Computer-assisted molecular modeling of benzodiazepine and thyromimetic inhibitors of the HepG2 iodothyronine membrane transporter.

T3 cellular uptake is inhibited in the presence of benzodiazepines (BZs). The structure-activity relationship of BZ inhibition correlates strongly with halogen substitution of the nonfused phenyl ring and indicates that this ring is required for activity. A structure-activity series of thyromimetic (TH) inhibitors of the HepG2 iodothyronine transporter further point out the critical importance of the amino group of the alanine side chain, its L-stereo configuration, and the size of the substituents of the inner and outer phenyl rings. A third series of compounds, reported to interact at related sites, were inactive as HepG2 iodothyronine transport inhibitors, and therefore the potent inhibitors were restricted to the BZ and TH compounds. Using both of these BZ and TH structure-activity series along with computer-assisted molecular modeling techniques, we determined which chemical structural components were important at the transporter interaction site. By superimposing structures from active chemicals, excluding residues from poor inhibitors, and incorporating molecular electropotential data, we developed a five-point model of BZ conformational similarity to the endogenous transporter ligand, L-T3: the alkyl substitution at the N1 of the BZ ring seems to simulate the alanine side chain of T3, and the electro-negative halogen and oxygen atoms of substituents at R3/R7/R2'/R4' of BZ form a pyramidal pharmacophore that seems to correspond with the 3-l/5-l/3'-l/4'-OH substituents of T3, respectively. These points, suggesting a tilted cross-bow formation, may be sites for ligand interaction with the iodothyronine transporter.

Benzodiazepines inhibit temperature-dependent L-[125I]triiodothyronine accumulation into human liver, human neuroblast, and rat pituitary cell lines.

L-T3 (T3) accumulates into cells in a temperature-dependent saturable manner through a purported iodothyronine membrane carrier protein. We report energy-dependent uptake of picomolar [125I]T3 into differentiated cell lines derived from human liver, human neuroblast, and rat pituitary malignancies. Furthermore, this cellular uptake is inhibited by classical and nonclassical benzodiazepine-type drugs (BZs); the apparent half-maximal inhibitory concentrations range from 50 nM to 50 microM, varying with drug and cell type. The site of this T3-BZ interaction was explored with cross-competitive radioligand binding to rat liver cell fractions. No interaction was seen in experiments cross-competing unlabeled T3 (10(-9)-10(-5) M) against [3H]Ro5 4864, a peripheral BZ receptor ligand, for binding sites in a crude rat liver mitochondrial fraction. As well, lormetazepam and triazolam, BZs that potently inhibit cellular uptake of [125I]T3, have no effect on [125I]T3 binding to rat liver nuclear sites. Studies of [3H]diazepam and [3H]Ro5 4864 show very little temperature-dependent uptake into HepG2 cells (less than 0.5% over 90 min) and no effect from coincubation of unlabeled T3 (1 microM). Thus, the possibility that BZs are substrates for the T3 carrier protein and are causing the reduced cellular hormonal accumulation via competitive uptake and dilution of the radiolabeled cellular T3 is unlikely. In summary, 1) drugs from the BZ class inhibit high affinity temperature-dependent cellular accumulation of thyroid hormone into cell lines from rat and human species; 2) the site of action of BZ inhibition does not involve direct antagonism of the T3 nuclear receptor, nor is it likely that the peripheral BZ receptor is the iodothyronine carrier. BZs could be interacting with the purported iodothyronine carrier protein itself to block uptake.

Sex differences in plasma cocaine levels and subjective effects after acute cocaine administration in human volunteers.

Gender differences after acute cocaine administration have received little attention in spite of the fact that males and females respond differently to many drugs. Seven male and seven female occasional cocaine users received both an intranasal dose of cocaine hydrochloride (0.9 mg/kg) and placebo powder in a randomized order and reported subjective effects via an instrumental joystick device and various questionnaires. Blood samples were withdrawn at 5-min intervals to assess pharmacokinetic differences. Male subjects achieved the highest peak plasma cocaine levels (144.4 +/- 17.5 ng/ml), detected cocaine effects significantly faster than females and also experienced a greater number of episodes of intense good and bad effects. Women studied during the follicular phase of their menstrual cycle had peak plasma cocaine levels of 73.2 +/- 9.9 ng/ml, which was significantly higher than when they were studied during their luteal phase (54.7 +/- 8.7 ng/ml), but there were no differences in their subjective reports of cocaine effects. In spite of the different cocaine blood levels and subjective effects, peak heart rate increases did not differ between males and females suggesting that women may be more sensitive than males to the cardiovascular effects of cocaine. These data suggest that there are significant gender and menstrual cycle differences in the response to acute intranasal cocaine administration and these differences may have implications for the differential abuse of this drug.

Theophylline--an alternative therapy for bradyarrhythmia in the elderly.

Treating elderly debilitated patients with symptomatic bradyarrhythmia may be difficult. Traditional therapy includes use of limited resources (intensive care unit monitoring) and expensive interventions (permanent pacemaker insertion). When a pacemaker is not ideal or appropriate, yet intervention is desired to improve quality of life, theophylline may be a safe and effective alternative therapy. We expanded this use of theophylline in a series of 11 patients admitted 14 consecutive times with hemodynamically compromising bradyarrhythmias, including atropine-resistant complete heart block with idioventricular rhythm. Three patients experienced deterioration of status and recurrence of bradycardia with the withdrawal or reduction of theophylline, and subsequent recovery of heart rate and conduction with its resumption.

Requisite structural characteristics for benzodiazepine inhibition of triiodothyronine uptake into a human liver cell line.

Previously, we reported potent inhibition of triiodo-L-thyronine (T3) cellular uptake into a human liver cell line (HepG2) by central and peripheral receptor specific benzodiazepine (BZ) compounds and our working hypothesis of BZ's as direct competitors for the iodothyronine transporter, displacing T3 but not acting as a substrate for transport. In this report, we list other reported uptake inhibitors and compare them to 23 benzodiazepine receptor ligands, in their potency to inhibit cellular uptake of T3. The most potent inhibitors are restricted to the benzodiazepine class. From the BZ structure-activity relationship (SAR) for inhibition, we see that the nonfused phenyl ring may be essential for activity and the strongest relationship is seen with substitution at R2' where Cl greater than F greater than H. Substitution at R4' and hydroxyl substitution at R3 enhances potency as will alkyl groups at R1 or on the imidazole group in the 1,2-annelated series. With R7 substitution, Cl is preferred over NO2 but not necessarily H when R4' = Cl; this may reflect a slightly different orientation of the molecule with large aliphatic R1 groups and/or R4' substitution. The carbonyl at R2 in the 1,4 benzodiazepine series, enhances their potency. The resultant structure-activity relationship highlights the importance of the halogen-substituted nonfused phenyl ring and seems unique relative to other described benzodiazepine sites and/or effects.

Altered peripheral benzodiazepine receptor binding in cardiac and liver tissues from thyroidectomized rats.

We measured the density and affinity of the peripheral benzodiazepine receptor (PBR) ligand, [3H]4'-Cl-diazepam, in cardiac ventricular and liver homogenates from thyroidectomized (TX) Holtzman adult male rats, and compared these data to sham-operated controls. When data from hypothyroid tissues were compared to those of controls, the densities of PBRs were decreased in cardiac ventricles but not in liver tissue. This reduction in cardiac PBR density is opposite to what has been reported for ventricular calcium channel density in hypothyroidism. PBR affinity for the ligand was increased in both the liver and ventricular homogenates from the hypothyroid tissues, but this difference was not seen in membranes prepared from the liver homogenates. Although 4'-Cl-diazepam affinity is reported to vary between tissues from different species, this is the first report of an in vivo hormone treatment induced change in the benzodiazepine type PBR affinity. Liver tissues from both groups failed to show any interaction when radiolabeled [3H]4'-Cl-diazepam was tested against competing concentrations of thyroid hormone analogs.

Measures of skeletal muscle calcium channels and acetylcholine receptors in thyroidectomized rats.

Hypothyroidism frequently presents with muscle complaints. No consistent histopathology nor electrophysiology explains these symptoms and signs. As well, no previous study shows specific changes in components of the nerve-muscle synapse nor excitation-contraction coupling in adult muscles, but changes are seen in hormone-treated embryonic myoblasts. In this study, adult male Holtzman rats underwent thyroidectomy and their age-matched euthyroid controls were simultaneously subjected to sham operation. Thirty days post-operative, animals were sacrificed for anterior tibialis muscles harvest. Muscle dihydropyridine type calcium channel (isradipine) and acetylcholine receptor (alpha-bungarotoxin) binding were measured and compared between experimental treatment groups. There were no significant differences in either the affinity or density of isradipine binding. However, hypothyroid muscles showed a nearly 50% reduction in acetylcholine receptor density when compared to control muscles. Thyroidectomy is associated with specific effects on components of neuromuscular transmission in adult fast twitch muscle.

Acetylcholinesterase activity in fast-twitch skeletal muscle from thyroidectomized rats.

Hypothyroidism is associated with muscle weakness and slowed movements. Hormone-deficient muscles do show changes in contractile proteins and sarcoplasmic calcium pumps, however effects on the components of the neuromuscular junction are less clear. In this study, we examined cholinesterase activity in adult fast-twitch muscle from hypothyroid and control rats. Male Holtzman rats underwent thyroidectomy and their age-matched euthyroid controls were simultaneously subjected to sham operation. Thirty days post-operative, animals were sacrificed for anterior tibialis muscles harvest. Muscle cholinesterase isoform activity was measured and compared between experimental treatment groups. Butyrylcholinesterase activities and peak I and III acetylcholinesterase (AchE) activities were similar in euthyroid and hypothyroid groups. However, hypothyroid muscles exhibited half the peak II AchE isoform (G4) activity, as compared to control muscles. Hypothyroidism specifically affects AchE isoform expression in rodent fast-twitch muscle.

Stoichiometry and apparent dissociation constant of the calcium-arsenazo III reaction under physiological conditions.

In vitro and in situ tests have been run to characterize the reaction of the mettalochromic indicator, arsenazo III, with calcium. Job plots as well as plots of indicator absorbance vs. [Ca2+] at different indicator concentrations show a 1:1 reaction stoichiometry. Equilibrium analysis and analysis using Adair's equation are also consistent with 1:1 complexes being formed and give estimates of 34 and 45 muM for the apparent dissociation constant. In situ tests were carried out using giant neurons from Archidoris monteryensis, a marine gastropod mollusc. Dye absorbance changes were measured during voltage clamp pulses which produced a fixed calcium influx. The dependence of absorbance change on total dye concentration is consistent with the formation of a 1:1 complex of Ca with ArIII if measurements are made during the initial period of the loading pulse, less than 300 ms, although the apparent dependency changes with longer delay in measurements from the onset of the pulse.

Rat cardiac calcium channels and their relationships with beta-adrenergic and muscarinic receptors in hypothyroidism.

Thyroid hormone deficiency is associated with changes in the cardiovascular system. No one has reported the measures of both atrial and ventricular calcium channel density and function, in association with measures of negative and positive channel modulators, in hypothyroid hearts. Hormonally-induced modulation of calcium channels has clinical significance in the development and application of therapeutic agents in dysthyroid states. We thyroidectomized male rats and sham-operated euthyroid controls, in order to measure radioligand binding to ventricular and atrial membrane Ca2+ channels ([3H]-isradipine), beta-adrenoceptors ([125I]-iodocyanopindolol) and atrial muscarinic receptors ([3H]-quinuclidinyl benzilate) and related these data to contraction and heart rate responses to isoproterenol, carbachol and calcium. When data from hypothyroid tissues were compared to those of controls, the densities of calcium channels increased 50% in ventricles, but no differences were seen in atrial homogenates. In both atria and ventricles, beta-adrenoceptors decreased modestly with no change in affinity. Atrial muscarinic receptor density was also unchanged. Dose response curves of left atrial contractions showed: decreased sensitivity (increased EC50 value) but equal maximal responsiveness to extracellular calcium; an increased carbachol sensitivity (decreased EC50 value); and no significant difference in isoproterenol response. Comparisons of within-individual preparation ratioed EC50 values confirm the changed calcium and carbachol sensitivities. Heart-rate dose response curves displayed: increased maximal heart rate responsiveness to calcium associated with increased EC50 values; isoproterenol sensitivity was decreased nearly 3-fold. There was no significant difference in heart rate response to carbachol; however, ratioed values of carbachol and calcium EC50s were significantly different. These data are consistent with a tissue-level state of enhanced negative chronotropism and inotropism occurring in hypothyroid myocardia. We also confirm an earlier controversial finding of increased calcium channel density in ventricles from hypothyroid rats.