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Inhalation therapy for infectious lung diseases, such as tuberculosis, is currently being explored, with microspheres being used to target alveolar macrophages. One method of drug encapsulation into polymeric microspheres to form hydrophobic ion-paired (HIP) complexes, and then coprecipitate the complex and polymer using supercritical fluid methodology. For the potent antituberculosis drug, isoniazid (isonicotinic acid hydrazide, INH), to be used in this fashion, it was modified into an ionizable form suitable for HIP. The charged prodrug, sodium isoniazid methanesulfonate (Na-INHMS), was then ion paired with hydrophobic cations, such as alkyltrimethylammonium or tetraalkylammonium. The logarithms of the apparent partition coefficients (log P') of various HIP complexes of INHMS display a roughly linear relationship with the numbers of carbon atoms in the organic counterions. The water solubility of the tetraheptylammonium-INHMS complex is about 220-fold lower than that of Na-INHMS, while the solubility in dichloromethane exceeds 10 mg/mL, which is sufficient for microencapsulation of the drug into poly(lactide) microspheres. The actual logarithm of the dichloromethane/water partition coefficient (log P) for tetraheptylammonium-INHMS is 1.55, compared to a value of - 1.8 for the sodium salt of INHMS. The dissolution kinetics of the tetraheptylammonium-INHMS complex in 0.9% aqueous solutions of NaCl was also investigated. Dissolution of tetraheptylammonium-INHMS exhibited a first-order time constant of about 0.28 min(-1), followed by a slower reverse ion exchange process to form Na-INHMS. The half-life of this HIP complex is on the order of 30 min, making the enhanced transport of the drug across biological barriers possible. This work represents the first use of a prodrug approach to introduce functionality that would allow HIP complex formation for a neutral molecule.

The ability of stoichiometric amounts (based on charged groups) of ionic detergents to bind to oppositely charged ionic compounds has been recently reviewed. These hydrophobic ion-paired (HIP) complexes display altered solubility properties. Most of the work to date on HIP compelxes has focused on basic drugs and anionic detergents. It would be extremely useful to extend this approach to acidic compounds, including DNA and RNA. However, most cationic detergents are relatively toxic. It is hypothesized that detergents constructed from naturally occurring or well tolerated components, coupled by labile linkages, will be less toxic and still able to form strong HIP complexes. This study describes the synthesis and characterization of long chain alkyl esters of arginine. This class of cationic detergents, which have not been reported previously, are less cytotoxic than alkyltrimethylammonium detergents, possibly making them more acceptable in drug delivery applications. These arginine esters exhibit detergent-like properties. For example, the dodecyl ester of arginine has a critical micelle concentration of 0.07 mM, while being approximately 5-10 fold less toxic than tetradecyltrimethylammonium bromide. The arginine dodecyl ester forms stable HIP complexes with plasmid DNA. The complex is sufficiently stable to allow some modest level of transfection with Cos-7 cells in a time- and concentration-dependent fashion. This work demonstrates that arginine-based cationic detergents are effective ion-pairing agents, appear to be less toxic than alkyltrimethylammonium compounds, and form stable complexes with DNA.

Pigmented (C57BI) and nonpigmented (balb/c) mice, 25 days of age, were treated intraperitoneally with [3H]-nicotine (4 mg/kg, 555 dpm/ng) or [3H]-flunitrazepam (1 mg/kg, 2200 dpm/ng) daily for three days. After 21 days, shaved back hair was digested at 37 degrees C for 24 h with either 1 M sodium hydroxide or 1 M sodium sulfide. With both drugs, sodium sulfide extraction removed the same amount of radioactivity as sodium hydroxide from nonpigmented hair. However, sodium sulfide removed significantly more radioactivity from pigmented hair than did sodium hydroxide. In pigmented hair, sodium sulfide solubilized 35% and 74% of the flunitrazepam- and nicotine-associated radioactivity, respectively. Of this, 12% and 43%, respectively, could be partitioned into ethyl acetate. Microscopic examination of residual pellets after digestion demonstrated a more thorough dissolution of the hair shaft with sodium sulfide with only melanosomes remaining. The results demonstrate the significant interaction of flunitrazepam and nicotine with melanins and the utility of sodium sulfide in increasing drug recovery.

1,3-Butadiene (BD) is a gas used widely in the rubber and plastics industry as an intermediate in production processes and has been detected in automobile exhaust and cigarette smoke. BD requires metabolic activation to exert toxicity and has been shown to be carcinogenic in rodents. IARC has classified BD as a group 2A (probably carcinogenic to humans) carcinogen. The initial oxidation of BD to butadiene monoxide (BMO) occurs primarily via cytochrome P450 2E1 and two stereoisomers of BMO (R and S) can be formed. (R) and (S)-BMO are metabolized differently and demonstrate markedly different toxicities in isolated rat hepatocytes. This work examined the generation of (R) and (S)-BMO from BD by cytochrome P450 2E1 from rabbit, rat and human. BMO level was measured by GC-MS analysis and enantiomeric composition was determined by GC-FID. The greatest rate of formation of BMO from BD was obtained with rabbit cytochrome P4502E1 followed by human and then by rat. Enantiomeric distribution of R and S-BMO produced by the three species demonstrated no significant differences.

1,3-Butadiene (BD) is a gas used heavily in the rubber and plastics industry. BD and its epoxide metabolites have been shown to be carcinogenic and mutagenic in rodents, and BD has been classified by IARC as a group 2A carcinogen. We have examined the role of stereochemistry in species-dependent metabolism and toxicity of BD. Diastereo- and enantioselective synthetic routes to butadiene monoxide (BMO), butadiene bisoxide (BBO), and 3,4-epoxybutane-1,2-diol isomers have been developed. These routes have allowed the development of chiral gas chromatographic and GC/MS analytical procedures for quantitation of these metabolites in biological experiments. We have utilized hepatic microsomes from male B6C3F1 mice and hepatic microsomes and intact hepatocytes from male Sprague-Dawley rats as experimental systems. At 30 min, BMO production from BD was two times higher in mouse hepatic microsomes than in rats, and stereoselective analysis was used to determine the relative formation of (R)- and (S)-BMO. Formation of BBO from both (R)- and (S)-BMO was characterized in rat and mouse microsomal systems. As expected, more BBO was formed in mouse hepatic microsomes (3-4-fold) than in rat hepatic microsomes. No difference in total BBO formed from either isomer was observed in rat microsomes, but in mouse microsomes significantly more BBO was produced from (S)-BMO than from (R)-BMO. The cytotoxicity of each BMO and BBO enantiomer was examined in freshly isolated rat hepatocytes. (R)-BMO showed greater cytotoxicity than (S)-BMO. Stereospecific cytotoxicity was also observed using BBO enantiomers and (meso)-BBO was more cytotoxic than either the (R:R) or the (S:S)-BBO. The results show that stereochemistry plays an important role in BD metabolism and cytotoxicity and for the purposes of risk assessment needs to be compared across species.

1,3-Butadiene (BD), a gas used in the production of rubber and plastics, induces a high incidence of leukemias and lymphomas in B6C3F1 mice. Because of the potential involvement of the hematopoietic system in response to BD, we have examined metabolism of BD by B6C3F1 mouse and human bone marrow and by purified human myeloperoxidase (MPO), an enzyme rich in bone marrow. BD was metabolized to butadiene monoxide (BMO) by MPO and by mouse and human bone marrow cells. In all of these systems metabolism was stimulated by hydrogen peroxide suggesting a peroxidase-mediated process. In B6C3F1 mouse bone marrow cell lysates, hydrogen peroxide but not NADPH stimulated metabolism suggesting that cytochrome P450 was not involved in BMO formation. Metabolism of BD to BMO in hydrogen peroxide-fortified mouse bone marrow cell lysates was more than two orders of magnitude lower than in either NADPH-fortified rat or mouse hepatic microsomes. Experiments using both mouse and human bone marrow cells showed that cells from both sources could generate BMO from BD. These data show that BD can be converted to BMO in a target organ of BD carcinogenicity.

In order to ensure the stability of protein pharmaceuticals, human serum albumin (HSA) is often added as an excipient, frequently in large excess. This makes chromatographic analysis of the stability of the active protein difficult. In the case of interleukin-4 (IL-4), separation from HSA can be achieved to some degree by size exclusion chromatography, but some HSA co-elutes with the IL-4. Hydrophobic ion pairing provides a method for selective precipitation of IL-4 from HSA. Hydrophobic ion pairing involves the electrostatic interaction of ionic detergents with oppositely charged polypeptides. Even when HSA is present in fifty-fold excess (w/w), the resulting precipitate contains greater than 70% of the IL-4. Selective precipitation with SDS produces enhancements in IL-4 over HSA of more than 2000-fold. This approach permits subsequent facile analysis of IL-4 by conventional reverse phase HPLC.

The metabolism of trans,trans-muconaldehyde (MA), a highly reactive alpha,beta-unsaturated dialdehyde, was examined in vitro using purified yeast alcohol and aldehyde dehydrogenases (ADH and ALDH, respectively). In the presence of NAD(+)-fortified ALDH, the mono-oxidation product (acid/aldehyde) was the primary metabolite formed with trace amounts of the dioxidation product (trans,trans-muconic acid). In NADH-fortified reactions with ADH, both the mono- and direduction products (hydroxy/aldehyde and dihydroxy, respectively) were readily detected. Oxidation and reduction products of MA were formed in incubates containing both dehydrogenases together with either NAD+ or NADH. Unexpectedly, an additional metabolite was detected, which was a major product in both NAD(+)- and NADH-fortified systems containing ALDH and ADH in combination and whose formation could be inhibited by pyrazole (an ADH inhibitor). ALDH-mediated oxidation of a synthetic standard of the hydroxy/aldehyde derivative of MA resulted in formation of this new metabolite, which was also a major product formed by rat hepatocytes incubated with MA. Using HPLC/photodiode array detection, the new metabolite was found to cochromatograph and have a uv spectrum identical to that of a synthetic standard of the hydroxy/acid derivative of MA. The metabolite was confirmed as the hydroxy/acid derivative of MA after preparative HPLC, TMS derivatization, and GC/MS analysis. The hydroxy/acid metabolite was not formed during ADH-mediated reduction of the mono-oxidation product of MA, suggesting that this metabolite was formed by yeast dehydrogenases via a primary reduction of MA and subsequent oxidation of the hydroxy/aldehyde to the hydroxy/acid. These data show that the hydroxy/acid derivative is a novel metabolite of MA, which arises from the interaction of both oxidative and reductive routes of metabolism.

Involvement of glutamate neurotransmission in the differential response of long-sleep (LS) and short-sleep (SS) mice to acute ethanol was examined by measuring the effect of centrally administered glutamate receptor agonists and antagonists on blood ethanol concentration (BEC) at loss of righting response following intragastric administration of ethanol. NMDA coinjected with glycine, and quinolinic acid (QA), decreased sensitivity to ethanol in both lines of mice. SS mice were more sensitive to QA than were LS. The NMDA antagonists 2-amino-5-phosphonovaleric acid (APV), MK-801 and an inhibitor of glutamate synthesis, methionine sulfoximine, increased sensitivity to ethanol in both lines of mice. MK-801 effects were line dependent with SS being more sensitive. In addition, coinjection of APV, Mg++ or Zn++ with QA blocked the decrease in sensitivity seen with QA alone. These results demonstrate that NMDA agonists and antagonists alter the acute hypnotic response to ethanol in both LS and SS mice, and support the hypothesis that ethanol exerts its effects in part by altering glutamatergic neurotransmission.

The effect of chloride ion on the transport of [3H]dopamine into synaptic vesicles purified from rat striatum has been evaluated. The inclusion of 10 mM chloride ion in the incubation medium produced a 100% increase in temperature-sensitive [3H]dopamine uptake into synaptic vesicles from approximately 1800 pmol/mg to 3600 pmol/mg of protein. Half-maximal effects were observed with chloride ion at 4 mM concentration. The anion selectivity of stimulation supports the presence of anion channels within the membranes of dopaminergic storage organelles. Low concentrations of thiocyanate ion (less than 10 mM), 4-acetamido-4'-isothiocyano-2-2'-disulfonic acid stilbene (100 microM), and duramycin (5 micrograms/ml) selectively blocked the chloride ion stimulated accumulation of [3H]dopamine. Higher concentrations of these agents are required to significantly reduce [3H]dopamine uptake in the absence of chloride ion. These results suggest that both components of the proton electrochemical gradient (delta psi and delta pH) are important for dopamine uptake by brain vesicles. This article presents the first demonstration that chloride ion plays a role in the transport of dopamine into vesicles isolated from the CNS.

To explore drug-melanin interactions, we examined the in vitro tyrosinase-mediated formation of melanin from tyrosine in the presence of the 3H-cocaine (3H-COC), 3H-flunitrazepam (3H-FLU), and 3H-nicotine (3H-NIC) at 10-100,000 ng/mL. Polymerization in the presence of 10 or 100 ng/mL of each drug resulted in almost complete drug incorporation into the melanin pellet. Only 12% (3H-NIC) to 28% (3H-FLU) of the pellet-associated radioactivity could be released upon treatment with 6 M HCl. At 1000-100,000 ng/mL, between 20 and 50% of label became melanin-associated. In each case a significant percentage of melanin-associated radioactivity was resistant to treatment with 6 M HCl. Nicotine-associated radioactivity in the polymer was subject to much greater quenching than was 3H-COC or 3H-FLU, suggesting a much tighter association with the melanin. The subsequent demonstration of a covalent adduct of a melanin intermediate and nicotine has demonstrated the utility of this polymerization system as a model for further chemical characterization of drug-melanin interactions.

Pigmentation is a major factor in the incorporation of many drugs into hair. In an attempt to elucidate potential mechanisms of drug-melanin interaction, melanin was synthesized in vitro in the presence of nicotine, which we have shown to have a substantial interaction with melanin, and cotinine, a primary nicotine metabolite. L-DOPA, a precursor of eumelanin, was oxidized and oligomerized with tyrosinase. Nicotine, cotinine, and/or their deuterated analogues were added to the oligomerization reaction mixture in a 10:1 L-DOPA:drug ratio. A black precipitate formed within 60 min. Aliquots were removed from the incubation mixture at 60, 120, and 360 min. MALDI-TOF MS determinations were carried out on each sample to provide a mean and standard error for the masses of interest. Internal calibration allowed accurate mass measurement of the products. A careful comparison of the spectra of samples prepared both with and without drug indicated the presence of masses corresponding to the protonated drug, melanin oligomers, and nicotine or continine adducts of the monomeric melanin intermediate dopaquinone (DOPAQ). Additional support for the presence of drug-melanin adducts was provided by employing deuterated analogues of nicotine and L-DOPA in the reaction and observing that the masses shifted accordingly. Structures of the adducts were further confirmed by select ion gating and postsource decay analysis.

With very low concentrations of anionic detergents, such as sodium dodecyl sulfate (SDS) and Aerosol OT (AOT), it is possible to solubilize proteases in organic solvents, while retaining enzymatic activity. For example, the SDS-subtilisin BPN' complex catalyzes transesterification of Ac-Phe-OMe in ethanol with a kcat/Km of 36 M-1 s-1 for mutant M1 and 39 M-1 s-1 for the wild type. By comparison, M1 suspended in ethanol is approximately 1000-fold less active, with a kcat/Km of 0.03 M-1 s-1. Similarly, AOT complexes of alpha-chymotrypsin were found to be approximately 1000 times more active (kcat/Km = 100-350 M-1 s-1) than the suspended enzyme.

Stabilization of proteins through proper formulation is an important challenge for the pharmaceutical industry. Two approaches for stabilization of proteins in solution are discussed. First, work describing the effect of additives on the thermally induced denaturation and aggregation of low molecular weight urokinase is presented. The effects of these additives can be explained by preferential exclusion of the solute from the protein, leading to increased thermal stability with respect to denaturation. Diminished denaturation leads to reduced levels of aggregation. The second approach involves stoichiometric replacement of polar counter ions (e.g., chloride, acetate, etc.) with anionic detergents, in a process termed hydrophobic ion pairing (HIP). The HIP complexes of proteins have increased solubility in organic solvents. In these organic solvents, where the water content is limited, the thermal denautration temperatures greatly exceed those observed in aqueous solution. In addition, it is possible to use HIP to selectively precipitate basic proteins from formulations that contain large amounts of stabilizers, such as human serum albumin (HSA), with a selectivity greater than 2000-fold. This has been demonstrated for various mixtures of HSA and interleukin-4. (c) 1995 John Wiley & Sons, Inc.

It has previously been reported that isolated rat hepatocytes rapidly and completely metabolize high concentrations of 4-hydroxy-2,3-(E)-nonenal (4-HNE). However, until this report, the degree to which oxidative-reductive and nonoxidative metabolic pathways function in the depletion of 4-HNE by isolated rat hepatocytes has been speculative. The objective of the present study was to quantitate the extent to which cellular aldehyde dehydrogenases (ALDH; EC 1.2.1.3.), alcohol dehydrogenase (ADH; EC 1.1.1.1.), and glutathione S-transferases (GST; EC 2.5.1.18) function simultaneously during hepatocellular metabolism of 4-HNE. Hepatocytes were incubated with varying concentrations of 4-HNE (50, 100, 250 microM) and reversed-phase HPLC was used to quantitate 4-HNE and the oxidative and reductive metabolites, 4-hydroxy-2-nonenoic acid and 1,4-dihydroxy-2-nonene, respectively. Conjugative metabolism of 4-HNE was determined from the depletion of cellular reduced glutathione (GSH) and concomitant formation of a GSH-4-HNE adduct detected as 2,4-dinitrofluorobenzene derivatives measured by reversed-phase HPLC. Hepatocellular elimination of 4-HNE was estimated at rates of 1.666, 0.902, and 0.219 nmol min-1 10(6) hepatocytes-1 for 50, 100, and 250 microM aldehyde, respectively. At aldehyde concentrations of 50, 100, and 250 microM the maximal concentrations of oxidative (acid) metabolites formed were 5.9, 12.7, and 28.9 nmoles 10(6) hepatocytes-1, whereas the concentrations of the reductive (diol) metabolite were 0.4, 12.6, and 42.3 nmoles 10(6) hepatocytes-1, respectively. The presence of 4-methylpyrazole or cyanamide abolished formation of the reductive metabolite 1,4-dihydroxy-2-nonene or the oxidative metabolite 4-hydroxy-2-nonenoic acid in hepatocyte suspensions. At all 4-HNE concentrations evaluated, hepatocellular glutathione was not completely depleted by the aldehyde and the depletion of cellular reduced GSH corresponded to the production of the GSH-4-HNE conjugate. Metabolism by the alcohol/aldehyde dehydrogenase pathways accounted for approximately 10% of the 4-HNE elimination, while bioconversion by GST represent 50-60% of the total 4-HNE removal by hepatocytes. The enzymatic pathways responsible for the remaining 40% of 4-HNE metabolism remain to be identified. Taken together these results describe the quantitative and dynamic importance of oxidative, reductive, and nonoxidative routes in the metabolism and detoxification of 4-HNE.

Long-sleep (LS) and short-sleep (SS) mice, genetically selected for their differential CNS sensitivity to ethanol, have also been shown to differ in their response to other sedative-hypnotics, including the barbiturates. We have applied a gas-chromatographic method of analysis of brain barbiturate concentrations following IP administration of either the water-soluble barbiturate diethylbarbital (DB) or the lipid-soluble barbiturate secobarbital (SB). Brain barbiturate levels were assessed at loss of righting response, and at regaining righting response (waking). In addition, latency to loss of righting response and duration of loss of righting response were measured following IP barbiturate administration. We have observed a differential sensitivity of LS and SS mice to the sedative effects of DB, with LS mice having greater sensitivity compared to SS. This differential sensitivity to DB, as measured by a lower concentration of DB which caused loss of righting in LS, was accompanied by an equal rate of water-soluble barbiturate brain distribution and elimination in the two lines. With the lipid-soluble barbiturate SB, LS and SS mice did not differ in brain SB concentration at loss of righting response or at waking. However, sleep time was much longer in SS mice than LS due to slower brain clearance of the barbiturate in SS. Therefore, duration of loss of righting (sleep time) did not adequately reflect central sensitivity to the lipid-soluble barbiturate. These data suggest the importance of quantifying brain concentrations at loss of righting reflex when assessing central sensitivity to sedative-hypnotic agents. Furthermore, it appears important to consider differences in physico-chemical properties of a particular pharmacological agent before generalizing findings to an entire class of such agents.

A stereospecific route to (R)- and (S)- [ 13 C]-butadiene monoxide was developed using (R)- or (S)-glycidaldehyde and a polymer-supported Wittig reagent.

In order to assess the importance of the aromatic rings in inhibition of neuronal amine uptake produced by tricyclic antidepressant drugs, derivatives of imipramine (IMI) and dcsipramine (DMI) were prepared in which either one (IMIH, DMIH) or both (IMIH2, DMIH2) of the aromatic rings were fully reduced to cyclohexane rings. The relative abilities of these compounds to inhibit the uptake of l-[ 3 H ]- norepinephrine (NE), [ 3 H]-dopamine (DA) and [ 3 H]-5-hydroxytryptamine (5-HT) into chopped tissue of rat cerebral cortex were determined. Reduction of one or both aromatic rings did not alter significantly the inhibition of uptake of [ 3 H]-DA or [ 3 H]5-HT produced by either IMI or DMI ( IC 50 values 25–80 μM). However, saturation of one or both rings abolished the selectivitv of DMI for inhibition of NE uptake ( IC 50 0.12 μM). decreasing potency 150-fold ( IC 50 18.3 μM) and 250-fold ( IC 50 29.4 μM) respectively. The effect of aromatic ring reduction on the IMI-induced inhibition of NE uptake was much less pronounced. The results suggest that hydrophobic rather than π-electron attractive forces are involved in the interaction of DMI or IMI with DA or 5-HT uptake sites. However, the loss in selectivity for inhibition of NE uptake upon reduction of DMI may reflect loss of π-electron interactions in the binding of DMI to the NE uptake site, or may reflect increased sensitivity to spatial disposition of the hydrophobic binding areas of the drug relative to that found at the DA or 5-HT amine uptake sites.

The cocaine sensitivity of male and female long-sleep (LS) and short-sleep (SS) mice, which have been selectively bred for differential ethanol-induced "sleep-time," was examined in a battery of behavioral and physiological tests. Differences between these two mouse lines were subtle and were seen primarily at high doses. At high doses, SS mice were more sensitive than LS mice, particularly to cocaine-induced hypothermia; however, significant hypothermia was not seen except at doses which were very near to the seizure threshold. During a 60-min test of locomotor activity, LS mice showed greater stimulation of Y-maze activity by 20 mg/kg cocaine than SS mice. Consistent with the finding of subtle differences in sensitivity to low doses of cocaine. LS and SS mice did not differ in sensitivity to cocaine inhibition of synaptosomal uptake of [3H]-dopamine, [3H]-norepinephrine or [3H]-5-hydroxytryptamine. However, consistent with the finding of differential sensitivity to high doses of cocaine, SS mice were more sensitive to the seizure-producing effects of the cocaine and lidocaine, a local anesthetic. It is hypothesized that the differential sensitivity of these mouse lines to high doses of cocaine is due to differential sensitivity to cocaine's actions on systems that regulate local anesthetic effects. Selective breeding for differential duration of alcohol-induced "sleep-time" may have resulted in differential ion channel structure or function in these mice.

Four conformationally defined analogs of amphetamine were synthesized and studied for their ability to potentiate the action of levarterenol on the isolated vas deferens from reserpine-treated rats. The compounds also were studied for indirect adrenergic agonist activity in the same test system. A definite stereochemical correlation was demonstrated in each case, the exo -isomers being considerably more active than their endo -counterparts both in potentiation and in indirect activity. The more active isomers of each pair correspond to an anti -periplanar conformation of amphetamine. The compounds are probably acting by inhibition of the neuronal amine uptake mechanism, since none of the compounds was a direct-acting agonist itself. These results are discussed in relationship to other previously reported, conformationally defined, amphetamine analogs.

The ATP-stimulated accumulation of L-[3H]glutamate by whole brain synaptic vesicle preparations from long-sleep and short-sleep mice, lines selectively bred for difference in sleep time response to acute ethanol administration, was examined. L-[3H]Glutamate accumulation in vesicles from short-sleep mice was approximately twice that observed in vesicles from longsleep mice at three glutamate loading concentrations. The vesicular content of endogenous L-glutamate in crude and enriched vesicle preparations from short-sleep mice was approximately 1.5-fold higher than in vesicles from long-sleep mice. The data suggest that L-glutamate associated with synaptic vesicles may serve a role in glutamate neurosecretion.

The MgATP-stimulated accumulation of (-)- 3 H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K + concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)- 3 H-NE (Km 1 × 10 −6 M) in this system (37°) was examined under saturating (10 −5 M) and non-saturating (2 × 10 −7 M) concentrations of NE. At 10 −5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated in vivo ) and 6.0±0.79 (without MgATP). At 2×10 −7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2 x 10 −7 M).

The 30 mM K + -induced release of endogenous glutamate has been examined in slices of cortex, hippocampus, striatum and cerebellum from "short sleep" and "long sleep" mice, lines genetically selected for differential response to acute ethanol administration. In cortex and straitum, glutamate release was significantly higher in "short sleep" mice, while in hippocampus the difference was reversed. No differences were observed in glutamate release from cerebellum. The release was Ca ++ -dependent, being inhibited by lanthanum and verapamil.

Synaptic vesicles from rat brain were immobilized on glass microspheres covalently coated with poly-L-lysine. Using a potassium tartrate perfusion medium, the vesicular accumulation and methamphetamine-induced release of (L)-3H-norephinephrine could be conveniently monitored in a flow experiment.

The effect of acute (10-75 min) exposure of isolated spontaneously beating rat atria to L-triiodothyronine (T3)(12.9-1290 nM) on the positive chronotropic response to (-)-norepinephrine (NE) 10(-10)-10(-4)M) was examined. Preincubation (50 min) with 129 nM T3 increased the maximal response to (-)-NE from 128 +/- 10 to 176 +/- 11 beats/min. This effect was temporally biphasic, being maximal at 50 min pre-exposure. The results demonstrate an effect of acute in vitro exposure to T3 on the noradrenergic responsiveness of the heart.

The accumulation of 3H-dopamine by synaptic vesicles from rat striatum was significantly stabilized in a membrane impermeant medium. The characteristics of dopamine accumulation by striatal vesicles were quite similar to those reported for dopamine accumulation by a whole brain vesicle preparation in the same medium, and were significantly different from the characteristics previously reported for vesicular accumulation of norepinephrine.

Leucine-enkephalin produces a dose related decrease in the contractile response of spirally cut strips of rat aorta to norepinephrine (NE). The maximal effect (55.0 +/- 1.0 percent reduction in developed tension) occurs at a leucine-enkephalin concentration of 10(-10) M. The attenuation of contractile response is reversed by naloxone.

Potential interactions between opiate peptides and catecholamines in mammalian heart were examined using isolated spontaneously beating rat atria as a test system. Methionine-enkephalin (ME), leucine-enkephalin (LE), phe-met-arg-phe amide (FMRFamide), D-ala2, N-methyl-phe4, met (O)5-ol-enkephalin (FK 33-834), methionine-enkephalin arg6 arg7 (ME arg6 arg7) and beta-endorphin had no effect on basal beating rate of isolated atria at all concentrations up to 10(-5) M. The positive chronotropic effect of norepinephrine (NE) on atrial rate is, however, significantly attenuated by enkephalin peptides. Thus, the maximal chronotropic effect of NE (an increase from 317 +/- 7.0 to 473 +/- 7.3 beats per minute (bpm) in 250 gm rats at a dose of 10(-5)M NE) is decreased by 42% in the presence of 10(-7)m ME. The action of ME is completely blocked by addition of 10(-7)M naloxone, which by itself has no effect on NE-induced positive chronotropy or basal beating rate. The dose-effect curve for ME attenuation of NE-induced positive chronotropy is bell-shaped, i.e., both 10(-8) M and 10(-5) M ME have no significant effect on NE positive chronotropy. Other enkephalin peptides acted in a similar manner to ME; LE (10(-7) M) and FK 33-834 (10(-8) M) decreased maximal NE-induced positive chronotropy 42 and 27%, respectively. The molluscan cardioexcitatory peptide FMRFamide (10(-7) M) also decreased maximal NE positive chronotropy, about 30%. In contrast, beta-endorphin did not significantly affect NE stimulation of atrial rate. We conclude that enkephalins can modulate the noradrenergic responsiveness of rat atria in vitro. The possible physiological relevance of this interaction is discussed.

The spontaneous photooxidation of thiothixen (Navane) to 2-(N,N-dimethyl-sulfonamido)-9-thioxanthone is described. The corresponding sulfoxide is demonstrated to be the fluorescent species obtained upon permanganate oxidation of thiothixene in the fluorometric assay procedure of Mjorndal and Oreland.

3H-Dopamine (DA) accumulation by storage vesicles from whole rat brain was significantly stablized in a buffer system based upon the membrane-impermeant D-potassium tartrate. 3H-DA uptake saturated by twenty minutes (Km 2.1 X 10(-5)M) and remained stable for periods of 40-60 minutes. Accumulated DA was rapidly exchangeable with exogenous DA. Total levels of accumulation (pmol/mg protein) were 41.7 +/- 2.9 (37 degrees), 11.9 +/- 2.5 (4 degrees), 31.3 +/- 1.8 (absence of ATP), 26.3 +/- 2.7 (reserpine, 10(-6)M), 26.1 +/- 0.67 (no ATP + reserpine 10(-6), and 14.6 +/- 2.4 (carbonylcyanide-p-triflouromethoxyphenylhydrazone, FCCP, 10(-6)M). Depletion of endogenous DA levels by pretreatment of the animals with alpha-methyl-p-tyrosine greatly diminished the reserpine-insensitive DA accumulation. After depletion of endogenous DA, ATP-independent uptake was significantly retarded, but eventually reached near-control levels. This uptake was abolished in the presence of FCCP (10(-6)M). The results suggest that endogenous levels of DA and ATP contribute to the reserpine- and ATP-insensitive DA accumulation observed in vesicles from untreated animals. HPLC analysis demonstrated no conversion of DA to norepinephrine (NE) in the course of the experiments.

Rat brain synaptic vesicles suspended in a medium comprised of potassium tartrate displayed saturable accumulation of L-[ 3 H] glutamic acid at 37° (Km 2.0 × 10 −4 M; 311±13 pmol/mg protein), which was stable for periods up to 60 min. The accumulation was temperature sensitive and partially ATP-dependent, uptake levels being reduced to 18.7±0.8 pmol/mg protein at 4°, and to 141±4 pmol/mg protein in the absence of ATP. Fractionation of a crude vesicle preparation on a discontinuous sucrose gradient demonstrated the accumulation to be specifically associated with the synaptic vesicle fraction.

Exposure of rat atrial slices to 10 −5 M norepinephrine (NE) for 10 minutes increases 45 Ca ++ accumulation from 1.64 ± 0.10 to 2.23 ± 0.06 nmol/mg tissue. In the presence of leucine enkephalin (10 −8 M), NE-stimulated 45 Ca ++ uptake is reduced to 1.44 ± 0.10 nmol/mg tissue. The effect of leu-enkephalin is reversed in the presence of 10 −7 M naloxone, NE-stimulated 45 Ca ++ uptake being increased to 2.17 ± 0.15 nmol/mg tissue. The results support a direct interaction of leuenkephalin with β-agonist-stimulated Ca ++ flux in rat atria, and correlate with the previously reported enkephalin antagonism of NE-induced positive chronotropy in the same tissue.

Norepinephrine (NE) (10 −5 M) increases the beating rate of isolated, spontaneously beating guinea-pig atria 78 ± 3 beats per minute. Leucineenkephalin (10 −7 M) increases the maximal chronotropic response to NE by 38%, i.e., by 30 beats per minute. In the presence of 10 −7 M leucineenkephalin and 10 −7 M naloxone, the chronotropic response to NE is reduced to 38 ± 3 beats per minute, a value observed in the presence of naloxone alone. Neither naloxone nor leucine enkephalin significantly altered the inotropic response to NE, or significantly altered the basal beating rate. Parallel effects were observed when 45 Ca ++ uptake by atrial tissue was examined. Incubation of atrial slices with 10 −5 M NE for 10 minutes minimally stimulated 45 Ca ++ uptake from 1.27 ± 0.04 to 1.45 ± 0.17 nmol/mg tissue. In the presence of 10 −7 M leucine-enkephalin, 45 Ca ++ uptake was increased to 1.95 ± 0.14 nmol/mg tissue. 45 Ca ++ uptake was reduced to control values (1.19 ± 0.09 nmol/mg tissue) in the presence of NE, leucine-enkephalin and naloxone (10 −7 M). The data are consistent with a leucine-enkephalin augumentation of NE-induced chronotropy in guinea-pig atria due to an enhancement of NE-dependent Ca ++ accumulation. This effect of leucine-enkephalin is opposite that previously reported for rat atria, in which leucine-enkephalin inhibits both NE-induced positive chronotropy and Ca ++ influx.

The relative ability of cocaine, tropacocaine and amphetamine to inhibit the uptake of [3H]norepinephrine (NE), [3H]dopamine (DA) and [3H]5-hydroxytryptamine (5HT) was examined in whole brain synaptosomes from BALB, C3H, C57BL and DBA inbred mouse strains. With inhibition of [3H]NE uptake, synaptosomes from BALB and C57 mice were substantially more sensitive to cocaine inhibition than those from DBA or C3H. Moreover, with BALB and C57 tissue, amphetamine was as potent as cocaine, whereas with C3H and DBA, amphetamine and tropacocaine were much less potent inhibitors of [3H]NE uptake. With respect to [3H]DA accumulation, synaptosomes from BALB, C57 and DBA were equally sensitive to cocaine inhibition, while C3H synaptosomes were significantly less sensitive. In each of the four strains, amphetamine was more potent than cocaine, and tropacocaine far less potent. The relative potencies of the three drugs varied significantly among the four strains. With [3H]5HT accumulation, synaptosomes from DBA were exquisitely sensitive to cocaine inhibition, followed by BALB and lastly by C57 and C3H. In each of these strains, amphetamine and tropacocaine were equipotent at [3H]5HT inhibition, and less potent than cocaine. The results suggest that there are pronounced genetic differences in sensitivity to monoamine uptake inhibition by cocaine, which may arise from genetic differences in either carrier topology or other site of cocaine interaction. The results further suggest that genetic behavioral differences to cocaine and amphetamine may involve complex neurotransmitter interactions.

In order to examine possible selectivity of amine uptake by synaptic vesicles, the ATP-stimulated accumulation of 3 H-5-hydroxytryptamine (5HT) by synaptic vesicles from rat whole brain was examined in a medium comprised largely of membrane-impermeant anions (d-tartrate). Such media have previously been shown to stabilize vesicular accumulation of several neurotransmitters. Accumulation of 3 H-5HT did not occur in tartrate medium alone, but was increased biphysically with increasing concentrations of both potassium phosphate and potassium bicarbonate. At optimal concentrations of each anion (10 mM), stable accumulation of 3 H-5HT was observed at 37° (26.1 ± 1.2 pmol/mg protein; Km 6×10 −7 M), which was reduced by > 95% in the absence of K 2 ATP, at 4°C, in the presence of 10 −6 M reserpine, or in the presence of the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Uptake was significantly antagonized by millimolar concentrations of Na + , Mg ++ or Cl − , but was unaffected by ouabain (10 −5 M). Pretreatment of animals with 5, 7-dihydroxytryptamine (5, 7-DHT) (200 ωg, intraventricular) 10 days prior to sacrifice reduced endogenous 5HT levels by 70%, while levels of endogenous norepinephrine (NE) and dopamine (DA) were unaffected. Accumulation of 3 H-5HT, examined in the presence of 10 −6 M NE to block 3 H-5HT accumulation by vesicles from noradrenergic nerve endings, was reduced by 40% in vesicles from treated animals. Vesicular accumulation of 3 H-(−)-NE and 3 H-DA was unaffected by 5, 7-DHT treatment. The data suggest the possibility of preferential accumulation of 3 H-5HT by vesicles arising from serotonergic nerve endings.

Leucine-enkephalin (LE) at 10 −8 M reduces the maximum chronotropic response of isolated spontaneously beating rat atria to exogenously added (−)-norepinephrine (NE) by approximately 27%, with no effect on the NE ED 50 (1.5 × 10 −7 M) for positive chronotropy. This modulatory effect of LE is completely blocked by addition of 10 −7 M naloxone, and seems to be catecholamine-receptor specific, since the positive chronotropic response to forskolin is unaltered in the presence of LE. Isoproterenol (ISO)-induced positive chronotropy is also attenuated by LE. This effect is markedly dependent on the extracellular calcium concentration: LE actually causes a greater than two-fold enhancement of the positive chronotropic effect of ISO at low (0.5 mM) extracellular calcium concentration. A possible role for enkephalins to modulate catecholamine action on the heart via an alteration of catecholamine-induced inward calcium flux is discussed.

The effects of cocaine on Y-maze activity and heart rate have been examined in four inbred strains of mouse (BALB, C57BL, C3H and DBA). In addition, brain [3H]-cocaine concentrations were measured at the time of maximal response to cocaine. Cocaine produced a dose-related increase in Y-maze cross activity in C3H, DBA and C57BL, with C3H mice being considerably more sensitive than DBA or C57BL. Cocaine was without effect on Y-maze cross activity in BALB mice. Cocaine produced a biphasic effect on rearing activity in C3H mice, a dose related depression in BALB mice, and was without effect on C57BL and DBA mice. At the highest dose studied (15 mg/kg), cocaine produced a small decrease in heart rate in C3H mice. Strain differences in behavior were maximal 15 minutes after a dose of 5 mg/kg, IP. At this dose and time interval, brain [3H]-cocaine concentrations were not significantly different among the four strains of mice. The results suggest a genetically-determined difference in CNS sensitivity to cocaine.