The quantal secretion of catecholamines is impaired by the accumulation of β-adrenoceptor antagonists into chromaffin cell vesicles

Background and purpose:

The delayed onset of certain effects of antagonists of β-adrenoceptors (β-blockers), such as lowering arterial blood pressure (several days), cannot be explained solely by their effects on β-adrenoceptors, an action that occurs within minutes. Although several mechanisms have been proposed, none of them explain this temporal delay. This work aimed at providing a new explanation based on the interference of these drugs with the functional accumulation of catecholamines within neurosecretory vesicles.

Experimental approach:

We used the simultaneous on-line monitoring of catecholamine and labetalol release from bovine isolated chromaffin cells and from rat perfused adrenal glands, as well as single cell amperometry, intracellular electrochemistry, patch amperometry and HPLC.

Key results:

Using amperometry, three β-blockers, labetalol, atenolol and propranolol, reduced the quantal size of secretory events in chromaffin cells, accompanied by a slowing down of exocytosis. By patch amperometry, we found that treatment with β-blockers also increases the chromaffin vesicle volume, thereby creating a functional dilution of catecholamines. Experiments with intracellular electrochemistry show that vesicles cannot uptake new catecholamines. There was progressive accumulation of labetalol in secretory vesicles of bovine adrenal chromaffin cells, and this β-blocker was co-released with catecholamines from rat and bovine chromaffin tissues.

Conclusions and implications:

We propose that β-blockers are progressively concentrated into sympathetic secretory vesicles, and interfere with the storage of catecholamines and are co-released with the natural transmitters, resulting in a decrease in the sympathetic tone. This could explain the delayed onset of the hypotensive effects of β-blockers.     

Evidence of carrier‐mediated transport in the penetration of donepezil into the rat brain

The objective of this study was to characterize the mechanism that controls the transport of donepezil into the brain. The apparent brain uptake clearance (CL_app,br) was decreased as a function of the dose of donepezil, suggesting an involvement of a saturable transport process via transporter(s) in the penetration across the blood–brain barrier (BBB). Consistent with in vivo results, the uptake of substrates for organic cation transporters was significantly reduced by donepezil in both MBEC4 cells (i.e., a model for BBB) and HEK 293 cells expressing the transporters found in the brain, indicative of the involvement of organic cation transporters in the transport of the drug. Furthermore, donepezil transport was enhanced (p < 0.01) in HEK 293 cells expressing rOCNT1, rOCTN2, or rCHT1. The CL_app,br was reduced up to 52.8% of the control in rats that had been pretreated with choline, while the CL_app,br was unaffected with pretreatments with organic cations other than choline, suggesting that choline and donepezil share a common transport mechanism in the penetration across the BBB in vivo. Taken together, these observations suggest that the transport of donepezil across the BBB is mediated by organic cation transporters such as choline transport system(s). © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1548–1566, 2010     

Does the patient or pathology exist?

For a long time, the guidelines of medical conduct represented a useful, but not binding, model for adapting in order to avoid professional liability. In this sense, the idea of relying solely on these guidelines raises serious concerns, as very often there is no real “evidence” to scientifically support the recommendations. The authors have conducted a review of the literature to draw the attention of the scientific community to the highlights and shadows of this dangerous Italian prospect.     

Does transbilayer diffusion have a role in membrane transport of drugs?

The existing consensus on coexistence of transbilayer diffusion and carrier-mediated transport as two main mechanisms for drugs crossing biological membranes was recently challenged by a systems biology group. Their transporters-only hypothesis is examined in this article using published experimental evidence. The main focus is on the key claim of their hypothesis, stating that 'the drug molecules cross pure phospholipid bilayers through transient pores that cannot form in the bilayers of cell membranes, and thus transbilayer drug transport does not exist in cells'. The analysis shows that the prior consensus remains a valid scientific view of the membrane transport of drugs.     

Does the Addition of Pindolol Accelerate or Enhance the Response to Selective Serotonin Reuptake Inhibitor Antidepressants?

Although in the last few years new antidepressants have become available in the United States, responses in many patients differ, from none, to partial, to delayed therapeutic response to the agents. If the antidepressant properties of these drugs could be enhanced or accelerated, it might be possible to improve patient functioning and quality of life. Pindolol, a serotonin 1A autoreceptor antagonist, accelerates and augments the therapeutic effects of antidepressants, especially selective serotonin reuptake inhibitors.     

Does QT widening in the Langendorff-perfused rat heart represent the effect of repolarization delay or conduction slowing?

It has been suggested that class I antiarrhythmic drugs and ischemia can widen the QT interval in the Langendorff-perfused rat heart preparation as a consequence of slowed ventricular conduction. If this were so, it would undermine the clinical relevance of the preparation and its effectiveness as an antiarrhythmic bioassay. To test this, the authors determined whether three different class I drugs could prolong QT in the preparation and whether this effect was augmented by ischemia and elevation of the potassium (K+) content of the perfusion solution. Baseline drug-free QT intervals correlated inversely with the K+ content (3 microM vs. 5 mM). QT intervals widened during the first 3-5 minutes of ischemia (P < 0.05), then returned gradually to baseline. Lidocaine (3.88 microM and 12.93 microM) had no effect on the QT interval before or during ischemia, whereas quinidine (7.90 microM but not 0.79 microM) and flecainide (1.48 microM but not 0.74 microM) caused QT widening before and during ischemia (P < 0.05). Elevating perfusion solution K+ content from 3 microM to 5 mM reduced the QT-widening effects of quinidine and flecainide (P < 0.05). Because lidocaine, a relatively selective sodium (Na+) channel blocker, failed to widen QT interval whereas quinidine and flecainide (combined Na+ and K+ channel blockers) did so, and because K+ elevation reduced rather than potentiated the drug-induced QT widening, it is unlikely that Na+ channel blockade and conduction slowing play any role in ischemia- or class I drug-induced QT widening in this model.     

Ghosts of chromaffin granules accumulate biogenic amines according to a “pump and leak system” without contribution of carrier-mediated efflux

Summary Unidirectional inward transport into, accumulation by and loss of biogenic amines from ghosts of bovine chromaffin granules were studied to determine whether a carrier-mediated process contributes to the outward passage of amines across the granule membrane.In the presence of ATP-Mg²⁺, incubated ghosts (30°C; pH 7.3) showed a reserpine-sensitive (IC₅₀ 2–5 nmol/l), unidirectional, inward transport of catecholamine (CA = 70% adrenaline/30% noradrenaline), 5-hydroxytryptamine (5-HT) and tyramine with different K_m values (mol/l: tyramine 2; 5-HT 5; CA 8) but with the same V_max [20 nmol/(mg protein · min)] during the first 3 min of incubation. During longer incubation, the rate of unidirectional inward transport declined rapidly with time to about 30 to 40%, at which level it stayed nearly constant from 50 to 100 min of incubation. As the decline of unidirectional transport was independent of the amine accumulated in the ghosts, it is concluded that it reflects ageing of the membrane vesicles. During incubation for up to 100 min with CA, 5-HT or tyramine (10- to 30-fold K_m) in the presence of ATP-Mg²⁺, the amine content of the ghosts increased, approaching a steady-state content (nmol amine/mg protein: CA 400–500, 5-HT 250, tyramine 60), which was negatively correlated with the lipid solubility of the amine (tyramine > 5-HT > CA), whereas the rate of approach to steady state (t1/2 in min: CA 20–30, 5-HT 7–10, tyramine < 5) was positively correlated. Low concentrations of reserpine ( 25 nmol/l) caused net loss of amine from amine-loaded ghosts by inhibition of inward transport. However, reserpine did not reduce the fractional rate of loss (FRL) of CA-loaded ghosts induced by NH₄ ⁺ or uncoupling agents. Accelerated exchange diffusion was not found to occur at the granule membrane, as addition of high concentrations of 5-HT or dopamine to CA-loaded ghosts did not result in a higher FRL of CA than did blockade of inward transport by reserpine.Analysis of steady-state kinetics revealed the following features of the granule transport. The approach to steady state (t1/2) was independent of the rate of inward transport. The steady-state amine content of ghosts approached a limiting value as the external concentration of the amine was increased; it was determined by the same kinetic constants (K_m and the time-dependent V_max) as found for the rate of carrier-mediated inward transport. In addition, the steady-state content was inversely related to the FRL of amine after blockade of inward transport by reserpine. When the steady state at low external concentration had been reached, the distribution ratio of the accumulated amine (amine_internal/amine_external) showed a maximum. This ratio declined and approached zero with increasing external concentrations of amine. Each of these features is compatible with a single transport system having the kinetic properties of a pump and leak system, i.e. the unidirectional carrier-mediated inward transport of amines into ghosts is counteracted by efflux due to simple diffusion only.Supported by the Deutsche Forschungsgemeinschaft (Bu 296/4-3)A preliminary account was presented to the Deutsche Pharmakologische Gesellschaft (Burger and Michalke 1989) Send offprint requests to A. Burger at the above address     

Does the effect of herbicide pulse exposure on aquatic plants depend on Kow or mode of action?

The highest concentrations of herbicides measured in flowing surface waters are often only present for short periods of time. These herbicide pulses can reach concentrations that would affect aquatic plants if present over a long time. The aim of this study was to assess the effect of a 3-h herbicide pulse relative to the effects of long-term (4 and 7 days) exposure of six herbicides with different sites of action and different K(ow) on the growth of the floating macrophyte Lemna minor. The herbicides were the two photosynthetic inhibitors: diquat and terbuthylazine, the inhibitors of acetolactate syntase (ALS), imazamox and metsulfuron-methyl and the microtubule assembly inhibitors propyzamide and pendimethalin. The log K(ow) ranged from -4.6 to 5.2. For imazamox, metsulfuron-methyl, propyzamide and pendimethalin a 3-h pulse induced the effect on area-specific growth as did a 4-day exposure at an approximate 10-fold higher concentration. For diquat and terbuthylazine a concentration closer to a factor of 100 or more was needed for a 3-h pulse to induce an effect similar to that of a 4-day exposure. For diquat, the low pulse-effect was most likely due to a slow uptake of the hydrophilic ion (log K(ow) = -4.6), as no effect was observed on chlorophyll fluorescence within 8 h after exposure. The chlorophyll fluorescence parameters are expected to respond quickly to a PSI inhibitor as diquat. For terbuthylazine, fluorescence measurements showed an effect on photosynthesis within 1h of exposure, and reached a minimum after 3 h. Recovery was fast, and initial fluorescence was restored within 24 h. Hence, the small pulse effect on area-specific growth was due to rapid recovery of photosynthesis. In contrast to terbuthylazine, the stop in area-specific growth observed for the ALS-and microtubule assembly inhibitors, took up to 4 days to recover from. Such a long recovery time after a pulse of only 3 h indicate that at realistic pulse exposures of up to a day or two, pulse-effects will approach the effects obtained in long-term studies. When investigating the effects of pulse exposures on aquatic plants, we should therefore focus more on non-photosynthetic inhibitors, which might not appear in pulses in as large concentrations as the PSII inhibitors investigated up till now, but whose effect, even in a shorter pulse, can be more damaging.     

Does Drug Use Inhibit Crime Deceleration or Does Crime Inhibit Drug Use Deceleration? A Test of the Reciprocal Risk Postulate of the Worst of Both Worlds Hypothesis

Background: Prior research has shown that ongoing drug use inhibits the commonly reported maturing out of crime process and that ongoing crime inhibits the commonly reported maturing out of drug use process. Objectives: To test the predictive efficacy of drug use for crime deceleration and of crime for drug deceleration using a prospective analysis of data on a group of 524 male California Youth Authority parolees to see if both effects exist simultaneously. Method: A two-equation regression analysis of Year 3 arrests and illicit drug use was performed, controlling for age, race, marital status, employment status, and number of months free in the community. Results: It was determined that illicit drug use at Year 2 predicted an increase in arrests between the first and third years of the analysis and arrests at Year 2 predicted an increase in illicit drug use over this same time period. Alcohol use, on the other hand, failed to predict a change in arrests and arrests failed to predict a change in alcohol use. Conclusions: The results of this study suggest that illicit drug use may inhibit the natural maturing out of crime process observed in emerging adults whereas involvement in crime may inhibit the natural maturing out of illicit drug use process. These findings not only support the reciprocal risk postulate from the Worst of Both Worlds hypothesis but also have implications for risk prediction, risk management, and treatment.     

Does gender, food or grapefruit juice alter the pharmacokinetics of primaquine in healthy subjects?

AIMS: To evaluate the effects of gender, food and grapefruit juice on the pharmacokinetics of primaquine in healthy subjects. METHODS: In a randomized, two-phase cross-over study, 10 male and 10 female healthy Vietnamese subjects were administered 30 mg primaquine in the fasting state or with food, followed by administration of primaquine with grapefruit juice. RESULTS: The pharmacokinetics of primaquine were comparable between male and female subjects, with geometric mean ratios of Cmax = 0.89 [95% confidence interval (CI) 0.65, 1.22] and AUC = 0.80 (95% CI 0.56, 1.15). The mean CL/F of primaquine was slightly higher in males than in females [0.52 l h(-1) kg(-1)vs. 0.43 l h(-1) kg(-1), mean difference of 0.09 (95% CI -0.10, 0.28), P = 0.32]. When compared with fasting state values, food increased the geometric mean Cmax of primaquine by 26% (95% CI 12, 40) and the AUC by 14% (95% CI 3, 27). Similarly, grapefruit juice increased the geometric mean Cmax by 23% (95% CI 4, 45) and the AUC by 19% (95% CI 4, 37). CONCLUSIONS: The disposition of primaquine was comparable between genders, suggesting no need to modify the dose of primaquine for malaria treatment or prophylaxis. Food increased the oral bioavailability of primaquine, which may lead to higher antimalarial efficacy. Grapefruit juice increased the bioavailability of primaquine, with marked interindividual differences suggesting that people should not take primaquine with grapefruit juice.     

Reactive oxygen species: Destroyers or messengers?

Abundant evidence leaves no doubt that reactive oxygen species (ROS) are not only inevitable by-products of oxygen metabolism but also play a role in cellular signaling. ROS are produced by a family of NADPH oxidases for signaling purposes and mediate or augment the effects of insulin, growth factors, cytokines and G-protein-coupled receptors. Disturbances of ROS signaling leading to overproduction of these intermediates inflict oxidative damage of cell components in the course of various diseases. Restoration of proper ROS signaling, especially inhibition of cellular sources of ROS, may thus provide new ways of therapy.     

Novel classes of antibiotics or more of the same?

The world is running out of antibiotics. Between 1940 and 1962, more than 20 new classes of antibiotics were marketed. Since then, only two new classes have reached the market. Analogue development kept pace with the emergence of resistant bacteria until 10–20 years ago. Now, not enough analogues are reaching the market to stem the tide of antibiotic resistance, particularly among gram-negative bacteria. This review examines the existing systemic antibiotic pipeline in the public domain, and reveals that 27 compounds are in clinical development, of which two are new classes, both of which are in Phase I clinical trials. In view of the high attrition rate of drugs in early clinical development, particularly new classes and the current regulatory hurdles, it does not seem likely that new classes will be marketed soon. This paper suggests that, if the world is to return to a situation in which there are enough antibiotics to cope with the inevitable ongoing emergence of bacterial resistance, we need to recreate the prolific antibiotic discovery period between 1940 and 1962, which produced 20 classes that served the world well for 60 years. If another 20 classes and their analogues, particularly targeting gram-negatives could be produced soon, they might last us for the next 60 years. How can this be achieved? Only a huge effort by governments in the form of finance, legislation and providing industry with real incentives will reverse this. Industry needs to re-enter the market on a much larger scale, and academia should rebuild its antibiotic discovery infrastructure to support this effort. The alternative is Medicine without effective antibiotics.

LINKED ARTICLES

This article is part of a themed issue on Respiratory Pharmacology. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-1     

Proton pump inhibitors: the beginning of the end or the end of the beginning?

Despite the dramatic success of pharmacological acid suppression in healing peptic ulcers (PUs) and managing patients with gastro-esophageal reflux disease (GERD) a number of challenges remain in the management of acid-related disorders. Several new drugs are currently being investigated to provide a significant advance over current treatments. These include new drug formulations, novel proton pump inhibitors (PPIs) as well as potassium-competitive acid blockers (P-CABs), which have already reached clinical testing. Some others (like NO-releasing antisecretory compounds) are still in preclinical development and require proof of concept in humans. While H(2)-receptor antagonists (especially soluble or OTC formulations) will become the 'antacids of the third millennium' and will be particularly useful for on-demand symptom relief, clinicians will continue to rely on PPIs to control acid secretion in GERD and other acid-related diseases. Since an increasing proportion of patients fail to respond to the best PPI treatment, more potent and long-acting drugs and more effective regimens are needed.     

Does the Number of Irradiated Cells Influence the Spatial Distribution of Bystander Effects?

There is growing evidence that the radiation effects at low doses are not adequately described by a simple linear extrapolation from high doses, due, among others, to bystander effects. Though several studies have been published on this topic, the explanation of the mechanisms describing the bystander effects remains unclear. This study aims at understanding how the bystander signals are or can be propagated in the cell culture, namely if the number of irradiated cells influences the bystander response. An A549 cell line was exposed to several doses of α-particles, being the bystander response quantified in two non-irradiated areas. The radius of irradiated areas differs by a factor of 2, and the non-irradiated areas were optimally designed to have the same number of cells. Our results show evidence for bystander effects occurring in cells far away from the irradiated ones, meaning that bystander signals can easily spread throughout the cell culture. Additionally, our study highlights that the damage caused by radiation on the surrounding of irradiated areas could be different according to the number of irradiated cells, i.e., for the same dose value; the overall cellular damage could be different.