. The channel's sensitivity to lanthanides is reminiscent of stretch-sensitive channels which, in turn, suggests a physiological role for this ion channel as a mechanotransducer during syncytiotrophoblast-volume regulation.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR), and of at least one other member of the ATP-binding cassette family of transport proteins, P-glycoprotein, is associated with the electrodiffusional movement of the nucleotide ATP. Evidence directly implicating CFTR expression with ATP channel activity, however, is still missing. Here it is reported that reconstitution into a lipid bilayer of highly purified CFTR of human epithelial origin enables the permeation of both Cl−and ATP. Similar to previously reported data for in vivo ATP currents of CFTR-expressing cells, the reconstituted channels displayed competition between Cl−and ATP and had multiple conductance states in the presence of Cl−and ATP. Purified CFTR-mediated ATP currents were activated by protein kinase A and ATP (1 mM) from the “intracellular” side of the molecule and were inhibited by diphenylamine-2-carboxylate, glibenclamide, and anti-CFTR antibodies. The absence of CFTR-mediated electrodiffusional ATP movement may thus be a relevant component of the pleiotropic cystic fibrosis phenotype.

Apx, the amphibian protein associated with renal amiloride-sensitive Na+ channel activity and with properties consistent with the pore-forming 150-kDa subunit of an epithelial Na+ channel complex initially purified by Benos et al. (Benos, D. J., Saccomani, G., and Sariban-Sohraby, S. (1987) J. Biol. Chem. 262, 10613-10618), has previously failed to generate amiloride-sensitive Na+ currents (Staub, O., Verrey, F., Kleyman, T. R., Benos, D. J., Rossier, B. C., and Kraehenbuhl, J.-P. (1992) J. Cell Biol. 119, 1497-1506). Renal epithelial Na+ channel activity is tonically inhibited by endogenous actin filaments (Cantiello, H. F., Stow, J., Prat, A. G., and Ausiello, D. A. (1991) Am. J. Physiol. 261, C882-C888). Thus, Apx was expressed and its function examined in human melanoma cells with a defective actin-based cytoskeleton. Apx-transfection was associated with a 60-900% increase in amiloride-sensitive (Ki = 3 μM) Na+ currents. Single channel Na+ currents had a similar functional fingerprint to the vasopressin-sensitive, and actin-regulated epithelial Na+ channel of A6 cells, including a 6-7 pS single channel conductance and a perm-selectivity of Na+:K+ of 4:1. Na+ channel activity was either spontaneous, or induced by addition of actin or protein kinase A plus ATP to the bathing solution of excised inside-out patches. Therefore, Apx may be responsible for the ionic conductance involved in the vasopressin-activated Na+ reabsorption in the amphibian kidney.

Recent studies from our laboratory indicate that members of the ATP-binding cassette (ABC) family of transporters, including P-glycoprotein and cystic fibrosis transmembrane conductance regulator (CFTR), are ATP-permeable channels. The physiological relevance of this novel transport mechanism is largely unknown. In the present study, intra- and extracellular ATP content, cellular ATP release, and O2 consumption before and after adenosine 3',5'-cyclic monophosphate (cAMP) stimulation were determined to assess the role of CFTR in the transport of ATP under physiological conditions. The functional expression of CFTR by the stable transfection of mouse mammary carcinoma cells, C1271, with human epithelial CFTR cDNA resulted in a stimulated metabolism, since both basal and cAMP-inducible O2 consumption were increased compared with mock-transfected cells. The stimulated (but not basal) O2 consumption was inhibited by diphenyl-2-carboxylic acid (DPC), a known inhibitor of CFTR. CFTR expression was also associated with the cAMP-activated and DPC-inhibitable release of intracellular ATP. The recovery of intracellular ATP from complete depletion after metabolic poisoning was also assessed under basal and cAMP-stimulated conditions. The various maneuvers indicate that CFTR may be an important contributor to the release of cellular ATP, which may help modify signal transduction pathways associated with secretory Cl- movement or other related processes. Changes in the CFTR-mediated delivery of nucleotides to the extracellular compartment may play an important role in the onset and reversal of the cystic fibrosis phenotype.

Two cationic channels present in the microsomal fraction from Echinococcus granulosus protoscoleces of the sheep strain were studied in planar bilayer reconstitution experiments. A whole-worm homogenate was subjected to differential centrifugation and the postmitochondrial supernatant was laid on the top of a discontinuous sucrose gradient. The 15-30% (w/v) membrane fraction, enriched in NADPH-cytochrome c reductase, exhibited the highest fusion rate, two cationic channels being most frequently reconstituted. Both of them were highly cation-selective and had high conductances (244 and 107 pS in symmetrical 150 mM KCl). In most experiments, none of them displayed voltage dependence. The 244-pS channel was activated by Ca 2+ and blocked by Ba 2+ , both in the micromolar range, thus partially resembling the Ca 2+ -activated K + channel from more highly evolved animals. The 107-pS channel exhibited a Cs + ≅ K + > Na + > Li + > Ca 2+ selectivity sequence (as measured by permeability ratios) and, most frequently, a high open probability (>0.9) irrespective of the experimental conditions used, therefore sharing many properties of Schistosoma mansoni outer tegumental membrane cation channels.

The association between fibrinogen measured in healthy individuals and subsequent development of ischemic heart disease is well established, but studies reporting fibrinogen levels in coronary heart disease patients are scarce. Plasma fibrinogen was determined for 5729 men and 728 women (aged 45 to 74) with established coronary heart disease, screened for participation in the Bezafibrate Infarction Prevention study, with the following lipid profile at the time of the first screening visit: total serum cholesterol ⩽270 mg/dl, high density lipoprotein cholesterol ⩽45 mg/dl and triglyceride ⩽ 300 mg/dl. Increased age was associated with augmented plasma fibrinogen values. Age-adjusted fibrinogen levels were higher in women than in men. A direct association was found between mean fibrinogen levels and low density lipoprotein cholesterol. On the other hand, the correlation with high density lipoprotein cholesterol was inverse. Fibrinogen was also associated with body mass index, behavioral variables and severity of coronary heart disease. In a multivariable linear regression analysis performed, risk factors considered explained merely 6 and 4% of fibrinogen variation for men and women, respectively. Therefore, most of the fibrinogen level variability in coronary heart disease patients is accounted for by factors that remain to be established by further research.

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a superfamily of proteins implicated in the transport of ions, proteins, and hydrophobic substances. Recent studies have demonstrated that CFTR is a protein kinase A-sensitive anion channel regulated by ATP. In the present study, patch-clamp techniques were used to assess the role of CFTR in the transport of Cl- and ATP. The stable transfection of mouse mammary carcinoma cells, C127i, with the cDNA for human CFTR resulted in the appearance of a diphenylamine-2-carboxylate-inhibitable Cl- channel, which was activated by cAMP under whole-cell and cell-attached conditions and by protein kinase A plus ATP under excised, inside-out conditions. CFTR expression was also associated with the electrodiffusional movement of ATP as indicated by the cAMP activation of ATP currents measured under whole-cell conditions. In excised, inside-out patches, it was demonstrated that ATP currents were mediated by ATP-conductive channels, which were also activated by protein kinase A and blocked by the Cl- channel blocker diphenylamine-2-carboxylate under excised, inside-out conditions. Single-channel currents observed in the presence of asymmetrical Cl-/ATP concentrations indicated that the same conductive pathway was responsible for both ATP and Cl- movement. Thus, CFTR is a multifunctional protein with more than one anion transport capability and may modify signal transduction pathways for Cl- or other secretory processes by the selective delivery of nucleotides to the extracellular domain.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel activated by protein kinase A and regulated by ATP in a complex manner. We have applied patch-clamp techniques to C127i mouse mammary carcinoma cells transfected with human CFTR to assess the role of external ATP in the modulation of CFTR function. Extracellular ATP was sufficient to activate non-rectifying, Cl(-)-selective whole-cell currents in CFTR-transfected, but not mock-transfected cells. The ATP-mediated activation of CFTR was independent of protein kinase A since channel activation by ATP was preserved in cells that were (a) depleted of intracellular ATP, (b) incubated with the cAMP antagonist Rp-cAMPS, or (c) exposed to the protein kinase A inhibitor, 5-24 amide. In each of these conditions, 8-Br-cAMP was no longer capable of activating CFTR. The possibility that the extracellular ATP activation of Cl- currents in CFTR-expressing C127i cells was mediated by a P2-type purinergic receptor was supported by studies in which the effect of external ATP on the Cl- currents was mimicked by the ATP analogs, ATP gamma S and beta,gamma-methylene ATP, but not the uridine nucleotide, UTP. Single-channel analysis of ATP-activated Cl -currents under both cell-attached and excised, inside-out patch-clamp configurations indicated that this channel is only present in CFTR-transfected cells and indistinguishable from CFTR. External ATP also activated ATP currents in CFTR-transfected cells, a novel function of CFTR. These findings are consistent with the presence of a purinergic receptor signal transduction mechanism in C127i cells whose activation by external ATP is linked to the activation of CFTR in a cAMP-independent manner. The data provide additional support for the use of ATP and its analogs as alternative therapies in cystic fibrosis.

Von 245 Patienten, die nach ihrer Prasentation in unserer Klinik auf eine Therapie mit einem Angiotensin-Konversionsenzym (ACE)-Hemmer gesetzt wurden, uber-pruften wir die Krankenblatter, um diejenige

Myocardial ischemia during daily life can be induced by increased demand and by increased coronary tone. The purpose of this study was to assess the mechanism of action of mibefradil, a new T-channel calcium blocker that is a vasodilator with negative chronotropic properties. Included in this study were 114 patients with chronic stable angina pectoris and ischemic episodes during baseline 48-hour ambulatory ECG monitoring (AEM). After a placebo run-in period patients received 50 mg, 100 mg, or 150 mg of mibefradil per day and repeat 48 hours AEM was performed. Ischemic episodes were divided into 2 categories: Type I is those in which an increase in heart rate10% preceded the development of 1 mm ST depression; Type II is those withor = 10% increase in heart rate. Of the 625 ischemic episodes recorded at baseline, 83% were Type I and 17% were Type II. At 50 mg mibefradil dose, there was a significant decrease in the number of Type I ischemic episodes but not of Type II. At doses of 100 mg and 150 mg/day, there was a significant decrease in frequency of both types of ischemic episodes. At a low dose of 50 mg/day, mibefradil reduces ischemia predominantly by preventing an increase in heart rate, while at higher doses of 100 mg and 150 mg/day, it also acted as a vasodilator suppressing episodes associated with increased coronary tone.

Ischemic heart disease (IHD) is women is characterized by a higher morbidity and mortality in the peri-infarction and coronary bypass peri-operative periods. These epidemiological data strengthen our impression that the health system unintentionally "ignores" the high proportion of females with IHD. The process of investigating chest pain, diagnosing IHD, and the subsequent treatment and rehabilitation, seem to differ between the genders. Time elapsed from beginning of chest pain to diagnosis of IHD seems to be longer in women than in men. Personal, educational and social factors are contributory. Although time elapsed between diagnosis and rehabilitation is usually similar in the genders, peri-operative morbidity and mortality are higher in women. It may be that the higher rates in women are caused by delay in diagnosis and treatment, which allows worsening of the disease in women before treatment. This delay can occur during the time needed for evaluation of chest pain, from the door of the physician to diagnosis and treatment. In our retrospective study we determined the difference in referral of men and women with chest pain to the emergency department (ED) and the attitude of physicians in the ED and medical department to chest pain in men and in women, including final diagnosis on discharge. 615 patients over 18 years referred to the ED for chest pain during 3 randomly chosen, consecutive months were studied. We found that women constituted only 39.5% of the referred patients, but the proportion hospitalized was similar to that in men. Hospitalized women were older (57.7 +/- 18.4 versus 49.7 +/- 17.8 years in men), and had more risk factors (4 versus 2 in men). Proportions of specific diagnoses on discharge from hospital were equal in the genders. To bridge the differences and to implement education in prevention, investigation and treatment of IHD in women, we established the "Female Heart" clinic. The objective of this clinic is to reduce differences in the first step, in the process of evaluating chest pain in women, by educating and encouraging them to present early to their physicians, and by changing physicians' attitudes in the investigation of chest pain in women. We plan to determine in a prospective study if these goals are reached.

To confirm the safety of prostaglandin E ( 1 ) (PGE ( 1 ) ) when administered in 100 mL normal saline to patients with severe peripheral occlusive disease (PAOD; Fontaine class III or IV) and concomitant compensated chronic congestive heart failure (CHF) and to explore possible hemodynamic benefits of PGE ( 1 ) in CHF.PGE ( 1 ) has been found to be effective in the treatment of severe PAOD. The agent may beneficially affect left ventricular performance or hemodynamics in patients with CHF. However, it must be administered intravenously (in saline diluent, adding potential hazard in patients with volume CHF).In a randomized, double-blinded protocol, 50 patients received intravenous (i.v.) infusion of either 60 microg PGE ( 1 ) or placebo, each dissolved in 100 mL saline solution administered over 2 hours each day for 14 days. During the succeeding 14 days, i.v. PGE ( 1 ) was administered to all patients in open-label fashion. Safety was assessed by clinical evaluation of symptoms and signs of CHF or other adverse events, by catheter-based and echocardiographic search for objective cardiac functional influences, and by echocardiogram monitoring for cardiac rhythm. PAOD status also was defined.No evidence of clinical or objective cardiac functional influence was detected. With the usual dosage approved in PAOD, no significant influence on cardiac performance was observed.PGE ( 1 ) is safe for treatment of PAOD in patients with concomitant chronic, compensated CHF.

Prostaglandin E ( 1 ) (PGE ( 1 ) ), the active ingredient of the drug alprostadil-alpha-cyclodextrin, has been effective in mitigating the clinical manifestations of peripheral arterial occlusive disease (PAOD). PGE ( 1 ) often is administered to patients with the potential for developing serious arrhythmias, presenting potential safety hazards if the drug caused or potentiated arrhythmias. However, PGE ( 1 ) has antiadrenergic properties and, theoretically, might have an antiarrhythmic action. Therefore, the effect of PGE ( 1 ) on frequency and severity of atrial and ventricular arrhythmias was evaluated from 48-hour electrocardiographic recordings in patients receiving PGE ( 1 ) therapy for severe PAOD. No significant effects on arrhythmia frequency or severity, and no evidence of proarrhythmia, was apparent after PGE ( 1 ) administration.

This study assesses the impact of infarct location on immediate (in-hospital) and 1- and 5-year mortality among patients with reinfarction during the year following discharge from the initial episode of myocardial infarction. The analysis included 192 patients with a second myocardial infarction who were compared in four infarction location groups. The in-hospital mortality associated with reinfarction was higher in patients with a second anterior (32%) than with a second inferior (18%) location, irrespective of the first infarction location (p = 0.03). At 5 years of follow-up, the mortality (65%) tended to be higher in patients with a first anterior-second anterior infarction as compared with patients with all other combinations of location.

A 107-pS (symmetrical 150 mm KCl), nonselective cation channel was reconstituted from a microsomal membrane fraction of the larval stage of the tapeworm Echinococcus granulosus. Most of the time, it displayed a high open probability (>0.95) irrespective of either the applied voltage, Ca2+, Ba2+, or tetraethylammonium concentration. Nevertheless, in contrast with this ``leaklike'' behavior, less frequently this ``all-the-time-open'' channel reversibly entered two different kinetic modes. One of them was characterized by lower P o values and some voltage sensitivity (V ½≅ 129 mV, and an equilibrium constant for channel closing changing e-fold per 63-mV change) the kinetic analysis revealing that it resulted from the appearance of voltage-sensitivity in the mean closed times and a sixfold increase in the equilibrium constant for channel closing at 0 mV. The other mode was characterized by a very fast open-close activity leading to poorly resolved current levels and a P o around 0.6–0.7 which, occasionally and in a voltage-sensitive manner, entered a long-lived nonconducting state. However, the rare nature of these mode-shifting transitions precluded a more detailed analysis of their kinetics. The conductive properties of the channel were not affected by these switches. Model gating alone does not seem to ensure any physiological role of this channel and, instead, some other channel changes must occur if this phenomenon were to be of regulatory importance in vivo. Thus, mode-shifting might constitute an alternative target for channel activity modulation also in tapeworms.

We describe the first successful reconstitution of placental ionic channels on planar lipid bilayers. An apical plasma membrane-enriched vesicle fraction from human syncytiotrophoblast at term was prepared by following isotonic agitation, differential centrifugation, and Mg2+-induced selective precipitation of nonapical membranes, and its purity was assessed by biochemical and morphological marker analysis. We have already reported that, unlike previous patch-clamp studies, nonselective cation channels were incorporated in most cases, a result consistent with the higher permeability for cations as compared with Cl- and with the low apical membrane potential difference at term revealed by fluorescent probe partition studies, and microelectrode techniques. In this paper, we report that Cl--selective channels were incorporated in 4% of successful reconstitutions (14 out of 353) and that their analysis revealed two types of activity. One of them was consistent with a voltage-dependent, 100-pS channel while the other was consistent with the lateral association of 47-pS conductive units, giving rise to multibarrelled, DIDS-sensitive channels of variable conductance (300 to 650 pS). The latter displayed a very complex behavior which included cooperative gating of conductive units, long-lived substates, voltage-dependent entry into an apparent inactivated state, and flickering activity. The role of the reported Cl- channels in transplacental ion transport and/or syncytium homeostasis remains to be determined.

We evaluated the early and long-term prognosis of patients with a first non-Q-wave acute myocardial infarction (AMI) in relation to infarct site. Among 4,314 patients with a first AMI, 610 (14%) had a non-Q-wave AMI. Of them, 248 patients with anterior wall AMI were compared with 327 patients with inferior/lateral AMI. Baseline clinical characteristics were similar in both groups except for higher mean age in the anterior wall group. In-hospital complications were more common among patients with anterior wall AMI than in the inferior/lateral group. Patients with anterior wall AMI also had higher rates of in-hospital (15%), 1-year (12%), and 5-year (36%) postdischarge mortality compared with the inferior/lateral infarction group (10%, 6%, and 22%, respectively). The 1-year cardiac event rate (recurrent AMI and cardiac death) was significantly higher among the anterior wall AMI group than the inferior/lateral AMI group (14.2% and 4.8% respectively, p = 0.001). After adjustment for age, gender, systemic hypertension, diabetes mellitus, prior angina, and treatment with various medications, an increased risk for 1-year (odds ratio 1.31, 95% confidence interval [CI] 0.62 to 2.78) and 5-year mortality (relative risk 1.29, 95% CI 0.90 to 1.85) was observed, but it did not reach statistical significance. Anterior wall AMI location emerged as a predictor for higher 1-year cardiac event rate (odds ratio 3.15, 95% CI 1.59 to 6.78). These findings suggest that AMI location is an important prognostic variable for risk stratification of patients with a first non-Q-wave AMI.

Objective: To characterize the 24-h arterial pressure (AP) profile and the left ventricular (LV) structures and functions in women with pregnancy-associated hypertension. Methods: Twenty nulliparous pregnant women after 20 weeks' gestation, 10 normotensive and 10 hypertensive women matched for gestational age, were hemodynamically investigated using 24-h AP monitoring and Doppler echocardiography to determine LV structures and functions, both systolic and diastolic. Results: The hypertensive women had significantly higher AP determinations throughout the 24 h, with no change in diurnal variation, i.e. nocturnal decline and early morning peaks. Their LV mass was greater and it was accompanied by a slight reduction in contractility and a significant reduction in LV relaxation. The increased AP was due to peripheral vasoconstriction, while cardiac output was preserved. Conclusions: It appears that pregnancy-associated hypertension is caused mainly by arterial vasoconstriction and not by higher cardiac output. The hypertension increases the LV mass, which is associated with a fall in LV relaxation.

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a superfamily of proteins implicated in the transport of ions, proteins, and hydrophobic substances. Recent studies have demonstrated that CFTR is a protein kinase A-sensitive anion channel regulated by ATP. In the present study, patch-clamp techniques were used to assess the role of CFTR in the transport of Cl- and ATP. The stable transfection of mouse mammary carcinoma cells, C127i, with the cDNA for human CFTR resulted in the appearance of a diphenylamine-2-carboxylate-inhibitable Cl- channel, which was activated by cAMP under whole-cell and cell-attached conditions and by protein kinase A plus ATP under excised, inside-out conditions. CFTR expression was also associated with the electrodiffusional movement of ATP as indicated by the cAMP activation of ATP currents measured under whole-cell conditions. In excised, inside-out patches, it was demonstrated that ATP currents were mediated by ATP-conductive channels, which were also activated by protein kinase A and blocked by the Cl- channel blocker diphenylamine-2-carboxylate under excised, inside-out conditions. Single-channel currents observed in the presence of asymmetrical Cl-/ATP concentrations indicated that the same conductive pathway was responsible for both ATP and Cl- movement. Thus, CFTR is a multifunctional protein with more than one anion transport capability and may modify signal transduction pathways for Cl- or other secretory processes by the selective delivery of nucleotides to the extracellular domain.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel activated by protein kinase A and regulated by ATP in a complex manner. We have applied patch-clamp techniques to C127i mouse mammary carcinoma cells transfected with human CFTR to assess the role of external ATP in the modulation of CFTR function. Extracellular ATP was sufficient to activate non-rectifying, Cl(-)-selective whole-cell currents in CFTR-transfected, but not mock-transfected cells. The ATP-mediated activation of CFTR was independent of protein kinase A since channel activation by ATP was preserved in cells that were (a) depleted of intracellular ATP, (b) incubated with the cAMP antagonist Rp-cAMPS, or (c) exposed to the protein kinase A inhibitor, 5-24 amide. In each of these conditions, 8-Br-cAMP was no longer capable of activating CFTR. The possibility that the extracellular ATP activation of Cl- currents in CFTR-expressing C127i cells was mediated by a P2-type purinergic receptor was supported by studies in which the effect of external ATP on the Cl- currents was mimicked by the ATP analogs, ATP gamma S and beta,gamma-methylene ATP, but not the uridine nucleotide, UTP. Single-channel analysis of ATP-activated Cl -currents under both cell-attached and excised, inside-out patch-clamp configurations indicated that this channel is only present in CFTR-transfected cells and indistinguishable from CFTR. External ATP also activated ATP currents in CFTR-transfected cells, a novel function of CFTR. These findings are consistent with the presence of a purinergic receptor signal transduction mechanism in C127i cells whose activation by external ATP is linked to the activation of CFTR in a cAMP-independent manner. The data provide additional support for the use of ATP and its analogs as alternative therapies in cystic fibrosis.

214 patients with angina pectoris were randomized to placebo or isosorbide-5-mononitrate (ISMN) rapid release 5, 10 or 20 mg b.i.d. at 8 a.m. and 3 p.m. Exercise tests were performed between 8 a.m. and 10 p.m., before and 2 and 7 h after each dose on days 2 and 14 and before and 2 h after the morning dose on days 7 and 21. All doses of ISMN increased exercise duration significantly more than placebo, and this effect lasted throughout most of the day. It was maximal (73 s; 24%) 2 h after the morning dose, slightly attenuated but still significant at 7 h, increased 2 h after the second dose and attenuated but still greater than with placebo at 7 h. The increase was even greater at 3 weeks (99 s; 29%), perhaps due to a training effect. Similar improvement was observed in other exercise parameters. There was no significant dose response. There were 100% more anginal attacks in the placebo than in the 20-mg treatment group. No rebound (assessed by comparing exercise duration before the morning dose between the groups) was observed.ISMN b.i.d. eccentrically has an antianginal effect throughout most of the day, peaking at 2 h. This effect is sustained during chronic therapy, without tolerance or rebound.

Ventricular fibrillation in humans is generally sustained (SVF), but it can be also transient (TVF), reverting spontaneously to sinus rhythm. In previous studies we have shown that: a) TVF appears in all young mammals and varies according to age and species; b) it requires synchronization of myocardial cell activity; c) infusion of certain drugs may change the type of ventricular fibrillation from sustained into transient. We hypothesize that the synchronization required for TVF depends on the electrical conductivity of intercellular structures. These intercellular couplings differ among species and decrease with age. Comparison between the inter- and intra-specific variations of intercellular connective structure described in the literature with the type of ventricular fibrillation found in our previous studies on various animals of different ages showed a clear relationship between these histological variations and the changes in the type of ventricular fibrillation. In this study we examined intercellular connective structures ultrastructurally in 3 groups of cats: a. control, untreated cats exhibiting sustained ventricular fibrillation; b. untreated cats exhibiting transient ventricular fibrillation; c. treated cats exhibiting sustained ventricular fibrillation before infusion of a defibrillating drug and transient ventricular fibrillation thereafter. It was found that the intercellular connective structure in cats exhibiting sustained ventricular fibrillation differs significantly from that in cats exhibiting transient fibrillation. In hearts exhibiting sustained ventricular fibrillation, many intercellular connective structures are widened and the degree of widening is pronounced, forming a continuous line, while in hearts exhibiting transient ventricular fibrillation the widened junctions are rare and isolated and the widening is relatively small. These preliminary results strongly support our above-mentioned hypothesis, providing an explanation for the origin of transient ventricular fibrillation and a tool for the development of new defibrillating drugs.

The files of 172 consecutive hypertensive patients who received captopril or enalapril have been reviewed and the patients questioned on the development of chronic dry cough, persisting for at least two months. Forty patients had cough that was attributed to the drugs. Thirteen of them discontinued the drugs because of this adverse effect. In 15 of the 27 patients (55%) who continued receiving ACE inhibitors (7 males, 8 females, aged 65.4 +/- 9.9 years) the cough had spontaneously disappeared after 3.9 +/- 1.9 months of continued unaltered administration of these drugs and without any treatment aimed against this symptom. All patients were followed for at least four months after disappearance of cough, without recurrences. This finding may discourage withdrawal of ACE inhibitors from many patients who develop cough. Continuation of ACE inhibitors for at least several months, despite cough, (if the cough is not too severe) is probably justifiable.

The safety of pregnancy after myocardial infarction remains a significant dilemma for both the obstetrician and the cardiologist. Only 20 cases of pregnancy after myocardial infarction have been reported. To clarify this problem, we add our experience of four such cases in which conception occurred 9 months to 9 years after myocardial infarction with no previous consultation. Each woman had an uneventful pregnancy with no cardiac or obstetric complications related to the myocardial infarction. All patients were under the strict supervision of an obstetrician and a cardiologist during pregnancy in our conjoined antepartum-cardiologic clinic. The mode of delivery in all patients was related to the obstetric indications. Our experience and the accumulated experience in the literature demonstrate good prognosis for patients who conceive after myocardial infarction.

Nitrates are widely used for the treatment and prevention of heart failure and angina pectoris. Even the traditionally most rapid method of administration, the sublingual tablet, takes at least 2 min until the onset of effect. Usually this takes longer because 2 min is the time required from the moment of dissolution by nitrates, administered in the form of sublingual tablets, to reach peak plasma levels [1]. An additional period is, however, required for dissolution of the tablet, which must take place at least partially before absorption begins. This period shows interpatient variability, which may involve further delay of the onset of action.

An iatrogenic burn is an unexpected burn that results from a diagnostic or therapeutic procedure. Modern therapy and research, while achieving new standards of cure rate, likewise has the potential for causing serious and, at times, hazardous complications. This report describes an illustrative case of an unusual iatrogenic burn.

The safety of pregnancy after myocardial infarction remains a significant dilemma for both the obstetrician and the cardiologist. Only 20 cases of pregnancy after myocardial infarction have been reported. To clarify this problem, we add our experience of four such cases in which conception occurred 9 months to 9 years after myocardial infarction with no previous consultation. Each woman had an uneventful pregnancy with no cardiac or obstetric complications related to the myocardial infarction. All patients were under the strict supervision of an obstetrician and a cardiologist during pregnancy in our conjoined antepartum-cardiologic clinic. The mode of delivery in all patients was related to the obstetric indications. Our experience and the accumulated experience in the literature demonstrate good prognosis for patients who conceive after myocardial infarction.

The purpose of this study was to assess the effectiveness of using the microcounseling model in the training of rehabilitation counselors to respond appropriately to the sexual concerns of spinal cord injured women. The study demonstrated the effectiveness of utilizing the microcounseling approach especially when compared to a traditional lecture and discussion teaching format. Microcounseling offers the trainee a more involved learning stimulus that involves both modeling and demonstration role-play. The implications point to the utilizing of microcounseling techniques in assisting potential counselors to develop effective counseling response skills in relating to the sexual concerns of spinal cord injured women.

The permeability of secondary E. granulosus cysts to [ 14 C]mebendazole was studied. The cysts were obtained by transplanting secondary cysts raised in mice into rats. The permeability to [ 14 C]mebendazole was established by two different experiments: uptake and washout of the drug. The cyst wall permeability to [ 14 C]mebendazole was found to be 1·33 × 10 −4 cm s −1 , which is of the same order as the diffusion permeability coefficient to water (1·88 × 10 −4 cm s −1 , Rotunno, Kammerer, Perez Esandi & Cereijido, 1974). The drug readily permeates through the cyst wall and experimental data suggest that it moves across the barrier by simple diffusion.

Reisin I.L. , Rabito C.A. and Cantiello H.F. 1981. Water and electrolyte balance in protoscoleces of Echinococcus granulosus incubated in vitro : effect of metabolic inhibitors. International Journal for Parasitology 11: 405–410. The effects of metabolic inhibitors on the Na + , K + , Cl − and water balance of protoscoleces of Echinococcus granulosus (sheep strain) were studied in vitro . The protoscoleces were incubated at 37°C in Ringer Krebs solution for 3 h in the presence of iodoacetate, 3 mM (IA); potassium cyanide, 3 m m (KCN); 2−4 dinitrophenol, 0.2 m m (DNP); ouabain, 10 − M or ethacrynic acid 0.5 m m . The effects of IA and/or KCN on the water and electrolyte balance were tested at high (0.95 × 10 5 Pa) and low (0.05 × 10 5 Pa) oxygen tensions. Inhibitors produced a decrease in K + as well as an increase in Na + contents. At both high and low O 2 tensions the Na + balance was greatly altered by IA, the action of which could be already observed during the first hour of treatment. The cations did not reach a steady state balance during 3 h of incubation. At high oxygen tension Na + and K + balance was also altered by KCN or DNP though their actions were not as marked as that of IA. Ouabain affected the Na + and K + contents that reached new steady state distribution between 1.5 and 3 h of treatment while water and electrolyte contents were not modified by ethacrynic acid. In all the experiments no changes in Cl − and water contents were observed. It is concluded that the energy required to maintain the Na−K balance mechanisms within protoscoleces is largely provided by the anaerobic glycolytic pathway and that the aerobic oxidative pathway contribution to the energy balance is only accessory.

The localization of the outermost barrier to chloride influx in the abdominal skin of Leptodactylus ocellatus was investigated by a technique developed by Kidder et al. The method analyses the transient changes in transepithelial electrical potential differences produced when an impermeable anion (SO4(2) or gluconate) is rapidly replaced by Cl in the external bathing solution. The experimental results indicate that the Cl barrier is at the same level as the external Na barrier, that is, at the outward facing membrane of the cells of the stratum granulosum. Further experiments demonstrate that Br behaves like Cl, whereas I seems to behave as an impermeable anion, and that Na is needed for activating the anion permeation mechanism at the external barrier of the epithelium.

1. The content and distribution of sodium in the epithelium of the frog skin (Leptodactylus ocellatus L.) have been studied.2. The inulin space, the (22)Na exchange, and the amounts of water and sodium were measured in samples of connective tissue. The results indicate that the necessary assumptions generally made to calculate the sodium and water contents of the epithelial cells as the difference between the total content in the tissue and the amounts contained in the inulin space are not valid in the frog skin.3. The mean concentration of sodium in the epithelium has been obtained from direct measurements of sodium and water in samples of epithelium. To measure the water content of the epithelium a new technique has been developed. When the skin is bathed with Ringer solution containing 115 mM-Na on both sides, the mean concentration of sodium in the epithelium is 79 mM. When the concentration of sodium in the Ringer is 1 mM the mean concentration in the epithelium is 25 mM. When the skin is bathed with Ringer with 1 mM-Na on the outside and 115 mM-Na on the inside-a situation which resembles the natural condition in the skin-the mean concentration of sodium in the epithelium is 52 mM.4. The compartmentalization of Na was studied by comparing the sodium content and the degree of exchange with (22)Na in the bathing solutions. In these experiments the skins were exposed to Ringer solutions with different concentrations of sodium, and (22)Na on one or both sides.5. The results indicate that the epithelium has a compartment of sodium which is not exchangeable in 40-80 min and whose size is not appreciably changed by a threefold change in the Na content in the epithelium and a hundredfold change in the concentration of the bathing solution.6. Sodium exchangeable in 40-80 min seems to be contained in two different compartments: (a) a large one that contains fixed sodium is mainly connected to the inside, and does not appear to participate directly in sodium transport across the frog skin; (b) a small one, that is bounded on the inside by a Na-impermeable barrier, and that seems to comprise the sodium involved in active transport. When the skin is bathed with Ringer solutions with 115 mM-Na on the inside and 1 mM-Na on the outside, the transporting compartment contains some 13% of the total sodium in the epithelium.7. The results are interpreted on the basis of a model recently proposed by Cereijido & Rotunno (1968). The major feature of this model is that the sodium transporting compartment is confined to the plasma membrane of the epithelial cells.

Sodium-rich frog muscles are found to extrude sodium and reaccumulate potassium at 0 degrees C. The uptake of potassium by these muscles is studied at three different temperatures as a function of external potassium concentration, K(ex). The steady-state potassium content of the tissue is related to K(ex). by a sigmoidal cooperative curve at all temperatures. These results are compared with findings on a mammalian smooth muscle.

The aim of this paper is to obtain information on the number, nature and location of the barriers to Na movement across the frog skin, and on the size and location of the Na-pool that might be contained between these barriers. On the basis that Na penetrates passively across an outer barrier, and is actively extruded across an inner barrier which is impermeable to passive movements of Na, we expected to detect at least the Na-pool of a single cell layer containing some 10−8 moles per cm2 of epithelium (i.e., in a cell layer 5 μ thick and with 21mm Na). Yet no Na-pool with these characteristics was found. The method employed could have detected a Na-pool at least an order of magnitude smaller than the one expected. It is concluded that either a Na-pool does not exist (except for the Na bound to the mechanisms operating the translocation), or else that the Na-pool is contained between barriers with different characteristics than the ones assumed above. In the first case, Na transportacross the epithelium would consist of a translocation across a single asymmetrical functional “barrier”. In the second case, the experimental results would require that ouabain either directly (by inhibiting an active step) or indirectly (through a mediated decrease of the Na permeability of the outer barrier) prevents Na penetration at the outer border.