Search

The brainstem reticular formation is an area important to the control of rapid eye movement (REM) sleep. The antagonist of GABA-type A (GABA(A)) receptors, bicuculline methiodide (BMI), injected into the rat nucleus pontis oralis (PnO) of the reticular formation resulted in a long-lasting increase in REM sleep. Thus, one factor controlling REM sleep appears to be the number of functional GABA(A) receptors in the PnO. The long-lasting effect produced by BMI may result from secondary influences on other neurotransmitter systems known to have long-lasting effects. To study this question, rats were surgically prepared for chronic sleep recording and additionally implanted with guide cannulas aimed at sites in the PnO. Multiple, 60 nl, unilateral injections were made either singly or in combination. GABA(A) receptor antagonists, BMI and gabazine (GBZ), produced dose-dependent increases in REM sleep with GBZ being approximately 35 times more potent than BMI. GBZ and the cholinergic agonist, carbachol, produced very similar results, both increasing REM sleep for about 8 h, mainly through increased period frequency, with little reduction in REM latency. Pre-injection of the muscarinic antagonist, atropine, completely blocked the REM sleep-increase by GBZ. GABAergic control of REM sleep in the PnO requires the cholinergic system and may be acting through presynaptic modulation of acetylcholine release.

The adenosine agonist cyclohexaladenosine injected into the medial pontine reticular formation of the rat induces a long-lasting increase in rapid eye movement sleep. To investigate the adenosine receptor-subtype(s) mediating this effect, the dose-response relationships for increasing rapid eye movement sleep by two highly selective adenosine receptor agonists were compared. Rats were surgically prepared for chronic sleep recording and bilateral guide cannulae were aimed at medial sites in the caudal, oral pontine reticular formation. Injections were made unilaterally in 60 nl volumes within 1 h after lights-on. The adenosine agonists used were A1-selective cyclohexaladenosine (10(-6)-10(-4) M) and A2a-selective CGS 21680 (10(-7)-10(-3) M). Each animal also received a series of three, paired-consecutive injections of the muscarinic receptor antagonist atropine (4x10(-3) M) followed by the lowest effective dose of each agonist or saline as control. The A2a receptor agonist, CGS 21680, was one order of magnitude more potent than the A1 receptor agonist, cyclohexaladenosine, in inducing rapid eye movement sleep increases. Preinjection of atropine at a dose that did not itself affect rapid eye movement sleep resulted in antagonism of CGS 21680, but not cyclohexaladenosine-induced rapid eye movement sleep. The differential sensitivity of these ligands to antagonism by atropine supports the conclusion that both A1 and A2a adenosine receptor subtypes in the reticular formation subserve agonist-induced rapid eye movement sleep and that they do so by independent mechanisms. The A2a mechanism requires the cholinergic system and may act through the increased release of acetylcholine. The A1 mechanism operates at a different locus possibly through an inhibition of GABA neurotransmission.

During rapid eye movement (REM) sleep, activity of non-retinal origin is propagated into central visual-system pathways in a manner similar, in pattern and intensity, to central visual-system activity that is exogenously generated in waking. It has been hypothesized that REM sleep, which is more abundantly represented early in life than later, functions to provide adjunct 'afferent' input for shaping synaptic connectivity during brain maturation. Here we present data that support this proposal. Recent studies have described a developmentally regulated form of in vitro long-term potentiation (LTP) in the visual cortex that is experience- and age-dependent. In immature rats, suppression of retinal activation of the visual system by removal of visual experience (dark rearing) extends the age when the developmentally regulated form of LTP can be produced. This study tests whether suppression of REM-state activation of the visual system also lengthens the developmental period in which this specific form of LTP can be elicited. Young rats were deprived of REM sleep by the multiple-small-platforms-over-water method during the typically latest week for induction of such LTP in slices of visual cortex. After this week, we could still induce LTP in slices from nearly all the REM-sleep-deprived rats (8/9) but not from age-matched rats that had not lost REM sleep (0/5). The control rats had been housed on large platforms that allow the animals to obtain REM sleep. Only body weights and the concentration of thyrotrophin-releasing hormone in the hypothalamus distinguished home-caged, normal-sleeping controls from both groups of platform animals. On all measures, stress levels were not dissimilar in the two platforms groups. After 7 days of behavioral suppression of REM sleep in immature rats, and consequent reduction of the intense, extra-retinal activity endogenously generated during this sleep state, we found that the period was extended in which developmentally regulated synaptic plasticity (LTP) could be elicited in slices of visual neocortex. These studies support the role of REM sleep and its associated neuronal activity in brain maturation.

Microinjection of cholinergic and adenosinergic agonists into the medial pontine reticular formation of rats produces long lasting increases in the time spent in rapid eye movement sleep. Several G-protein-coupled muscarinic and adenosinergic receptors share a common action of inhibition of adenylyl cyclase and inhibition of cyclic adenosine monophosphate. Inhibition of cyclic adenosine monophosphate has been implicated in the mechanism of rapid eye movement sleep induction in the cat. We sought to determine whether a direct inhibitor of adenylyl cyclase microinjected into the rat reticular formation at sites where muscarinic and adenosinergic agonists are effective in producing long lasting elevations in rapid eye movement sleep also result in similar effects on the sleep/wake cycle. The caudal, oral pontine reticular formation was unilaterally infused with 60 nl volumes of carbachol (0.1-1.1mM) and N(6)-cyclohexyladenosine (0.1mM) each within 1h of lights on. Sites effective for significantly elevating rapid eye movement sleep for the 8h following microinjection of both receptor agonists were additionally injected with the adenylyl cyclase inhibitor, SQ22,536 (0.1M). Pontine injections of SQ22,536 resulted in significant mean increases in rapid eye movement sleep time and rapid eye movement sleep period frequency at all of these sites. As with the receptor agonists, SQ22,536 did not alter latency to rapid eye movement sleep onset. Rapid eye movement sleep amounts were observed to be significantly elevated by SQ22,536 at two days, but not at four days, following a single microinjection. These data implicate inhibition of cyclic adenosine monophosphate in the pons of the rat as a mechanism involved in the long-term modulation of rapid eye movement sleep. This mechanism may underlie the homeostatic regulation exhibited by this sleep-state.

The cholinergic agonist carbachol (1.1 mM) and the adenosinergic agonist cyclohexyladenosine (0.1 mM) were microinjected (60 nl) into the region of the caudal, oral pontine reticular formation of the rat. Local intracerebral infusion of each receptor agonist resulted in significant, long-lasting (at least 8 h) elevations in rapid eye movement sleep without reduction in latency to onset. The effects of carbachol were reduced by the muscarinic receptor antagonist atropine, while those of cyclohexyladenosine were reduced by the adenosinergic receptor antagonist 8-cyclopentyltheophylline. Atropine failed to antagonize the long-term induction of rapid eye movement sleep following cyclohexyladenosine, but did appear to suppress increases in the first 2 h. Similarity of effects on sleep parameters and the lack of additivity when injected consecutively are consistent with these agonist ligands targeting the same cellular mechanisms through their respective receptors. These findings suggest that transitory increases in the pons of either acetylcholine or adenosine may underlie long-lasting elevations in the amount of rapid eye movement sleep. Adenosine may play a role in the increased rapid eye movement sleep following prolonged wakefulness, as well as following conditions of stress and learning.

Heme oxygenase (HO) catalyzes the oxidation of heme to produce carbon monoxide, which is considered to be a novel neuronal messenger in the brain and may play a role in neuronal development. The objective of this study was to determine the effects of in vitro, acute in vivo, and chronic in vivo ethanol exposure on HO activity in the hippocampus, frontal cerebral cortex, and cerebellum of the near-term fetal guinea pig. HO activity was determined using a gas chromatographic method to quantitate CO formation in the microsomal fraction of the homogenate of each selected brain region, incubated with saturating concentrations of heme, NADPH, and O2. Fetal body, brain, hippocampal, and cerebellar weights were recorded. In vitro ethanol exposure (25-100 mM) did not affect hippocampal, cerebral cortical, or cerebellar HO activity of the fetal guinea pig at gestational day (GD) 62 (term, about GD 68). Acute maternal oral administration of 4 g ethanol/kg maternal body weight at GD 62 did not affect HO activity in these three fetal brain areas compared with control fetuses (maternal administration of isocaloric sucrose or water). For chronic daily maternal oral administration of 4 g ethanol/kg maternal body weight throughout gestation, fetal body, brain, hippocampal, and cerebellar weights were decreased at GD 62 compared with isocaloric-sucrose/pair-fed and water treatment control groups. Furthermore, isocaloric-sucrose/pair-feeding treatment decreased fetal body and brain weights compared with water treatment. Chronic in vivo ethanol exposure did not alter HO activity in the near-term fetal hippocampus, frontal cerebral cortex, or cerebellum. This is the first study of the effect of ethanol exposure on HO activity in the developing brain of any species. The data demonstrate, for ethanol CNS teratogenesis in the guinea pig manifesting as fetal brain growth restriction, there is no associated change in HO activity in the hippocampus, frontal cerebral cortex, or cerebellum.

Introduction: This longitudinal study examined the prevalence and demographic correlates of unprotected insertive and receptive anal intercourse among HIV-positive gay and bisexual men who were aware of their serostatus., Methods: Participants (n = 395), sampled randomly at two HIV outpatient clinics in Los Angeles, completed two waves of self-administered questionnaires (separated by approximately 7-9 months) that measured sexual behaviors in the previous 60 days., Results: The cross-sectional prevalence of unprotected insertive anal intercourse was 11.2% at time 1 and 7.1% at time 2. Longitudinal analysis indicated that nearly 15% of the participants had engaged in that high-risk behavior either at time 1 or time 2 and approximately 4% had engaged in the behavior at each time period. Similar rates of unprotected receptive anal intercourse were observed. These high-risk activities were more prevalent with seropositive and unknown serostatus partners than with seronegative partners. The rate of anal intercourse risk behaviors was higher among asymptomatic men and among those who were exclusively gay., Conclusion: The findings demonstrate considerable differences in the prevalence of stable and occasional high-risk sexual behaviors among HIV-positive gay and bisexual men. Simple cross-sectional analyses cannot capture the stability or variation in behavior across time and, thus, may generate misleading conclusions about disease transmission, especially if the partner's HIV serostatus is not considered in the analysis. The findings indicate a need for focused safer-sex interventions for seropositive men. The HIV outpatient clinic is an ideal setting for such interventions.

Rat hepatic cytochrome P450 (P450) isozymes 1A1, 2C6, 2C11, 3A1 and 3A2 are targets for mechanism-based inactivation by the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC). It is of interest to determine whether similar P450 isozymes are targets of porphyrinogenic drugs in the chick embryo liver. The chick embryo expresses P450 2H1/2 isozymes, which are similar to the rat P450 2B1/2 isozymes, a polycyclic aromatic hydrocarbon-inducible P450 1A isozyme, and a pregnenolone 16 alpha-carbonitrile-inducible P450 3A isozyme. We have found previously that chick embryo hepatic P450 1A and 3A isozymes are targeted for in vitro mechanism-based inactivation by 4-ethyl DDC and by the sydnone 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS). Marked differences have been observed between the in vitro and in vivo effects of porphyrinogenic drugs on P450 isozymes. Thus, the first objective of this study was to determine whether chick embryo hepatic P450 1A and 3A isozymes are subject to in ovo inactivation by these porphyrinogenic compounds. Our second objective was to determine whether the chick embryo hepatic P450 2H isozyme(s) was subject to in ovo and in vitro inactivation by 4-ethyl DDC and TTMS. Using hepatic microsomes prepared from beta-naphthoflavone-, dexamethasone-, phenobarbital-, and glutethimide-induced 19-day-old chick embryos, we found that total P450 content was decreased significantly in microsomes prepared from all treatment groups following in ovo administration of 4-ethyl DDC and TTMS. Moreover, in ovo administration of both 4-ethyl DDC and TTMS caused a significant decrease of 7-ethoxyresorufin O-deethylase, erythromycin N-demethylase, and benzphetamine N-demethylase activities, which are selective catalytic markers for chick embryo hepatic P450 1A, 3A and 2H isozymes, respectively. In addition, in vitro administration of 4-ethyl DDC and TTMS caused mechanism-based inactivation of benzphetamine N-demethylase activity in microsomes from phenobarbital- and glutethimide-treated chick embryos, showing that the chick embryo hepatic P450 2H isozyme is a target for mechanism-based inactivation. Therefore, it was concluded that the chick embryo hepatic P450 1A, 2H and 3A isozymes serve as targets for both in ovo and in vitro mechanism-based inactivation by 4-ethyl DDC and TTMS.

Rat hepatic P4501A1 and 3A1/2 have been shown previously to be targets for mechanism-based inactivation by the 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), namely, 4-ethyl DDC and 4-isopropyl DDC. In this study we have shown that rat hepatic P4501A and P4503A are targets for mechanism-based inactivation by the sydnones, 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) and 3-(2-phenylethyl)-4-methylsydnone (PEMS). The dihydroquinoline, 2,4-diethyl-2-methyl-1,2-dihydroquinoline (DMDQ), caused mechanism-based inactivation of rat hepatic P4501A but not of P4503A. The P4501A isozyme(s) of chick embryo liver was found to share the ability of rat liver P4501A to serve as a target for mechanism-based inactivation by the dihydropyridines, 4-ethyl DDC and 4-isopropyl DDC, the sydnones, TTMS and PEMS, and the dihydroquinoline, DMDQ. A P4503A-like isozyme of chick embryo liver shared the ability of the rat liver P4503A isozyme(s) to serve as a target for mechanism-based inactivation by the dihydropyridines, 4-ethyl DDC and 4-isopropyl DDC, and the sydnone, TTMS, but not of the sydnone PEMS. The dihydropyridine, DDC, was found to serve as a mechanism-based inactivator of the chick embryo P4501A isozyme(s), but not of the P4503A isozyme(s), in contrast to its previously reported inactivity with both the rat hepatic P4501A1 and 3A1/2 isozymes.

Acetylcholine has been implicated as an important neurotransmitter in the mechanisms of thalamic activation. Cholinergic mechanisms are thought to directly underlie the high level of excitability observed in thalamic relay neurons during waking and rapid eye movement sleep. We sought to determine if the cholinergic responsiveness of neurons in the ventroposterior nuclei of the thalamus in rat is consistent with this view. Neurons in the chloral hydrate-anesthetized rat were studied with extracellular recording and microiontophoretic application of cholinergic agents. In most cases (63% of 63 cells), the ejection of the agonist, carbachol, had no observable effect on spontaneous activity. Facilitation (25%), inhibition (8%) and inhibition followed by facilitation (3%) were also observed. Carbachol ejections that by themselves were ineffective in altering spontaneous activity proved capable, in 93% of 28 cells, of antagonizing the uniformly facilitatory responses produced by glutamate ejection. The putative M1-selective, cholinergic agonist, McN-A-343, was also ineffective alone in altering spontaneous activity in the majority of cases (74% of 27 cells) and produced only inhibitory responses in the remaining seven neurons studied. Interacting applications of McN-A-343 and glutamate resulted, in all cases, in antagonism of glutamate facilitation (N = 12). The various responses to applied cholinergic agonists were all capable of being antagonized by muscarinic receptor-blocking agents. Both the high proportion of inhibitory responses and the antagonism of glutamate facilitatory responses suggest that ventroposterior neurons in the rat differ from other thalamocortical relay neurons in the rat and cat with regard to cholinergic responsiveness. Additionally, the lack of predominantly facilitatory responding renders it unlikely that cholinergic mechanisms directly underlie increases in excitability of ventroposterior neurons observed during waking and rapid eye movement sleep.

Carbon monoxide (CO) was employed to assess the role of ferrous hemoproteins in the vasodilation and biotransformation of glyceryl trinitrate (GTN) in rabbit aortic strips. These tissues were contracted submaximally with phenylephrine, exposed to CO for 5 min, and then incubated with 0.5 microM [1,3(14) C]GTN in the presence of CO for 2 min or 30 s. The [14C]GTN-induced relaxation of the strips was recorded, and [14C]GTN biotransformation to [14C]glyceryl dinitrate metabolites by the tissues was determined by thin-layer chromatographic-liquid scintillation spectrometric analysis. CO treatment inhibited neither GTN-induced relaxation nor GTN biotransformation to glycerol dinitrate metabolites. These results indicate that ferrous hemoproteins are probably not involved in the biotransformation of GTN that is associated with relaxation of the rabbit aortic strips.

A series of compounds that increase the activity of delta-aminolevulinic acid synthase (ALAS) in chick embryo hepatocyte cultures were studied for their effects on steady-state levels of mRNA for ALAS and phenobarbital-inducible cytochrome PB1 P450. N-Ethylprotoporphyrin IX (N-EtPP), which is believed to lower heme levels by inhibition of ferrochelatase (FC), had little effect on steady-state ALAS mRNA levels. 3,5-Diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4- isobutylpyridine (4-isobutyl DDC), which is believed to lower heme levels by repetitive destruction of the heme moiety of cytochrome P450, increased steady-state levels of ALAS mRNA levels approximately 2-fold. 3,5-Diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DCC) which inhibits FC activity and destroys the heme moiety of cytochrome P450, increased ALAS mRNA levels approximately 4-fold. A combination of N-EtPP and 4-isobutyl DDC produced a synergistic increase in ALAS mRNA levels to approximately 6-fold over control levels. The synergistic increase in ALAS activity observed previously with this combination can be explained, at least in part, by a synergistic increase in ALAS mRNA levels. Other porphyrinogenic agents, which function as mechanism-based inactivators of cytochrome P450 and elevate ALAS activity, were found to elevate ALAS mRNA. These compounds included 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS), 2,4-diethyl-2-methyl-1,2-dihydroquinoline (DMDQ), and 2,2,4-trimethyl-1,2,dihydroquinoline (TMDQ). The elevation of ALAS mRNA by these porphyrinogenic agents is probably due to their lowering of cellular heme levels by a combination of ferrochelatase inhibition and repetitive destruction of the heme moiety of cytochrome P450. The lowering of heme levels should result in an enhancement of ALAS mRNA half-life as it has been demonstrated by others that heme shortens the half-life of ALAS mRNA. It was of interest that some of these drug treatments also caused an elevation in steady-state levels of cytochrome PB1 P450 mRNA; the exception was TTMS, which along with its analogue 3-(2-phenylethyl)-4-methylsydnone (PEMS), did not alter cytochrome PB1 P450 mRNA levels. Increases in steady-state levels of cytochrome PB1 P450 mRNA subsequent to increases in steady-state levels of ALAS mRNA were observed with 4-ethyl DDC, 4-isobutyl DDC, DMDQ, and TMDQ. The data obtained with N-EtPP and a combination of N-EtPP and 4-isobutyl DDC on cytochrome PB1 P450 mRNA levels do not support the contention that heme functions as a positive regulator of cytochrome P450 gene expression.

The porphyrinogenicity of 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) is related to the process of mechanism-based destruction of cytochrome P450 (P450) heme, accompanied by conversion of heme to N-alkylprotoporphyrins (N-alkylPPs). Certain DDC analogues (4-isopropyl, 4-isobutyl, 4-hexyl) are weakly porphyrinogenic in comparison to the potent porphyrinogen, 4-ethyl DDC. We have examined the abilities of these DDC analogues to promote irreversible binding of radiolabeled heme to protein in rat liver microsomal preparations. The goals of this study were to determine whether DDC analogues with different porphyrinogenicities differ in the extents to which they cause heme adduct formation, and whether P450 isozymes differ in their capacities to catalyze heme covalent binding. Incubation of microsomes with NADPH alone promoted heme covalent binding, while loss of spectral P450 heme was minimal or absent. In microsomal incubations containing NADPH, the 4-ethyl, 4-isopropyl, and 4-isobutyl analogues caused heme covalent binding to extents which paralleled their P450 destructive activities. In contrast, 4-hexyl DDC caused less heme covalent binding as a function of P450 loss than the other analogues in microsomes from untreated and beta-naphthoflavone (beta NF)-treated rats. Thus, the weakly porphyrinogenic DDC analogues do not cause greater heme covalent binding than 4-ethyl DDC. Weak porphyrinogenicity, therefore, cannot be explained by diversion of the heme moiety of P450 from conversion to N-alkylPPs towards utilization for formation of heme-derived protein adducts. Treatment of rats with P450 inducing agents altered the degree to which DDC analogues caused heme covalent binding. The greatest heme adduct formation occurred in microsomes from untreated and dexamethasone (DEX)-treated rats, whereas treatment with phenobarbital and especially beta NF reduced heme covalent binding as a function of P450 loss. Thus, these microsomal studies suggest that constitutive P450 isozymes and members of the DEX-inducible P450IIIA subfamily appear to catalyze heme covalent binding, while beta NF-inducible forms such as P450IA1 (P450c) seem to be relatively inactive in this regard.

3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) and 3-(2-phenylethyl)-4-methylsydnone (PEMS) cause mechanism-based inactivation of rat hepatic microsomal cytochrome P-450 and the formation of N-alkylprotoporphyrins in rat liver. In the present study, we have shown that both TTMS and PEMS cause mechanism-based inactivation of chick embryo hepatic microsomal cytochrome P-450. TTMS also caused the inhibition of ferrochelatase activity, the accumulation of protoporphyrin IX, and an increase in the activity of delta-aminolevulinic acid synthase in chick embryo liver cell culture. PEMS was devoid of effect on ferrochelatase activity, porphyrin accumulation, and delta-aminolevulinic acid synthase activity. There are two possible explanations for the lack of effect of PEMS on heme biosynthesis: (1) the ring-A- and/or ring-B-substituted regiosomers of the N-phenylethyl- and N-phenylethenylprotoporphyrins which are produced during the mechanism-based inactivation of cytochrome P-450 by PEMS are too bulky to fit into the active site of ferrochelatase to inhibit its activity, in contrast to the N-vinylprotoporphyrin formed from TTMS; and (2) the N-alkylprotoporphyrins produced consist of the ring-C- and/or ring-D-substituted regioisomers, which are not inhibitors of ferrochelatase activity.

Herpes simplex virus type 2-transformed hamster embryo fibroblasts (333-8-9 cells) produce increased plasminogen activator (PA) compared with normal hamster cells. These cells produce undifferentiated fibrosarcomas at the inoculation site in newborn hamsters, and metastasize to the lungs. Using a direct PA assay, in which 125I-labeled plasminogen is cleaved, the optimum pH and osmolarity for detection of the 333-8-9 extracellular PA were pH 8.9 and approximately 150 mOsmol. Secretion of enzyme did not vary significantly on a per cell basis over cell densities from 0.1 to 8.0 X 10(7) cells/T-75 cm2 flask. This assay demonstrates that the 333-8-9 cells produce at least 20-fold greater levels of PA than normal cell counterparts. Based on the molecular weight (50-58 kDa) of secreted 333-8-9 cells PA and lack of fibrin stimulation, we conclude that it is a urokinase type PA. Subclonal lines of the 333-8-9 cells, selected for an increased PA phenotype were stable in culture, more tumorigenic and probably more metastatic. Correlation of these two events was examined by passaging 333-8-9 cells in vivo to select for greater tumorigenic potential and then determining the production of PA by the in vivo-derived sublines. The metastatic potential of the resulting cells was heterogeneous. Increased PA production upon increased passage in vivo did not always occur, whether the cells were passaged as subcutaneous tumors or as ascites tumors. Thus, while enzyme production correlated with tumorigenicity when selecting cells for an increased protease phenotype, this correlation was not observed when selecting for in vivo tumorigenicity. The results suggest that increased ability to make PA represents only one of multiple selective advantages for tumor growth.

Isosorbide dinitrate (ISDN) (at a concentration of 100 ng/ml) was incubated aerobically at 37 degrees in whole blood from five male and five female normal volunteers. Following incubation of the blood samples for 0, 30, 60, 120, 240 and 360 min, the samples were centrifuged and the plasma was assayed for ISDN. A linear relationship was observed between the logarithm of the concentration of ISDN remaining and incubation time, and there was a significant difference between the T1/2 of ISDN in blood from males (90.6 min) and females (161.4 min). Very little ISDN metabolism was observed when ISDN was incubated with plasma rather than whole blood. When erythrocytes, resuspended in saline, were incubated with ISDN, there was a time-dependent loss of ISDN from the saline incubation medium. Investigation of the soluble fraction obtained after hemolysis of these erythrocytes also showed a time-dependent loss of ISDN. The saline incubation medium contained sufficient concentrations of the two major ISDN metabolites (isosorbide 2- and 5-mononitrate) to account for the observed disappearance of ISDN. The results indicate that ISDN is metabolized in the cellular compartment of blood and that the metabolic rate in males is greater than that in females.

The levels of the cellular free heme pool in chick embryo hepatocyte culture were lowered using N-ethylprotoporphyrin IX (N-ethylPP) and analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), and the effect on delta-aminolevulinic acid synthase (ALAS) was examined. N-EthylPP, which lowers cellular heme levels by inhibiting ferrochelatase activity, produced an induction of ALAS activity to 444% of control at 3 hr after its administration. 4-Ethyl DDC, which lowers heme levels by destroying the heme moiety of cytochrome P-450 and lowering ferrochelatase activity, caused an induction of ALAS to 565% of control at 12 hr after administration. 4-Isobutyl DDC, which lowers heme levels by destroying the heme moiety of cytochrome P-450, induced the activity of ALAS to 289% of control at 3 hr after administration. This indicates that ferrochelatase inhibition is a more important mechanism of heme lowering than alkylation of cytochrome P-450 heme when both heme-depleting mechanisms are acting in chick embryo liver cells. It was anticipated that administration of a combination of 4-isobutyl DDC plus N-ethylPP would mimic the effect of 4-ethyl DDC. However, this combination induced ALAS activity to levels that were much greater than those observed after 4-ethyl DDC (1257% of control at 12 hr). This synergistic induction may be attributable to lowering of free heme levels to the point where transcription, translation, and translocation of ALAS are all derepressed.

A method employing a cannula-guidetube system with attached electrodes is described that permits infusions into discrete brain areas and simultaneous recording of multiunits or single units. This method can be used to determine the immediate local effects of drug infusion, and also to acquire electrophysiological feedback information in the process of placement and identification of cell populations at the injection site.

Our studies lead us to suggest that in order for an organic chemical to cause porphyrin accumulation in chick embryo hepatocyte culture, it must be: 1. lipophilic; 2. resistant to biotransformation by phase I or phase II reactions to compounds which are devoid of biological activity; and 3. capable of interacting with cytochrome P-450 with concomitant destruction of the heme moiety or the generation of reactive oxygen species. The interaction of porphyrin-inducing chemicals with cytochrome P-450 results in lowering of a regulatory "free heme" pool. The "free heme" pool may be lowered by inhibiting one or more of the enzymes of heme biosynthesis or by destruction of the heme moiety of cytochrome P-450. Specific chemical features enable organic chemicals to inhibit ferrochelatase activity or to destroy the heme moiety of cytochrome P-450. Chemicals with a wide variety of structures are able to inhibit uroporphyrinogen decarboxylase activity.

Uroporphyrin, heptacarboxylic acid porphyrin and coproporphyrin were the major porphyrins to accumulate when phenobarbital, nifedipine, 3,5-diethoxycarbonyl-2,4,6-trimethylpyridine (Ox-DDC) and 3,3',4,4'-tetrachorobiphenyl (TCBP) were added to chick embryo hepatocyte culture. This pattern of porphyrin accumulation is consistent with the demonstration that these chemicals inhibit uroporphyrinogen decarboxylase (UROG-D). The degree of UROG-D inhibition observed was: TCBP (39%), Ox-DDC (39%), nifedipine (25%) and phenobarbital (50%). Since significant UROG-D inhibition was observed when the bulk of the porphyrins in the crude enzyme preparation was removed by gel filtration, it is unlikely that porphyrins produce the enzyme inhibition. When succinylacetone, a potent inhibitor of delta-aminolevulinic acid dehydratase, was coadministered with Ox-DDC, phenobarbital, TCBP and nifedipine, UROG-D inhibition was not observed. These results suggest that heme biosynthesis must proceed in order for xenobiotic mediated UROG-D inhibition to occur.