The Na<Superscript>+</Superscript>/Cl<Superscript>−</Superscript>-Coupled,Broad-Specific,Amino Acid Transporter SLC6A14 (ATB<Superscript>0,+</Superscript>): Emerging Roles in Multiple Diseases and Therapeutic Potential for Treatment and Diagnosis

Amino acids are essential building blocks of all mammalian cells, and amino acid transporters play a vital role in transporting them into cells and their further distribution among the various cellular compartments. There are ~?430 known transporters in the solute-linked carrier (SLC) gene family, divided into 52 distinct families. Eleven of these gene families contain one or more amino acid transporters. These transporters differ significantly from each other in terms of substrate specificity, ion dependence, and energetics. Given the variety of roles they fulfill in human physiology, it is not surprising that a number of diseases are associated with the malfunction of these transporters. In particular, as amino acids are critical for cell growth, survival, and proliferation, the role of amino acid transporters in cancer is gaining increasing attention in recent years. The present review primarily focuses on one particular amino acid transporter, SLC6A14 (also known as ATB^0,+), with regard to its relevance to specific diseases, including cancer, and the molecular mechanisms underlying the disease-related alterations in the expression of the transporter. Furthermore, the review highlights the possible utility of this transporter in drug delivery and also its therapeutic potential for the treatment and diagnosis of cancer.     

Stimulatory effect of Coca-Cola<Superscript>TM</Superscript> on gastroduodenal HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> secretion in rats

We examined the effect of various carbonated beverages, especially Coca-Cola^TM, on the HCO₃⁻ secretion in the rat stomach and duodenum. Under urethane anaesthesia, a chambered stomach or a proximal duodenal loop was perfused with saline, and HCO₃⁻ secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The amount of CO₂ contained in these beverages was about 4–7 g/mL. Coca-Cola^TM topically applied to the mucosa for 10 min significantly increased the HCO₃⁻ secretion in both the stomach and the duodenum. The HCO₃⁻ response in the duodenum was totally abolished by indomethacin and also partially inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Likewise, the response in the stomach was also markedly inhibited by either acetazolamide or indomethacin. The mucosal application of Coca-Cola^TM increased the PGE₂ contents in both the stomach and the duodenum. Other carbonated beverages, such as sparkling water, Fanta Grape^TM or cider, also increased the HCO₃⁻ secretion in these tissues. These results suggest that Coca-Cola^TM induces HCO₃⁻ secretion in both the stomach and the duodenum, and these responses may be attributable to both the intracellular supply of HCO₃⁻ generated via carbonic anhydrase, and endogenous PGs, probably related to the acidic pH of the solution. Received 4 August 2006; accepted 10 November 2006     

Cellular mechanism for herbal medicine Junchoto to facilitate intestinal Cl<Superscript>−</Superscript>/water secretion that involves cAMP-dependent activation of CFTR

Constipation is a common symptom frequently compromising the quality of daily life. Several mechanistically different drugs have been used to mitigate constipation, including Japanese herbal (Kampo) medicines. However, the mechanisms of their actions are often not well understood. Here we aimed to investigate the molecular mechanisms underlying the effects of Junchoto (JCT), a Kampo medicine empirically prescribed for chronic constipation. Cl^? channel activity was measured by the patch-clamp method in human cystic fibrosis transmembrane conductance regulator (CFTR)-expressing HEK293T cells and human intestinal Caco-2 cells. cAMP was measured by a luciferase-based assay. Cell volume change was measured by a particle-sizing and particle-counting analyzer and video-microscopic measurement. In both CFTR-expressing HEK293T and Caco-2 cells, JCT dose-dependently induced whole-cell currents showing typical biophysical and pharmacological features of CFTR. Robust expression of CFTR was confirmed by RT-PCR and Western blotting in Caco-2 cells. Luciferase-based measurement revealed that JCT increases intracellular cAMP levels. Administration of the adenylate cyclase inhibitor SQ22536 or CFTR inhibitor-172, or treatment with small interfering RNAs (siRNA) targeting CFTR, abolished JCT-induced whole-cell currents, suggesting that elevated intracellular cAMP by JCT causes activation of CFTR in Caco-2 cells. Finally, blockade of CFTR activity by CFTR inhibitor-172 or siRNA-knockdown of CFTR or application of SQ22536 markedly reduced the degree of cell volume decrease induced by JCT. JCT can induce a Cl^? efflux through the CFTR channel to promote water secretion, and this effect is likely mediated by increased cAMP production.     

Cardioprotective effects of the novel Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger-1 inhibitor KR-32560 in a perfused rat heart model of global ischemia and reperfusion: Involvement of the Akt-GSK-3β cell survival pathway and antioxidant enzyme

To investigate the cardioprotective effects and mechanism of action of KR-32560 {[5-(2-methoxy-5-fluorophenyl)furan-2-ylcarbonyl]guanidine}, a newly synthesized NHE-1 inhibitor, we evaluated the effects of KR-32560 on cardiac function in a rat model of ischemia/reperfusion (I/R)-induced heart injury as well as the role antioxidant enzymes and pro-survival proteins play these observed effects. In isolated rat hearts subjected to 25 min of global ischemia followed by 30 min of reperfusion, KR-32560 (3 and 10 μM) significantly reversed the I/Rinduced decrease in left ventricular developed pressure and increase in left ventricular enddiastolic pressure. In rat hearts reperfused for 30 min, KR-32560 (10 μM) significantly decreased the malondialdehyde content while increasing the activities of both glutathione peroxidase and catalase, two important antioxidant enzymes. Western blotting analysis of left ventricles subjected to I/R showed that KR-32560 significantly increased phosphorylation of both Akt and GSK-3β in a dose-dependent manner, with no effect on the phosphorylation of eNOS. These results suggest that KR-32560 exerts potent cardioprotective effects against I/Rinduced rat heart injury and that its mechanism involves antioxidant enzymes and the Akt-GSK-3β cell survival pathway.     

Unique excitation–contraction characteristics of mouse myocardium as revealed by SEA0400, a specific inhibitor of Na<Superscript>+</Superscript>–Ca<Superscript>2+</Superscript> exchanger

The functional role of the sodium–calcium exchanger in mouse ventricular myocardium was evaluated with a newly developed specific inhibitor, SEA0400. Contractile force and action potential configuration were measured in isolated ventricular tissue preparations, and cell shortening and Ca²⁺ transients were measured in indo-1-loaded isolated ventricular cardiomyocytes. SEA0400 increased the contractile force, cell shortening and Ca²⁺ transient amplitude, and shortened the late plateau phase of the action potential. -adrenergic stimulation by phenylephrine produced a sustained decrease in contractile force, cell shortening and Ca²⁺ transient amplitude, which were all inhibited by SEA0400. Increasing the contraction frequency resulted in a decrease in contractile force in the absence of drugs (negative staircase phenomenon). This frequency-dependent decrease was attenuated by SEA0400 and enhanced by phenylephrine. Phenylephrine increased the Ca²⁺ sensitivity of contractile proteins in isolated ventricular cardiomyocytes, while SEA0400 had no effect. These results provide the first pharmacological evidence in the mouse ventricular myocardium that inward current generated by Ca²⁺ extrusion through the sodium–calcium exchanger during the Ca²⁺ transient contributes to the action potential late plateau, that -adrenoceptor-mediated negative inotropy is produced by enhanced Ca²⁺ extrusion through the sodium–calcium exchanger, and that the negative staircase phenomenon can be explained by increased Ca²⁺ extrusion through the sodium–calcium exchanger at higher contraction frequencies.     

Cross-talk between β<Subscript>1</Subscript>-adrenoceptors and ET<Subscript>A</Subscript> receptors in modulation of the slow component of delayed rectifier K<Superscript>+</Superscript> currents

Delayed rectifier K⁺ currents (I_K) play a critical role in determining cardiac action potential duration (APD). Modulation of I_K affects cardiac excitability critically. There are three components of cardiac delayed rectifier, and the slowly activating component (I_Ks) is influenced strongly by a variety of stimuli. Plasma levels of noradrenaline and endothelin are elevated in heart failure, and arrhythmias are promoted by such humoral abnormalities through modulation of ion channels. It has been reported that protein kinase A (PKA) and protein kinase C (PKC) modulate I_Ks from human minK in a complex manner. In the present study, we coexpressed human minK with the human ₁-adrenoceptor (h₁AR) and the endothelin receptor subtype A (hET_AR) in Xenopus oocytes and investigated the effects of receptor activation on the currents (I_Ks) flowing through the oocytes. ET-1 modulated I_Ks biphasically: a transient increase followed by a decrease. The PKC inhibitor chelerythrine completely inhibited the effects of ET-1. Intracellular EGTA abolished the transient increase by ET-1 and partially inhibited the subsequent decrease in the currents. When I_Ks was increased by 10^–6 M isoproterenol (ISO), ET-1 did not increase but rather decreased the current to an even greater extent than under control conditions. In addition, the effects of ISO on I_Ks were suppressed by ET_AR stimulation. These data indicate that I_Ks can be regulated by cross-talk between the ET_AR and ₁AR systems in addition to direct regulation by each receptor system.     

Comparison of smoked marijuana and oral Δ<Superscript>9</Superscript>-tetrahydrocannabinol in humans

Abstract Rationale. Although smoked marijuana contains at least 60 cannabinoids, Δ⁹-tetrahydrocannabinol (Δ⁹-THC) is presumed to be the cannabinoid primarily responsible for many marijuana-related effects, including increased food intake and subjective effects. Yet, there has been no systematic comparison of repeated doses of oral Δ⁹-THC with repeated doses of smoked marijuana in the same individuals. Objective. To compare the effects of oral Δ⁹-THC and smoked marijuana in humans under controlled laboratory conditions. Methods. Eleven healthy research volunteers, who reported smoking an average of six marijuana cigarettes per day, completed an 18-day residential study. Marijuana cigarettes (3.1% Δ⁹-THC, q.i.d.) were smoked or Δ⁹-THC (20 mg, q.i.d.) was taken orally using a staggered, double-blind, double-dummy procedure for three consecutive days. Four days of placebo administration separated each active drug condition. Psychomotor task performance, subjective effects, and food intake were measured throughout the day. Results. Relative to placebo baseline, oral Δ⁹-THC and smoked marijuana produced similar subjective-effect ratings (e.g., "high" and "mellow"), although some effects of smoked marijuana were more pronounced and less prone to the development of tolerance. Additionally, participants reported "negative" subjective effects (e.g., "irritable" and "miserable") during the days after smoking marijuana but not after oral Δ⁹-THC. Both drugs increased food intake for 3 days of drug administration, but had little effect on psychomotor performance. Conclusion. These results indicate that the behavioral profile of effects of smoked marijuana (3.1% Δ⁹-THC) is similar to the effects of oral Δ⁹-THC (20 mg), with some subtle differences. Electronic Publication     

Chronic Δ<Superscript>9</Superscript>-Tetrahydrocannabinol Administration Reduces IgE<Superscript>+</Superscript>B Cells but Unlikely Enhances Pathogenic SIV<Subscript>mac251</Subscript> Infection in Male Rhesus Macaques of Chinese Origin

Delta9-tetrahydrocannabinol (Δ⁹-THC) is the major psychoactive component of the cannabis plant. Δ⁹-THC has been used in the active ingredient of Marinol as an appetite stimulant for AIDS patients. Its impact on progression of HIV-1 infection, however, remains debatable. Previous studies indicated that Δ⁹-THC administration enhanced HIV-1 infection in huPBL-SCID mice but seemingly decreased early mortality in simian immunodeficiency virus (SIV) infected male Indian-derived rhesus macaques. Here, we determine the chronic effect of Δ⁹-THC administration using 0.32 mg/kg or placebo (PBO), i.m., twice daily for 428 days on SIV_mac251 infected male Chinese-derived rhesus macaques. Sixteen animals were divided into four study groups: Δ⁹-THC⁺SIV⁺, Δ⁹-THC⁺SIV^?, PBO/SIV⁺ and PBO/SIV^? (n = 4/group). One-month after daily Δ⁹-THC or PBO administrations, macaques in groups one and three were challenged intravenously with pathogenic SIV_mac251/CNS, which was isolated from the brain of a Chinese macaque with end-staged neuroAIDS. No significant differences in peak and steady state plasma viral loads were seen between Δ⁹-THC⁺SIV⁺ and PBO/SIV⁺ macaques. Regardless of Δ⁹-THC, all infected macaques displayed significant drop of CD4/CD8 T cell ratio, loss of CD4⁺ T cells and higher persistent levels of Ki67⁺CD8⁺ T cells compared with uninfected animals. Moreover, long-term Δ⁹-THC treatment reduced significantly the frequency of circulating IgE⁺B cells. Only one Δ⁹-THC⁺SIV⁺ macaque died of simian AIDS with paralyzed limbs compared with two deaths in the PBO/SIV⁺ group during the study period. These findings indicate that chronic Δ⁹-THC administration resulted in reduction of IgE⁺B cells, yet it unlikely enhanced pathogenic SIV_mac251/CNS infection in male Rhesus macaques of Chinese origin.     

In vivo and ex vivo percutaneous absorption of [<Superscript>14</Superscript>C]-bisphenol A in rats: a possible extrapolation to human absorption?

Bisphenol A (BPA) is a monomer used mainly in the synthesis of polycarbonates and epoxy resins. Percutaneous absorption is the second source of exposure, after inhalation, in the work environment. However, studies on this route of absorption are lacking or incomplete. In this study, percutaneous BPA absorption was measured in vivo and ex vivo in the rat, and ex vivo in humans. An approximately 12-fold difference in permeability between rat skin and human skin was found, with permeability being higher in the rat. In addition, inter- and intra-individual variability of up to tenfold was observed in humans. No accumulation of BPA in the skin was found during exposure. The skin clearance rate following exposure was estimated at 0.4 μg/cm2/h. Ex vivo and in vivo percutaneous absorption fluxes of BPA in the rat were in the same range (about 2.0 μg/cm2/h), suggesting that extrapolation to the in vivo situation in humans may be possible. The European tolerable daily intake (TDI) of BPA is 50 μg/kg body weight. However, many research projects have highlighted the significant effects of BPA in rodents at doses lower than 10 μg/kg/day. A 1-h occupational exposure over 2,000 cm2 (forearms and hands) may lead to a BPA absorption of 4 μg/kg/day. This is 8% of the European TDI and is very close to the value at which effects have been observed in animals. This absorption must therefore be taken into account when evaluating risks of BPA exposure, at least until more relevant results on the toxicity of BPA in humans are available.     

Pharmacological evidence that tetraethylammonium-sensitive,iberiotoxin-insensitive K<Superscript>+</Superscript> channels function as a negative feedback element for sympathetic neurotransmission by suppressing ω-conotoxin-GVIA-insensitive Ca<Superscript>2+</Superscript> channels in the relaxation of rabbit facial vein

The facial vein isolated from various species relaxes in response to electrical field stimulation (EFS). EFS-elicited relaxation of the facial vein is mediated through the release of noradrenaline (NA) from sympathetic nerve endings and the subsequent activation of smooth muscle beta-adrenoceptors. The release of NA from sympathetic nerve endings in arterial tissues requires transmembrane Ca2+ influx, mediated predominantly by voltage-gated N-type Ca2+ channels. The present pharmaco-mechanical study was undertaken to determine whether the N-type channel is the exclusive pre-junctional Ca2+ channel mediating NA release from sympathetic nerve endings in the rabbit facial vein. Possible roles of K+ channels in the sympathetic neurotransmission were also examined, especially focusing on the contribution of voltage-dependent, Ca2+-activated K+ (BKCa) channels. An isolated ring preparation of the rabbit facial vein exhibited intrinsic myogenic tone which lasted for several hours when stretched. EFS produced frequency-dependent (0.25-2 Hz) relaxation in this preparation. EFS-elicited relaxation was abolished by tetrodotoxin (TTX, 1 microM), guanethidine (5 microM) or propranolol (1 microM), indicating that NA released from sympathetic nerve endings was mediating the relaxant response. NA-mediated neurogenic relaxation was almost eliminated by omega-conotoxin-GVIA (1 microM), an N-type Ca2+ channel blocker. On the other hand, tetraethylammonium (TEA, 2 mM) strongly potentiated EFS-elicited relaxation without affecting the relaxation induced by exogenously applied NA. This potentiation by TEA was not profoundly diminished by omega-conotoxin-GVIA (1 microM) alone or omega-conotoxin-GVIA (1 microM) plus omega-agatoxin IVA (10 nM, P-type channel blocker), but was almost abolished by omega-conotoxin-GVIA (1 microM) plus omega-agatoxin IVA (10 nM) plus omega-conotoxin-MVIIC (3 microM, N-, P- and Q-type channel blocker). The potentiating effect of TEA was not mimicked by iberiotoxin (100 nM) or charybdotoxin (3 microM), both of which block BKCa channels. These findings suggest that pre-junctional N-type Ca2+ channels play the predominant role in the sympathetic nerve transmission in the rabbit facial vein, as in peripheral arterial vascular beds. In addition, Ca2+ channels resistant to 1 microM omega-conotoxin-GVIA, most probably Q-type channels, appear to be present at the sympathetic nerve endings in the rabbit facial vein and contribute substantially to the regulation of NA release from the nerve endings. Prejunctional K+ channels, sensitive to TEA but pharmacologically distinct from iberiotoxin-sensitive BKCa channels, seem to be functionally coupled intimately with the omega-conotoxin-GVIA-resistant Ca2+ channels, and thus function as a negative feedback element in sympathetic neurotransmission in the rabbit facial vein.     

Comparison of the subjective effects of Δ<Superscript>9</Superscript>-tetrahydrocannabinol and marijuana in humans

RATIONALE: There has been controversy about whether the subjective, behavioral or therapeutic effects of whole plant marijuana differ from the effects of its primary active ingredient, Delta(9)-tetrahydrocannabinol (THC). However, few studies have directly compared the effects of marijuana and THC using matched doses administered either by the smoked or the oral form.OBJECTIVE: Two studies were conducted to compare the subjective effects of pure THC to whole-plant marijuana containing an equivalent amount of THC in normal healthy volunteers. In one study the drugs were administered orally and in the other they were administered by smoking.METHODS: In each study, marijuana users (oral study: n=12, smoking study: n=13) participated in a double-blind, crossover design with five experimental conditions: a low and a high dose of THC-only, a low and a high dose of whole-plant marijuana, and placebo. In the oral study, the drugs were administered in brownies, in the smoking study the drugs were smoked. Dependent measures included the Addiction Research Center Inventory, the Profile of Mood States, visual analog items, vital signs, and plasma levels of THC and 11-nor-9-carboxy-THC.RESULTS: In both studies, the active drug conditions resulted in dose-dependent increases in plasma THC levels, and the levels of THC were similar in THC-only and marijuana conditions (except that at the higher oral dose THC-only produced slightly higher levels than marijuana). In both the oral study and the smoking study, THC-only and whole plant marijuana produced similar subjective effects, with only minor differences.CONCLUSION: These results support the idea that the psychoactive effects of marijuana in healthy volunteers are due primarily to THC.     

A comparative autoradiographic study of the density of [<Superscript>3</Superscript>H]SR95531, [<Superscript>3</Superscript>H]MK-801 and [<Superscript>3</Superscript>H]cGMP binding in the locus coeruleus and central pontine grey of spontaneously hypertensive and Wistar–Kyoto rats

The Spontaneously Hypertensive rat (SHR) has been previously shown to have a host of neurochemical differences compared with their normotensive counterpart, the Wistar–Kyoto (WKY) rat. Using quantitative receptor autoradiography, the density of GABA_A and NMDA receptors and [³H]cGMP binding within the locus coeruleus (LC) and central pontine grey (CGPn) were compared in the SHR and WKY rat using the radioligands [³H]SR95531, [³H]MK-801 and [³H]cGMP respectively. It was found that [³H]SR95531 binding was significantly greater in both the LC and CGPn of the SHR compared with the WKY rat (unpaired t test; P<0.05). Greater binding densities of [³H]MK-801 and [³H]cGMP were also observed in the LC of the SHR compared with the WKY rat; however, no differences in the binding density of these two ligands were observed in the CGPn. It is suggested that these neurochemical differences within the LC of the SHR may relate to phenotypic differences between SHR and WKY rats that have previously been reported.     

Interactions between Δ<Superscript>9</Superscript>-tetrahydrocannabinol and <Emphasis Type="Bold">μ</Emphasis> opioid receptor agonists in rhesus monkeys: discrimination and antinociception

RATIONALE: Opioid receptor agonists can enhance some effects of cannabinoid receptor agonists, and cannabinoid receptor agonists can enhance some effects of opioid receptor agonists; however, the generality of these interactions is not established. OBJECTIVE: This study examined interactions between the discriminative stimulus and antinociceptive effects of mu opioid receptor agonists and Delta(9)-tetrahydrocannabinol (THC) in rhesus monkeys. RESULTS: Neither heroin nor morphine (intravenous (i.v.) or subcutaneous (s.c.)) altered the discriminative stimulus effects of THC in monkeys (n = 5) discriminating 0.1 mg/kg THC i.v. In contrast, THC (s.c.) markedly attenuated the discriminative stimulus effect of morphine and heroin in nondependent monkeys (n = 4) discriminating 1.78 mg/kg morphine s.c. Doses of THC that attenuated the discriminative stimulus effects of morphine in nondependent monkeys failed to modify the discriminative stimulus effects of morphine in morphine-dependent (5.6 mg/kg/12 h) monkeys (n = 4) discriminating 0.0178 mg/kg naltrexone s.c. THC also failed to modify the discriminative stimulus effects of naltrexone in morphine-dependent monkeys or the effects of midazolam in monkeys (n = 4) discriminating 0.32 mg/kg midazolam s.c. Doses of THC (s.c.) that attenuated the discriminative stimulus effects of morphine in nondependent monkeys enhanced the antinociceptive effects of morphine (s.c.) in nondependent monkeys. While mu receptor agonists did not alter the discriminative stimulus effects of THC, THC altered the effects of mu receptor agonists in a context-dependent manner. CONCLUSION: That the same doses of THC enhance, attenuate, or do not affect morphine, depending on the condition, suggests that attenuation of morphine by THC can result from perceptual masking rather than common pharmacodynamic mechanisms or pharmacokinetic interactions.     

Effects of Δ<Superscript>9</Superscript>-THC and WIN-55,212-2 on place preference in the water maze in rats

Abstract Rationale. Cannabinoids such as Δ⁹-tetrahydrocannabinol (Δ⁹-THC) or WIN-55,212–2 (WIN-2) have psychoactive effects on cognition. As a result, the reinforcing properties of Δ⁹-THC or WIN-2 may confound learning and memory tests with false negative results. It therefore seems advisable to assess the reinforcing properties of the drugs in the same behavioural model used for learning experiments. Objective. We therefore developed conditioned place preference protocols in the open-field water maze and tested both Δ⁹-THC (2 mg/kg) and WIN-2 (1 mg/kg and 3 mg/kg). Given that previous reports on cannabinoids have revealed conflicting data and that this was a novel behavioural test, we also tested the benzodiazepine receptor agonist diazepam (2.5 mg/kg). Some methodical refinements were appropriate in order to determine the behavioural strategy implemented by the animals. Methods. All animals were injected intraperitoneally 30 min prior to training/testing. In experiment 1, male hooded Lister rats injected with drug were repeatedly placed on the drug-related platform and subsequently tested for place preference. In experiment 2, rats were trained to swim to the drug platform on drug days and to the vehicle platform on vehicle days. A series of probe trials was introduced to delineate what had been learned. Experiment 3 studied the effect of WIN-2 on spatial learning in the water maze. Results. Neither WIN-2 nor Δ⁹-THC induced place preference in the water maze. When trained in the swim procedure, however, WIN-2 was neutral, but Δ⁹-THC resulted in place aversion. Conversely, diazepam consistently produced place preference in both procedures. WIN-2 (3 mg/kg), however, produced a small learning deficit in the spatial water maze task. Conclusion. It appears that the reinforcing properties of Δ⁹-THC and WIN-2 in the doses used here are different, despite them both being agonists at cannabinoid receptors within the central nervous system. The fact that Δ⁹-THC may be aversively related to a particular context has implications for previous work reporting deficits in spatial learning. Electronic Publication     

Binding of [<Superscript>3</Superscript>H]prazosin to α<Subscript>1A</Subscript>- and α<Subscript>1B</Subscript>-adrenoceptors,and to a cimetidine-sensitive non-α<Subscript>1</Subscript> binding site in rat kidney membranes

[(3)H]Prazosin bound to alpha(1A)- and alpha(1B)-adrenoceptors, as well as to a cimetidine-sensitive non-alpha(1)-adrenoceptor binding site in rat kidney membranes. An experimental design is presented where the alpha(1)-adrenoceptors are selectively exposed by blocking the non-alpha(1) binding site with 60 microM cimetidine. Conversely, the non-alpha(1) binding site can be selectively exposed by blocking the alpha(1)-adrenoceptors with 600 nM metitepine. The identity of the non-alpha(1) binding site for [(3)H]prazosin in the rat kidney, herein pharmacologically characterized by 33 competing substances, is still unknown.     

Cardiomyocyte Ca<Superscript>2+</Superscript> overload in atrial tachycardia: is blockade of L-type Ca<Superscript>2+</Superscript> channels a promising approach to prevent electrical remodeling and arrhythmogenesis?

Electrical remodeling paradigm has important implications for the understanding of atrial fibrillation (AF) and improvement of current treatment. Cardiomyocyte Ca²⁺ overload is generally accepted as the initiating signal for the tachycardia-induced changes in atrial electrical properties (electrical remodeling). The precise role of cardiomyocyte Ca²⁺ overload in AF-related ion channel alterations that contribute to AF maintenance is not fully understood. Clinically, patients with AF are often treated with Ca²⁺ channel blockers such as verapamil to control their ventricular rate and to improve the success rate of cardioversion procedures. However, verapamil may produce an increased L-type Ca²⁺ channel current (I_Ca,L) that may reinforce Ca²⁺ overload thereby promoting AF in the atrium. Ca²⁺ channel blockers which target T-type Ca²⁺ channels in addition to I_Ca,L (for instance, efonidipine) may be more efficient at preventing Ca²⁺ overload and arrhythmogenic electrical remodeling, but the potential benefits of these drugs have usually been tested in experimental models where drug administration preceded the initiation of electrical remodeling. Studies in animal models with established atrial tachycardia remodeling and in patients with AF are clearly warranted to prove the efficacy of Ca²⁺ channel blockers that additionally target T-type Ca²⁺ channels.     

Binding of (−)-[<Superscript>3</Superscript>H]-CGP12177 at two sites in recombinant human β<Subscript>1</Subscript>-adrenoceptors and interaction with β-blockers

To verify the hypothesis that the non-conventional partial agonist (–)-CGP12177 binds at two ₁-adrenoceptor sites, human ₁-adrenoceptors, expressed in CHO cells, were labelled with (–)-[³H]-CGP12177. We compared the binding affinity and antagonist potency of 12 clinically used -blockers against the cyclic AMP-enhancing effects of (–)-isoprenaline and (–)-CGP12177.(–)-[³H]-CGP12177 bound to a high affinity site (H; K_H=0.47 nM) and low affinity site (L); K_L=235 nM). (–)-[³H]-CGP12177 dissociated from the ₁-adrenoceptors with a fast component (k_off=0.45 min^–1), consistent with the L-site, and a slow component (k_off=0.017–0.033 min^–1), consistent with the H-site. (–)-Isoprenaline and (–)-CGP12177 caused 96-fold and 12-fold maximal increases in cyclic AMP levels with –logEC₅₀M of 8.2 and 7.6. (–)-CGP12177 antagonised the effects of (–)-isoprenaline with a pK_B of 9.9. The -blockers antagonised the effects of (–)-isoprenaline more than the effects of (–)-CGP12177 with potency ratios: (–)-atenolol 1,000, (±)-metropolol 676, (–)-pindolol 631, (–)-timolol 589, (±)-carvedilol 204, (±)-oxprenolol 138, (±)-sotalol 132, (–)-propranolol 120, (±)-bisoprolol 95, (±)-alprenolol 81, (±)-nadolol 68 and (–)-bupranolol 56. In intact cells the binding constants of -blockers, estimated from competition with 3–5 nM (–)-[³H]-CGP12177 (binding to the H-site), correlated with the corresponding affinities estimated from antagonism of the (–)-isoprenaline effects.We conclude that (–)-[³H]-CGP12177 binds at two sites in the recombinant ₁-adrenoceptor. (–)-CGP12177 is an antagonist of catecholamine effects through the H-site and a non-conventional partial agonist through the L-site. -blockers are more potent antagonists through the H-site than the L-site.     

HTLV-1 Infection and Neuropathogenesis in the Context of Rag1<Superscript>-/-</Superscript>γc<Superscript>-/-</Superscript> (RAG1-Hu) and BLT Mice

To date, the lack of a suitable small animal model has hindered our understanding of Human T-cell lymphotropic virus (HTLV)-1 chronic infection and associated neuropathogenesis defined as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The host immune response plays a critical role in the outcome of HTLV-1 infection, which could be better tested in the context of humanized (hu) mice. Thus, we employ here the Balb/c-Rag1^?/?γc^?/? or Rag1 as well as Bone marrow-Liver-Thymic (BLT) mouse models for engraftment of human CD34⁺ hematopoietic stem cells. Flow cytometry and histological analyses confirmed reconstitution of Rag1 and BLT mice with human immune cells. Following HTLV-1 infection, proviral load (PVL) was detected in the blood of Rag-1 and BLT hu-mice as early as 2 weeks post-infection (wpi) with sustained elevation in the subsequent weeks followed by Tax expression. Additionally, infection was compared between adult and neonatal Rag1 mice with both PVL and Tax expression considerably higher in the adult Rag1 mice as compared to the neonates. Establishment of peripheral infection led to lymphocytic infiltration with concomitant Tax expression and resulting myelin disruption within the central nervous system of infected mice. In addition, up-regulation in the expression of several immune checkpoint mediators such as programmed cell death-1 (PD-1), T-cell Ig and ITIM domain (TIGIT), and T cell Ig and mucin domain-3 protein (Tim-3) were observed on CD8⁺ T cells in various organs including the CNS of infected hu-mice. Collectively, these studies represent the first attempt to establish HTLV-1 neuropathogenesis in the context of Rag-1 and BLT hu-mice as potential novel tools for understanding HTLV-1 neuropathogenesis and testing of novel therapies such as immune checkpoint blockade in the amelioration of chronic HTLV-1 infection.     

Insulin Lung Deposition and Clearance Following Technosphere<Superscript>®</Superscript> Insulin Inhalation Powder Administration

Purpose

To determine distribution and deposition of Technosphere^® Insulin (TI) inhalation powder and the rate of clearance of fumaryl diketopiperazine (FDKP; major component of Technosphere particles) and insulin from the lungs.

Methods

Deposition and distribution of ^99mpertechnetate adsorbed onto TI immediately after administration using the MedTone® inhaler was quantified by gamma-scintigraphy. Clearance from the lungs was studied in a second experiment by serial bronchoalveolar lavage (BAL) after administration of TI inhalation powder and assay of the recovered fluid for FDKP and insulin.

Results

Following inhalation, ~60% of radioactivity (adsorbed on TI) emitted from the inhaler was delivered to the lungs; the remainder of the emitted dose was swallowed. Clearance from the lung epithelial lining fluid (ELF) of FDKP and insulin have a half-life of ~1 hour.

Conclusion

TI inhalation powder administered via the MedTone inhaler was uniformly distributed throughout the lungs; ~40% of the initial cartridge load reached the lungs. Insulin and FDKP are quickly cleared from the lungs, mainly by absorption into the systemic circulation. The terminal clearance half-life from the lung ELF, estimated from sequential BAL fluid measurements for both components, was ~1 hour. Since there is an overnight washout period, the potential for accumulation on chronic administration is minimal.