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Key PointsQuestionWhat are the benefits and risks of continuing aspirin in addition to P2Y12 receptor inhibition with ticagrelor among patients with acute coronary syndrome between 1 month and 12 months after percutaneous coronary intervention? FindingsIn this nonprespecified, post hoc analysis of the GLOBAL LEADERS randomized clinical trial, beyond 1 month after percutaneous coronary intervention in acute coronary syndrome, aspirin was associated with increased bleeding risk and appeared not to add to the benefit of ticagrelor on ischemic events. MeaningThe findings of this hypothesis-generating analysis pave the way for further trials evaluating aspirin-free antiplatelet strategies after percutaneous coronary intervention. ImportanceThe role of aspirin as part of antiplatelet regimens in acute coronary syndromes (ACS) needs to be clarified in the context of newer potent P2Y12 antagonists. ObjectiveTo evaluate the benefit and risks of aspirin in addition to ticagrelor among patients with ACS beyond 1 month after percutaneous coronary intervention (PCI). Design, Setting, and ParticipantsThis is a nonprespecified, post hoc analysis of GLOBAL LEADERS, a randomized, open-label superiority trial comparing 2 antiplatelet treatment strategies after PCI. The trial included 130 secondary/tertiary care hospitals in different countries, with 15991 unselected patients with stable coronary artery disease or ACS undergoing PCI. Patients had outpatient visits at 1, 3, 6, 12, 18, and 24 months after index procedure. InterventionsThe experimental group received aspirin plus ticagrelor for 1 month followed by 23-month ticagrelor monotherapy; the reference group received aspirin plus either clopidogrel (stable coronary artery disease) or ticagrelor (ACS) for 12 months, followed by 12-month aspirin monotherapy. In this analysis, we examined the clinical outcomes occurring between 31 days and 365 days after randomization, specifically in patients with ACS who, within this time frame, were assigned to receive either ticagrelor alone or ticagrelor and aspirin. Main Outcomes and MeasuresThe primary outcome was the composite of all-cause death or new Q-wave myocardial infarction. ResultsOf 15968 participants, there were 7487 patients with ACS enrolled; 3750 patients were assigned to the experimental group and 3737 patients to the reference group. Between 31 and 365 days after randomization, the primary outcome occurred in 55 patients (1.5%) in the experimental group and in 75 patients (2.0%) in the reference group (hazard ratio [HR], 0.73; 95% CI, 0.51-1.03; P=.07); investigator-reported Bleeding Academic Research Consortium-defined bleeding type 3 or 5 occurred in 28 patients (0.8%) in the experimental group and in 54 patients (1.5%) in the reference arm (HR, 0.52; 95% CI, 0.33-0.81; P=.004). Conclusions and RelevanceBetween 1 month and 12 months after PCI in ACS, aspirin was associated with increased bleeding risk and appeared not to add to the benefit of ticagrelor on ischemic events. These findings should be interpreted as exploratory and hypothesis generating; however, they pave the way for further trials evaluating aspirin-free antiplatelet strategies after PCI. Trial RegistrationClinicalTrials.gov identifier: NCT01813435. This secondary analysis of the GLOBAL LEADERS randomized clinical trial evaluates the benefit and risks of aspirin in addition to ticagrelor among patients with acute coronary syndrome beyond 1 month after percutaneous coronary intervention.

Calcium entry through voltage-dependent channels play a critical role in neuronal development. Using patch-clamp techniques we have identified the components of the macroscopic Ca 2+ current in acutely-isolated chick cochlear ganglion neurons and analysed their functional expression throughout embryonic development. With Ba 2+ as a charge carrier, the currents exhibited two main components, both with a high activation threshold but differing in their inactivation kinetics. One component showed inactivation with a time constant around 100 ms (transient) whereas the other hardly inactivated (sustained). The currents were sensitive to ω -Conotoxin GVIA and dihydropyridines, blocked by 20 μ M Cd 2+ , but unaffected by ω -Agatoxin IVA. In a few cases, only with Ca 2+ as a charge carrier, an additional component with low activation threshold and fast inactivation (time constant of 20 ms), was observed. Currents were first detected at day 7 of embryonic development. Current density (amplitude/cell capacitance) increased through embryonic day 9, when early synaptic contacts are established, and decreased thereafter to lower steady values. The effect of neurotrophin-3, a neurotrophic factor required for survival and differentiation of cochlear ganglion neurons, was also examined. Neurons isolated at embryonic day 7 or day 11 and maintained two days in culture with 2 ng/ml neurotrophin-3 showed a substantial increase in Ca 2+ current density, particularly in the transient component. These findings indicate that the expression of neuronal Ca 2+ channels is predominant at the time of synapse formation between transducing hair cells and their primary afferents. Besides its effects on survival and neuritogenesis, neurotrophin-3 enhances the expression of Ca 2+ channels in cultured neurons. Taken together these results suggest that the functional expression of Ca 2+ channels is regulated during embryonic development of cochlear neurons by the release of neurotrophin-3 from the differentiating sensory epithelium of the cochlea.

Cell Na activity, acNa, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and Na-selective microelectrodes. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular conductance, with NaNO3, Ringer on the apical surface. Under control conditions acNa averaged 8 +/- 2 mM (n = 9, SD). Apical addition of amiloride (20 microM) or Na replacement reduced acNa to 3 mM in 6-15 min. Sequential decreases in apical [Na] induced parallel reductions in acNa and cell current, Ic. On restoring Na after several minutes of exposure to apical Na-free solution Ic rose rapidly (approximately less than 30 sec) to a stable value while acNa increased exponentially, with a time constant of 1.8 +/- 0.7 min (n = 8). Analysis of the time course of acNa indicates that the pump Na flux is linearly related to acNa in the range 2-12 mM. These results indicate that acNa plays an important role in relating apical Na entry to basolateral active Na flux.

Na+, K+ and Cl− concentrations ( c j i ) and activities ( a j i ), and mucosal membrane potentials ( E m ) were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na+ and Cl− and 5.4 mM K+. Na+ and K+ concentrations were determined by atomic absorption spectrometry and Cl− concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO3. 14C-labelled inulin was used to determine extracellular volume. E m was measured with conventional open tip microelectrodes, a Cl i with solid-state Cl−-selective silver microelectrodes and a Na i and a K i with Na+- and K+-selective liquid ion-exchanger microelectrodes. The average E m recorded was −34 mV. c Na i , c K i and c Cl i were 51, 105 and 52 mM. The corresponding values for a Na i , a K i and a Cl i were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na+ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K+ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl−. a Cl i significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na+ and Cl− is implicated in intracellular Cl− accumulation in this tissue. This study permitted estimation of the steady-state transapical Na+ and Cl− electrochemical potential differences (ΔμNa and ΔμCl). ΔμNa (−7000 J · mol−1; cell minus mucosal medium) was energetically more than sufficient to account for ΔμCl (1000–2000 J · mol−1).

Intracellular C1, K and Na activities (aCli,aki andaNai) and transmucosal membrane potential (Em) in epithelial cells ofNecturus gallbladder were measured at different external Na concentrations ([Na]o), with liquid ion-exchanger and conventional microelectrodes. Bladders were mounted in a divided chamber at 23°C between identical HCO3-free Ringer solutions containing 5mm K. The pH was 7.2. Tris was substituted for Na. Measurements were made under steady-state conditions as determined by the constancy of the transepithelial potential difference. Both,aCli andaNai increased in a saturable fashion with [Na]o.Em did not change significantly. Average values (±sem) under normal conditions ([Na]o=100mm) foraCli,aNai andEm were 16.8±0.8mm (n=9), 9.7±0.6mm (n=10) and −52.6±0.6 mV (n=26), respectively. In Na-free mediaaCli declined to its equilibrium value.aKi (96±2mm;n=7) did not change when [Na]o was varied between 100 and 10mm but decreased to 80±3mm (n=4) in Na-free media.

A device is described that permits continuous measurement of electrophysiological parameters in epithelial tissues in the open-circuit mode. Transepithelial potential (VT) and microelectrode (either conventional or ion-selective) potential (VM) are directly measured. Application of transepithelial current pulses allows continuous monitoring of transepithelial resistance (RT) and the ratio between the changes in VM and VT induced by these pulses. Measurement of this ratio, which under some circumstances reflects the apical fractional resistance of the cellular pathway, is important in assessing membrane damage during microelectrode impalement and/or as an index that the microelectrode tip is inside a cell. This is particularly useful when the change in VM during impalement is small. Application of 0.5-nA current pulses through open-tip microelectrodes allows continuous recording of the microelectrode resistance (RM). In epithelia where the individual cells are electrically coupled this permits acceptable impalements (RM remains nearly constant) to be distinguished from those affected by tip potential artifacts due to plugging of the microelectrode tip (RM increases after penetration of the cell membrane). The device provides compensation for the IR voltage drop in the solution between the potential measuring salt bridges and the epithelial surfaces. The microelectrode electrometer has an input impedance greater than 10(15) and is provided with stray capacitance neutralization.

Mucosal hypertonicity produces a drop of transepithelial potential difference in isolated rat jejunum with a half time of about 15 s. The same effect is obtained when the osmorality of both bathing solutions is raised simultaneously. Serosal hypertonicity produces an increase of transepithelial potential difference an order of magnitude lower than the drop produced by mucosal hypertonicity. The change in the short circuit current parallels the one in potential difference. When the transepithelial potential difference is varied by adding different concentrations of glucose to the bathing media, the potential drop induced by mucosal hypertonicity is linearly related to the magnitude of the transepithelial potential difference just before the increase in osmolarity. The drop of potential can be explained by a decrease of the electrical resistance of the extracellular shunt pathway due to opening of the tight junctions. The results can be accounted for in terms of an equivalent electrical circuit proposed for small intestine. Using this equivalent circuit model it is possible to obtain estimates of the values of the diffusion potential and the salt gradient across the tight junction.

The sodium-selective ligand 1,1,1-tris[1(1)-(2(1)-oxa-4(1)-oxo-5(1)-aza-5(1)-methyl)dodecanyl]propane dissolved in 3-nitro-o-xylene containing a small amount of the lipophilic anion tetrachlorophenyl borate was used as a liquid ion-exchanger in sodium-selective microelectrodes. The microelectrodes gave rapid, stable responses that were linear functions of the logarithm of sodium activity. They were tested under conditions approximating those to be expected in the cell interior, and the results indicated that they can be used to measure intracellular sodium activity without significant interference from intracellular potassium.

Intracellular Na+, K+, and Cl− activities (a Na ,a K ,a Cl ) and transapical membrane potentials (V o) were measured with liquid ion-exchanger and open-tip microelectrodes in isolated short-circuited frog skins (R. pipiens) incubated at 23°C in normal amphibian Ringer's solution. Under control conditionsa Na =14±3mm,a K =132±10mm anda Cl =18±3mm (sd). The value ofa Cl is 4.4 times the value corresponding to electrochemical equilibrium for this ion. Thus, Cl− is actively accumulated by epithelial cells of the frog skin. Shortly after addition of amiloride (2–5 μm) to the apical bathing medium,a K ,a Na , anda Cl were essentially unchanged althoughV o had hyperpolarized by about 30–40 mV. During long-term exposure to amiloridea K anda Cl did not change significantly,V o depolarized by about 16 mV from the maximal value anda Na decreased to 8±3mm. Immediately after exposure to amiloride the transmembrane driving force for Na+ increased from 124 to 154 mV. During further exposure to amiloride, despite changes in bothV o anda Na , this driving force remained virtually constant. SinceI sc during this period was close to zero, it is suggested that the observed driving force for Na+ under these conditions approximates the maximal driving force generated by the Na+−K+ ATP-ase pump in the basolateral cell membrane.

Cell K activity, acK, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30-60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO3 Ringer on the apical surface. Under control conditions acK was nearly constant among skins (mean +/- SD = 92 +/- 8 mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 microA/cm2). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase in acK after inhibition of cell current was observed, on the average the change was not significant (98 +/- 11 mM after amiloride, 101 +/- 12 mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from -42 to -77 mV, hyperpolarized after inhibition of cell current, initially to -109 +/- 5 mV, then depolarizing to a stable value (-88 +/- 5 mV) after 15-25 min. At this time K was above equilibrium (EK = 98 +/- 2 mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport. The measurement of acK permitted the calculation of the passive K current and pump current under control conditions, assuming a "constant current source" with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.

In studies of apical membrane current-voltage relationships, in order to avoid laborious intracellular microelectrode techniques, tight epithelia are commonly exposed to high serosal K concentrations. This approach depends on the assumptions that high serosal K reduces the basolateral membrane resistance and potential to insignificantly low levels, so that transepithelial values can be attributed to the apical membrane. We have here examined the validity of these assumptions in frog skins (Rana pipiens pipiens). The skins were equilibrated in NaCl Ringer's solutions, with transepithelial voltageV t clamped (except for brief perturbations ΔV t) at zero. The skins were impaled from the outer surface with 1.5m KCl-filled microelectrodes (R el>30 MΩ). The transepithelial (short-circuit) currentl i and conductanceg t=−ΔI t/ΔV t, the outer membrane voltageV o (apical reference) and voltage-divider ratio (F o=ΔV o/ΔV t), and the microelectrode resistanceR el were recorded continuously. Intermittent brief apical exposure to 20 μm amiloride permitted estimation of cellular (c) and paracellular (p) currents and conductances. The basolateral (inner) membrane conductance was estimated by two independent means: either from values ofg i andF o before and after amiloride or as the ratio of changes (−ΔI c/ΔV i) induced by amiloride. On serosal substitution of Na by K, within about 10 min,I c declined andg t increased markedly, mainly as a consequence of increase ing p. The basolateral membrane voltage (V i(=−V o) was depolarized from 75±4 to 2±1 mV [mean±sem (n=6)], and was partially repolarized following amiloride to 5±2 mV. The basolateral conductance increased in high serosal K, as estimated by both methods. Essentially complete depolarization of the basolateral membrane and increase in its conductance in response to high [K] were obtained also when the main serosal anion was SO4 or NO3 instead of Cl. On clampingV t over the range 0 to +125 mV in K2SO4-depolarized skins, the quasi-steady-stateV o V t relationship was linear, with a mean slope of 0.88±0.03. The above results demonstrate that, in a variety of conditions, exposure to high serosal K results in essentially complete depolarization of the basolateral membrane and a large increase in its conductance.

Knowledge of the voltage dependencies of apical and basolateral conductances is important in determining the factors that regulate transcellular transport. To gain this knowledge it is necessary to distinguish between cellular and paracellular currents and conductances. This is generally done by sequentially measuring transepithelial current/voltage (It/Vt) and conductance/voltage (gt/Vt) relationships before and after the abolition of cellular sodium transport with amiloride. Often, however, there are variable time-dependent and voltage-dependent responses to voltage perturbation both in the absence and presence of amiloride, pointing to effects on the paracellular pathway. We have here investigated these phenomena systematically and found that the difficulties were significantly lessened by the use of an intermittent technique, measuring It and gt before and after brief (less than 10 sec) exposure to amiloride at each setting of Vt. I/V relationships were characterized by these means in frog skins (Rana pipiens, Northern variety, and Rana temporaria). Cellular current, Ic, decreased with hyperpolarization (larger serosa positive clamps) of Vt. Derived Ic/Vt relationships between Vt = 0 and 175 mV (serosa positive) were slightly concave upwards. Because values of cell conductance, gc, remained finite, it was possible to demonstrate reversal of Ic. Values of the reversal potential Vr averaged 156 +/- 14 (SD, n = 18) mV. Simultaneous microelectrode measurements permitted also the calculation of apical and basolateral conductances, ga and gb. The apical conductance decreased monotonically with increasing positivity of Vt (and Va). In contrast, in the range in which the basolateral conductance could be evaluated adequately (Vt less than 125 mV), gb increased with more positive values of Vt (and Vb). That is, there was an inverse relation between gb and cellular current at the quasi-steady state, 10-30 sec after the transepithelial voltage step.

The absorption of water, electrolytes, and nutrient substances by the small intestine has been a central concern of gastrointestinal physiology for many years. Beginning as early as the mid-seventeenth century, there is a more or less continuous history of experimental investigation in this field (Parsons 1968). In this area, as in many other areas of experimental science, the first half of this century witnessed a tremendous intensification of effort. This increase in the number of investigations designed to uncover the fundamental mechanisms of salt and water transport resulted in a concomitant proliferation of published results and the elaboration of a variety of ingenious hypotheses (see, e.g., Hober 1946; Schultz and Curran 1968). This activity has increased, in volume and intensity, until the present time and seems likely (given the survival of a reasonably supportive socioeconomic milieu) to continue increasing for many years to come. Despite all this, it is a salutory thought that, as pointed out by Schultz and Curran (1968), modern theories of intestinal absorption can still be described, in general terms, by Heidenhain’s (1894) hypothesis. That is, that the absorption process results from a combination of physicochemical (osmotic) driving forces and the intrinsic driving force (Triebkraft) generated by the absorptive cells.

It has been suggested that distribution of lateral interspace resistance in association with a highly conductive junction can significantly affect the measurement of outer membrane(o)/epithelial(t) voltage divider ratios (Fo = delta Vo/delta Vt), thereby leading to erroneous inferences regarding the outer membrane fractional resistance [fRo = Ro/Rc = Ro/(Ro + Ri)], where Ro and Ri are the outer and inner cell membrane resistance respectively and Rc is the total cell membrane resistance. We present here experimental evidence for this point of view. During seasons when frog skins were highly permeable to Cl, transepithelial conductance gt often exceeded 2 mS/cm2. High concentrations of external amiloride rapidly blocked cellular transport, but gt initially remained high and Fo remained appreciably less than 1.0. These values of Fo were found here to result from low junctional resistance Rj: increase of Rj, either gradually following the administration of amiloride, or abruptly with external replacement of Cl by other anions, was associated with increase of Fo to near unity, without effect on the membrane potential or significant change in the short-circuit current. Experimental results following amiloride validated a simple equivalent circuit model predicting near-linear increase in Fo with progressive decrease in gt and led to plausible values of Rj and lateral space resistance Rl. The possible influence of the paracellular resistance pattern on the evaluation of cell membrane resistances from voltage divider ratios is discussed.