[1-deamino-4-cyclohexylalanine] arginine vasopressin: a potent and specific agonist for vasopressin V1b receptors.

To date, there are no vasopressin (VP) agonists that exhibit a high affinity and selectivity for the VP V1b receptor with respect to the V1a, V2, and oxytocin receptors. In this study, we describe the synthesis and pharmacological properties of [1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha4]AVP). Binding experiments performed on various membrane preparations revealed that d[Cha(4)]AVP exhibits a nanomolar affinity for V1b receptors from various mammalian species (rat, bovine, human). It exhibits high V1b/V1a and V1b/oxytocin selectivity for rat, human, and bovine receptors. Furthermore, it exhibits high V1b/V2 specificity for both bovine and human vasopressin receptors. Functional studies performed on biological models that naturally express V1b receptors indicate that d[Cha4]AVP is an agonist. Like VP, it stimulated basal and corticotropin-releasing factor-stimulated ACTH secretion and basal catecholamine release from rat anterior pituitary and bovine chromaffin cells, respectively. In vivo experiments performed in rat revealed that d[Cha4]AVP was able to stimulate both ACTH and corticosterone secretion and exhibits negligible vasopressor activity. It retains about 30% of the antidiuretic activity of VP. This long-sought selective VP V1b receptor ligand with nanomolar affinity will allow a better understanding of V1b-mediated VP physiological effects and is a promising new tool for V1b receptor structure-function studies.

Prenatal development of sleep-wake patterns in sheep.

Chronic intrauterine monitoring of electrocortical activity, eye movements, and electromyographic activity of the nuchal muscles has permitted the use of established polygraphic criteria to study the ontogenetic development of organized sleep-wake patterns in the fetal lamb in utero. Sixty-five polygraphic recordings obtained from 21 fetal lambs ranging from 112 to 144 days of gestation (term = 145 days) were analyzed. The results show that the transition from disorganized to organized behavioral state patterns occurs at approximately 115-120 days (0.8 term). The physiologic characteristics of the arousal, quiet sleep (QS), and REM sleep (REMS) states are qualitatively similar to those in the adult. There is initially a high proportion of REMS (greater than 50%), with only 11% time spent in arousal. The direct transition from arousal to REMS without a QS period is frequently seen in the fetus at all ages. Quantitative changes in the distribution of behavioral states continue throughout gestation. There is a significant decrease in the incidence of REMS, with a small increase in arousal and QS. The duration of QS and REMS episodes increases with age until a steady level is achieved by 130 days. There is also an increase in the frequency of occurrence of arousal periods, with no change in episode duration.

Prenatal morphine exposure and sleep-wake disturbances in the fetus.

We have previously shown that acute exposure to low-dose morphine stimulates arousal and breathing movements in the fetal lamb. We now report on the effects of subacute low-dose morphine exposure on the regulation of fetal sleep-wake behavior and breathing patterns. Morphine was infused to 11 fetal lambs (121-129 days gestation) at a constant rate of 400 micrograms/h for 7 days via a mini osmotic pump implanted subcutaneously in the maternal flank and connected directly to an indwelling catheter in the fetal vena cava. On day 1, morphine resulted in a state of arousal in all fetuses, with loss of quiet sleep and rapid eye movement (REM) sleep. This response was greatly reduced by day 2 and was insignificant by day 3, despite continuous drug exposure. There was no decrease in plasma morphine levels. Two fetuses died during morphine exposure. Upon removal of the pump, all fetuses exhibited disturbances in their sleep cycles within 1-3 h, with an increase in arousal time and loss of REM sleep. The duration of the arousal and quiet sleep episodes was also greatly reduced. Such disturbances were noted for 3-4 days after termination of morphine infusion. Four fetuses were delivered prematurely (131-136 days) during this period. These results demonstrate the rapid onset of tolerance to low-dose morphine exposure in the fetus and the development of physical dependence, as manifested by a mild abstinence syndrome characterized by sleep-wake disturbances.

Amniotic fluid transfer of meperidine from maternal plasma in early pregnancy.

Concurrent samples of maternal plasma and amniotic fluid were collected from 40 subjects during the second trimester of pregnancy following a single intramuscular dose of meperidine (100 mg). Maternal meperidine plasma levels were highest in samples collected from 15 to 50 minutes after drug administration. Thereafter the level declined during the next 2 hours. Meperidine was not detected in amniotic fluid until 30 minutes after the intramuscular dose. We estimated that an apparent equilibrium was reached between plasma and amniotic fluid at 120 to 155 minutes after the drug was given to the mother. Normeperidine was not detected in either maternal plasma or amniotic fluid during the time course of this study.

Nociceptin/orphanin FQ increases blood pressure and heart rate via sympathetic activation in sheep.

This study was undertaken to examine the cardiovascular effects of nociceptin/Orphanin FQ (OFQ). Nociceptin/OFQ (10-300 nmol/kg, IV) stimulates an increase in mean blood pressure (MBP) and heart rate (HR) in chronically catheterized sheep. Pretreatment with phenoxybenzamine (5 mg/kg) attenuated the pressor response, consistent with sympathetically mediated vasoconstriction. Furthermore, the lack of a reflex bradycardia suggests either blunting of the baroreflex by nociceptin/OFQ or direct beta-adrenergic activation. The bradycardic response to norepinephrine (0.6 microg/kg, IV) remained intact after nociceptin/OFQ administration, demonstrating that nociceptin/OFQ does not blunt the baroreflex. Additionally, the increase in HR was completely reversed by pretreatment with propranolol. These data suggest that nociceptin/OFQ plays a role in cardiovascular regulation via sympathetic activation.

Respiratory depression after intravenous administration of delta-selective opioid peptide analogs.

We compared the effects of three micro-(DAMGO, DALDA, TNPO) and three delta-(DPDPE, DELT, SNC-80) opioid agonists on arterial blood gas after IV administration in awake sheep. None of the mu agonists altered pO2, pCO2 or pH. All three mu agonists decreased pO2 increased pCO2 and decreased pO2, and this effect was not sensitive to naloxone or TIPPpsi, a delta-antagonist, suggesting that it is not mediated by beta-opioid receptors. When administered to pregnant animals, there were significant changes in fetal pCO2 and pH. It may be possible to develop delta-selective opioid agonists which do not produce respiratory depression.

A distributed microcomputer-controlled system for data acquisition and power spectral analysis of EEG.

A relatively powerful and inexpensive microcomputer-based system for the spectral analysis of the EEG is presented. High resolution and speed is achieved with the use of recently available large-scale integrated circuit technology with enhanced functionality (INTEL Math co-processors 8087) which can perform transcendental functions rapidly. The versatility of the system is achieved with a hardware organization that has distributed data acquisition capability performed by the use of a microprocessor-based analog to digital converter with large resident memory (Cyborg ISAAC-2000). Compiled BASIC programs and assembly language subroutines perform on-line or off-line the fast Fourier transform and spectral analysis of the EEG which is stored as soft as well as hard copy. Some results obtained from test application of the entire system in animal studies are presented.

U50,488H-induced pressor effect in the ovine foetus is mediated by sympathetic activation and vasopressin.

The purpose of this study was to investigate the mechanism behind the increase in blood pressure observed after intravenous administration of U50,488H (trans-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide), a selective Kappa-opioid receptor agonist, to the ovine foetus. Intravenous administration of U50,488H (1.0 mg/kg) to the foetus resulted in an immediate increase in foetal blood pressure which lasted 15 min. Pretreatment with phentolamine (1.0 mg/kg i.v.) completely blocked the immediate (1-4 min) pressor effect of U50,488H, but not the subsequent increase in blood pressure after 5 min. In contrast, pretreatment with the vasopressin antagonist ([beta-mercapto-beta, beta-cyclopentamethylene-propionyl)-O-Me2-Tyr,Arg8]vasopressin, 0.06 mg/kg) did not affect the immediate pressor effect of U50,488H, but completely blocked the latter increase in blood pressure after 4 min. These data suggest that the immediate increase in blood pressure caused by U50,488H was mediated by sympathetic activation which was then further sustained by a release of vasopressin.

Cardiovascular and respiratory actions of U50,488H in the unanaesthetized ovine foetus.

In an effort to evaluate the feasibility of kappa-opioid receptor agonists for use in pregnancy, we have investigated the actions of U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide) on cardiovascular and respiratory control in the unanaesthetized ovine foetus. Intravenous administration of U50,488H (1.0 mg/kg) to the foetus resulted in an immediate increase in foetal blood pressure (P < 0.0001) and heart rate (P < 0.0001) which lasted 15 min, followed by a prolonged loss of heart rate variability for up to 3 h. There was also a significant suppression of foetal breathing movements for 2-3 h (P < 0.008). Pretreatment with naloxone (12 mg/h) completely blocked the hypertensive and tachycardiac response to U50,488H, but was unable to prevent the loss of variation in heart rate or respiratory depression. These data suggest that U50,488H can exert direct cardiovascular and respiratory actions in the ovine foetus via both opioid and non-opioid mechanisms. The naloxone-insensitive suppression of foetal breathing would severely limit the use of U50,488H as an obstetrical analgesic.

Opioid-induced stimulation of fetal respiratory activity by [D-Ala2]deltorphin I.

[D-Ala2]deltorphin I effects on fetal respiratory activity was characterized to determine the role delta-opioid receptors play in modulating fetal respiratory activity. [D-Ala2]deltorphin I, infused at 0.3 or 100 micrograms/h, intracerebroventricularly (i.c.v.), stimulated fetal respiratory activity without changing blood pH, PCO2 or PO2. Stimulation by 0.3 micrograms/h, but not 100 micrograms/h, was blocked by i.c.v. infusion of the delta-opioid receptor antagonist, naltrindole. Stimulation by 100 micrograms/h was blocked by the mu 1-opioid receptor antagonist naloxonazine. These data suggest stimulation of fetal respiratory activity by 0.3 micrograms/h [D-Ala2]deltorphin I are mediated specifically through delta-opioid receptors; while [D-Ala2]deltorphin I at 100 micrograms/h is no longer selective for the delta-opioid receptor, and the stimulation may be mediated through the mu 1-opioid receptor.

Behavioral states and their ontogeny: animal studies.

Despite numerous attempts in describing the many underlying events correlated with changes in behavioral states, the biological significance of state alternation remains unknown. There does not yet exist a satisfactory explanation for the biological need for such state changes, nor does there exist sufficient physiological evidence to explain the underlying processes leading to these state transitions. Nonetheless, the study of these ontogeny of behavioral states is important because it shows the progressive ability of the organism to integrate multiple neuronal systems. Available literature suggests that some rudimentary form of behavioral state is established in fetal life throughout the animal kingdom, although there is substantial difference in the degree and rate of development of organized behavioral states among the different animal species. Highly organized behavioral states that involve concurrence of many physiologic systems are present in the third trimester in the fetal lamb, while they are not seen until 2 to 3 weeks postnatally in the rat, rabbit, and cat. Subsequent maturation consists primarily of increased cycle length in all species. In addition, there is some evidence that a circadian rhythm may be superimposed on the ultradian rhythm of behavioral states in immature animals, but there is currently no information on the ontogenesis of this circadian rhythm. It is currently hypothesized that behavioral states cycling in the adult is generated by the interaction of multiple, anatomically distributed sets of neurons. Electrophysiological and pharmacological studies have suggested that both the cholinergic and aminergic neuronal systems play important roles in the maintenance of state cycling. Studies in the fetal lamb also support these proposed mechanisms in early development.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of opiates on fetal behavioral activity in utero.

The effects of methadone and morphine on fetal behavior were studied following their administration to either the mother (5micrograms/kg/min) or fetus (0.5micrograms/kg maternal wt./min) using the chronically-instrumented pregnant ewe model. Fourteen unanesthetized, unrestrained pregnant ewes between 130-141 days gestation (term 145 days) were studied 9-28 days following the surgical implantation of cannulae and electrodes to monitor fetal heart rate, blood pressure, breathing movements, eye movements, muscular and electrocortical activity. The three behavioral states of quiet sleep, rapid eye movement sleep and arousal could be distinguished using these parameters. Both drugs, by both routes, resulted in fetal arousal, largely at the expense of quiet sleep. While morphine and methadone were equipotent in suppressing quiet sleep following infusion to the fetus, methadone was three times more potent than morphine after maternal infusion. This potency difference is consistent with the results of prior pharmacokinetic studies demonstrating that fetal exposure to maternally administered methadone is approximately three times greater than that to maternally administered morphine.

The relationship between maternal and fetal plasma protein binding of methadone in the ewe during the third trimester.

The binding of methadone to maternal and fetal plasma proteins was determined throughout the third trimester in the pregnant ewe. Blood was sampled from chronic indwelling catheters placed in the maternal aorta and fetal aorta. Methadone binding was determined by use of equilibrium dialysis with (3H)-methadone. Maternal binidng ranged from 50.4 to 89.5%, with a mean of 76.2 +/- 1.3 (SE)%. Fetal binding was initially significantly lower than maternal binding, but increased rapidly in the last two weeks before parturition. Prior to 130 days gestation, the ratio of fetal binding to maternal binding was 0.40 +/- 0.03. This binding ratio increased to 0.82 +/- 0.08 in the last few days of pregnancy. Preliminary results suggested that maternal binding was higher in the early post-partum period. These results demonstrate that the relationship between maternal and fetal plasma binding of methadone changes rapidly towards the end of pregnancy, and fetal binding approaches maternal binding at parturition.

Effects of single and repeated marijuana smoke exposure on fetal EEG.

The effect of single and repeated marijuana smoke exposure on fetal EEG was investigated in the chronic fetal lamb model using power spectral analysis. Maternal inhalation of marijuana smoke (n = 9) resulted in a significant reduction in total power and power distribution in the delta (1-4 Hz) band, and an increase in power distribution in the faster frequencies in the first h after smoke inhalation. These EEG changes were not observed following maternal inhalation of placebo smoke (n = 5), nor in animals with 3-5 prior exposures to marijuana smoke (n = 5). These results suggest that the effects of marijuana smoke exposure on fetal EEG is short-lived and tolerance develops rapidly with repeated exposure.

Effects of the delta-opioid agonist, [D-Pen2,D-Pen5]-enkephalin, on fetal lamb EEG.

Opiates are known to exert biphasic effects on level of arousal, with excitation at low doses and depression at higher doses. It has been suggested that this dual excitatory and depressant actions of opiates may be mediated by different receptor subtypes. We have previously shown that activation of mu 1-opioid receptors evoked EEG activation in the fetal lamb. The purpose of the present study was to quantitate the effects of DPDPE, a highly selective delta-opioid agonist, on fetal EEG. When infused ICV (4.6-154 nmol/h), DPDPE elicited dose-dependent activation of fetal EEG, with a reduction in power distribution in the delta (1-4 Hz) band, and an increase in the beta (15-32 Hz) band. This activation was reflected by an increase in the spectral edge frequency. This EEG activation was greatly attenuated at DPDPE doses greater than 154 nmol/h, resulting in a U-shaped dose-response curve. The EEG activation was completely blocked by naloxone or naltrindole (delta antagonist), but not by naloxonazine (mu 1 antagonist). These results indicate that the activation of delta-opioid receptors will evoke EEG activation in the fetal lamb.

Maternal marijuana smoking alters respiratory timing in the fetal lamb.

The effect of single and repeated maternal marijuana smoke exposure on fetal breathing movements (FBMs) was investigated in 13 fetal lambs in the third trimester. These animals were surgically instrumented for long-term intrauterine recording of diaphragmatic electromyogram (EMG). Maternal inhalation of marijuana smoke [1.84% tetrahydrocannabinol (THC)] increased FBMs and resulted in a more continuous and regular breathing pattern. There was a significant increase in the number of breaths/h (p < 0.01) and the incidence of FBMs (p < 0.001) in the second hour. Breathing activity returned to presmoke level by the third hour. Duration of the longest breathing epoch was significantly increased from 16.8 +/- 3.3 min to 31.9 +/- 5.2 min (p < 0.005). Instantaneous breathing rate was much more stable in the second hour after marijuana exposure (p < 0.01). Inhalation of placebo smoke did not result in any significant change in either overall breathing activity or continuity and stability of the breathing rate. The effects of marijuana smoke on fetal breathing were not observed after repeated smoke exposure. These results suggest that tolerance develops rapidly to the respiratory stimulating effect of marijuana smoke in the fetus.

Serendipity and the discovery of novel compounds that restore mitochondrial plasticity.

The mitochondrial electron transport chain (ETC) plays a central role in energy generation in the cell. Mitochondrial dysfunctions diminish adenosine triphosphate (ATP) production and result in insufficient energy to maintain cell function. As energy output declines, the most energetic tissues are preferentially affected. To satisfy cellular energy demands, the mitochondrial ETC needs to be able to elevate its capacity to produce ATP at times of increased metabolic demand or decreased fuel supply. This mitochondrial plasticity is reduced in many age-associated diseases. In this review, we describe the serendipitous discovery of a novel class of compounds that selectively target cardiolipin on the inner mitochondrial membrane to optimize efficiency of the ETC and thereby restore cellular bioenergetics in aging and diverse disease models, without any effect on the normal healthy organism. The first of these compounds, SS-31, is currently in multiple clinical trials.

Mitochondrial pharmacology: its future is now.

Mitochondrial medicine is an evolving discipline whose importance derives from the central function of mitochondria in adenosine triphosphate (ATP) production, generation of reactive oxygen species, and cell death by necrosis or apoptosis. Consequently, mitochondrial dysfunction plays an important role in the progression of aging and the pathophysiology of many common diseases and off-target drug effects. This provides an impetus for the development of mitochondrial pharmacology, and some promising therapeutic targets for mitochondrial protective therapy have been identified.

Targeting mitochondrial cardiolipin and the cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis.

BACKGROUND AND PURPOSE: Cardiolipin plays an important role in mitochondrial respiration and cardiolipin peroxidation is associated with age-related diseases. Hydrophobic interactions between cytochrome c and cardiolipin converts cytochrome c from an electron carrier to a peroxidase. In addition to cardiolipin peroxidation, this impedes electron flux and inhibits mitochondrial ATP synthesis. SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH2 ) selectively binds to cardiolipin and inhibits cytochrome c peroxidase activity. Here, we examined whether SS-31 also protected the electron carrier function of cytochrome c. EXPERIMENTAL APPROACH: Interactions of SS-31 with cardiolipin were studied using liposomes and bicelles containing phosphatidylcholine alone or with cardiolipin. Structural interactions were assessed by fluorescence spectroscopy, turbidity and nuclear magnetic resonance. Effects of cardiolipin on electron transfer kinetics of cytochrome c were determined by cytochrome c reduction in vitro and oxygen consumption using mitoplasts, frozen and fresh mitochondria. KEY RESULTS: SS-31 interacted only with liposomes and bicelles containing cardiolipin in about 1:1 ratio. NMR studies demonstrated that the aromatic residues of SS-31 penetrated deep into cardiolipin-containing bilayers. SS-31 restored cytochrome c reduction and mitochondrial oxygen consumption in the presence of added cardiolipin. In fresh mitochondria, SS-31 increased state 3 respiration and efficiency of ATP synthesis. CONCLUSIONS AND IMPLICATIONS: SS-31 selectively targeted cardiolipin and modulated its interaction with cytochrome c. SS-31 inhibited the cytochrome c/cardiolipin complex peroxidase activity while protecting its ability to serve as an electron carrier, thus optimizing mitochondrial electron transport and ATP synthesis. This novel class of cardiolipin therapeutics has the potential to restore mitochondrial bioenergetics for treatment of numerous age-related diseases.