Abdulghani A. Houdi

High performance liquid chromatographic analysis of the pharmacologically active quinones and related compounds in the oil of the black seed (Nigella sativa L.)

An HPLC method for quantifying the putative pharmacologically active constituents: thymoquinone (TQ), dithymoquinone (DTQ), thymohydroquinone (THQ), and thymol (THY), in the oil of Nigella sativa seed is described. Extraction of the constituents from the oil was carried out using C18 PrepSep mini columns followed by quantification of the recovered constituents by HPLC on a reversed-phase muBondapak C18 analytical column, using an isocratic mobile phase of water:methanol:2-propanol (50:45:5% v/v) at a flow rate of 2 ml min(-1). UV detection was at 254 nm for TQ, DTQ, and THY, and at 294 nm for THQ. The above four compounds were separated with good resolution, reproducibility, and sensitivity under these conditions. This analytical method was used to quantify the above four constituents in a commercial sample of N. sativa seed oil, and provides a good quality control methodology for the pharmacologically active components in this widely used natural remedy.

Nicotine protects against μ-opioid receptor antagonism by β-funaltrexamine: Evidence for nicotine-induced release of endogenous opioids in brain

We have hypothesized that some effects of nicotine are mediated through endogenous opioids. This study was designed to demonstrate in rats that nicotine releases endogenous opioids in brain. In the control group, subcutaneous morphine (8 mg/kg) produced analgesia or antinociception as measured by prolongation of tail flick latency. Intracerebroventricular administration 24 h earlier of beta-funaltrexamine (beta-FNA, 2.5 micrograms), an antagonist which irreversibly alkylates opioid receptors, markedly reduced (66%) morphine analgesia. Subcutaneous administration of nicotine (0.1 mg/kg) prior to beta-FNA attenuated (31%) the inhibitory effect of beta-FNA on morphine analgesia. These data support our hypothesis that endogenous opioids released by nicotine bind to mu-opioid receptors in brain and protect them against inactivation by beta-FNA.

Opioid mediation of cocaine-induced hyperactivity and reinforcement

The mechanisms by which cocaine produces hyperactivity and reinforcement remain poorly understood. Since reinforcement is also a property of other drugs of abuse including opiates, we examined the possible mediation of these cocaine-induced behaviors by endogenous opioid peptides. In this study, we have confirmed reports that cocaine increases locomotor activity and conditioned place preference in rats. We have also demonstrated that opioid receptor blockade with naloxone antagonizes completely the locomotor-activating effect of cocaine and attenuates the strength of the place preference conditioning produced by cocaine. These data support the thesis that endogenous opioids are involved in mediation of cocaine-induced behavior.

Opioids intrinsically inhibit the genesis of mouse cerebellar granule neuron precursors in vitro: differential impact of μ and δ receptor activation on proliferation and neurite elongation

Although opioids are known to affect neurogenesis in vivo, it is uncertain the extent to which opioids directly or indirectly affect the proliferation, differentiation or death of neuronal precursors. To address these questions, the intrinsic role of the opioid system in neurogenesis was systematically explored in cerebellar external granular layer (EGL) neuronal precursors isolated from postnatal mice and maintained in vitro. Isolated neuronal precursors expressed proenkephalin‐derived peptides, as well as specific μ and δ, but negligible κ, opioid receptors. The developmental effects of opioids were highly selective. Morphine‐induced μ receptor activation inhibited DNA synthesis, while a preferential δ2‐receptor agonist ([d‐Ala2]‐deltorphin II) or Met‐enkephalin, but not the δ1 agonist [d‐Pen2, d‐Pen5]‐enkephalin, inhibited differentiation within the same neuronal population. If similar patterns occur in the developing cerebellum, spatiotemporal differences in endogenous μ and δ opioid ligand–receptor interactions may coordinate distinct aspects of granule neuron maturation. The data additionally suggest that perinatal exposure to opiate drugs of abuse directly interfere with cerebellar maturation by disrupting normal opioid signalling and inhibiting the proliferation of granule neuron precursors.

NICOTINE-INDUCED ALTERATION IN TYR-GLY-GLY AND MET-ENKEPHALIN IN DISCRETE BRAIN NUCLEI REFLECTS ALTERED ENKEPHALIN NEURON ACTIVITY

Nicotine acts in CNS, but the pathways and mechanisms of its actions are poorly understood. Recent studies suggest an interaction between brain nicotinic receptors and endogenous opioid peptides. Acute administration of nicotine may alter enkephalin release without affecting brain enkephalin level. Tyr-Gly-Gly has been shown previously to be an extraneuronal metabolite of opioid peptides derived from proenkephalin A. Concentrations of Tyr-Gly-Gly in brain were used to provide an index of enkephalin release in vivo. Thus we examined the thesis that nicotine alters brain neuronal enkephalin release, by measuring Tyr-Gly-Gly levels in specific brain nuclei from rats treated with nicotine 0.3 mg/kg SC 10 min before decapitation. Of 30 brain regions investigated, acute nicotine increased Tyr-Gly-Gly immunoreactivity in nucleus accumbens and in lower brain stem areas including dorsal raphe, pontine reticular formation, gigantocellular reticular formation, locus coeruleus, sensory trigeminal nucleus and the caudal part of ventrolateral medulla oblongata. Concomitantly, nicotine produced a significant decrease in native Met-enkephalin in central amygdala, flocculo-nodular lobe of cerebellum, caudal part of the ventrolateral medulla and intermediolateral cell column of the spinal cord. It is probable that the effects of nicotine to increase Tyr-Gly-Gly and alter Met-enkephalin concentration are mediated by nicotine-induced release of enkephalin at these brain sites. Furthermore, some of the physiologic and pharmacologic effects of nicotine may be mediated by such enkephalin release.

Nicotine-induced alterations in brain regional concentrations of native and cryptic Met- and Leu-enkephalin.

The distribution of cryptic forms (larger enkephalin-containing peptides) in neostriatum, hypothalamus, spinal cord T3-L1 and neurointermediate lobe of pituitary were determined by radioimmunoassay. Optimal conditions for enzymic hydrolysis of the cryptic enkephalins by trypsin and carboxypeptidase B were established. The proportion of total Met- and Leu-enkephalin represented by native pentapeptide varied markedly among these central nervous system regions. Also, the distributions of native and cryptic Met-enkephalin were distinct from that of Leu-enkephalin. Chromatographic separation by HPLC of immunoreactive Met-enkephalin peptides revealed only two peaks corresponding to Met-enkephalin and Met-enkephalin sulfoxide in rather equal amounts. Hydrolysis of cryptic Met-enkephalin also produced only two HPLC-separable peaks of immunoreactive Met-enkephalin, again corresponding to Met-enkephalin and Met-enkephalin sulfoxide. Bioactivity of cryptic striatal Met-enkephalin after hydrolysis was demonstrated by antinociception and catalepsy in rats following its intracerebroventricular injection. Repeated short-term administration of nicotine, 0.1 mg/kg IP six times at 30 min intervals, produced significant increases in native and cryptic Met-enkephalin in striatum, consistent with an increase in neuronal release of Met-enkephalin together with increases in synthesis and processing of proenkephalin A in this brain region. This regimen of nicotine also decreased levels of native Met-enkephalin and of both native and cryptic Leu-enkephalin in neurointermediate lobe, consistent with nicotine-induced release of both proenkephalin A- and prodynorphin-derived peptides from neurointermediate lobe.

Effect of nicotine use and withdrawal on brain preproenkephalin A mRNA

Although the effect of nicotine on brain neurotransmitters and behavior has been studied, the mechanism(s) by which nicotine contributes to tobacco use remains unclear. One transmitter that may relate to long-term nicotine use and its withdrawal is enkephalin, a five-amino acid opioid peptide derived from the proenkephalin A family. In the present study we determined the effect of acute and chronic nicotine treatment and its withdrawal on preproenkephalin A mRNA levels (PPE mRNA) in specific rat brain regions using Northern blot analysis. Acute treatment with nicotine produced a significant increase in PPE mRNA in striatum and hippocampus. Chronic treatment with nicotine caused a significant decrease in PPE mRNA in these brain regions. In both striatum and hippocampus there was a rebound increase in PPE mRNA 24 h after nicotine cessation which approached the saline level 7 days later. Nicotine withdrawal 24 h following nicotine cessation, caused a significant increase in PPE mRNA in both brain regions. These effects of nicotine were blocked by pretreating rats with the nicotinic antagonist, mecamylamine. These data strongly suggest that brain opioid system(s) are involved in mediating nicotinic responses and its withdrawal and may have clinical implications in treating nicotine addiction.

3-O-Acetylmorphine-6-O-sulfate: A potent, centrally acting morphine derivative

In view of the potent analgesia exhibited by the apparent structurally dissimilar morphine-6-O-glucuronide (M6G) and morphine-6-O-sulfate (M6S) conjugates of morphine, we have examined the effect of structural modification of M6S on analgesic activity, using the tail-flick test. Changes in the M6S structure were made that would affect the lipophilicity and polarity of the molecule. Subcutaneous (sc) and intracerebroventricular (ICV) administration of equimolar doses of morphine, M6S, 3-O-acetylmorphine-6-O-sulfate (M3A6S), 3-O-benzoylmorphine-6-O-sulfate (M3B6S), and 3-O-acetyl-N-methylmorphinium-6-O-sulfate (MM3A6S) were employed. M6S and M3A6S exhibited a longer duration of action and greater activity compared to morphine after SC and ICV administration. However, M3B6S and MM3A6S in doses equimolar to that of morphine were found to be inactive after both SC and ICV administration. In addition, M3A6S showed the highest potency in inhibiting electrically stimulated guinea pig ileum followed by M6S and M3B6S. Moreover, both M6S and M3A6S displayed a greater affinity than that of morphine to mu and kappa 3 receptor sites in guinea pig brain homogenate. In contrast, the nonanalgesic compounds M3B6S and MM3A6S showed weak receptor binding ability compared to morphine. These results indicate that lipophilicity alone is not a determinant of analgesic activity in these novel morphine derivatives. These modified effects of morphine by the conjugations at the 3- and 6-position, appear to be due to their altered interactions with opioid receptors.

Enantioselective metabolism during continuous administration of S-(−)- and R-(+)-nicotine isomers to guinea-pigs

Abstract— The S‐(−)‐ and R‐(+)‐nicotine isomers were administered subcutaneously via Alzet osmotic pumps to male Hartley guinea‐pigs (n = 5 with each isomer) over a 23‐day period. Estimated dosage rate throughout the experiment was 0.6 mg−1. Urine samples were collected over this time and the levels of urinary oxidative and N‐methylated nicotine metabolites were measured by cation‐exchange HPLC analysis. S‐(−)‐Nicotine formed only oxidative metabolites, whereas the R‐(+)‐isomer formed both oxidative and N‐methylated metabolites. 3′‐Hydroxycotinine and nicotine‐1′‐oxide were major metabolites of both enantiomers; cotinine and nornicotine were only minor metabolites. The major N‐methylated metabolite of R‐(+)‐nicotine was N‐methylnicotinium ion; N‐methylcotininium ion and N‐methylnornicotinium ion were also identified as metabolites of this nicotine isomer. Total N‐methylated quaternary ammonium metabolites accounted for 15 to 20% of the administered dose of R‐(+)‐nicotine. An interesting enantioselective reduction in the percent of oxidative urinary metabolites formed from S‐(−)‐nicotine was observed over 23 days. This may indicate the enantioselective induction of an uncharacterized metabolic pathway for this nicotine isomer.

Haloperidol-Induced Increase in Striatal Concentration of the Tripeptide, Tyr-Gly-Gly, Provides an Index of Increased Enkephalin Release In Vivo

A sensitive and specific radioimmunoassay has been developed for the tripeptide, Tyr‐Gly‐Gly, which has been shown previously to be an extraneuronal metabolite of opioid peptides derived from proenkephalin A. Using this assay, we found a regional variation in Tyr‐Gly‐Gly immunoreactivity in rat brain, with highest levels in striatum and lowest in cerebral cortex. Intracerebroventricular administration of the aminopeptidase inhibitor, bestatin, produced a threefold increase in Tyr‐Gly‐Gly immunoreactivity in rat striatum, whereas thiorphan, an enkephalinase inhibitor, produced a 45% reduction in striatal Tyr‐Gly‐Gly immunoreactivity. These data suggest that the tripeptide, Tyr‐Gly‐Gly, is in a dynamic state in the brain, and provide further support for the hypothesis that its concentration in specific brain areas may reflect the release of endogenous enkephalins in these brain areas. Further confirmation of the validity of measurements of brain Tyr‐Gly‐Gly as indices of enkephalin release under conditions of altered neuronal activity was provided by our demonstration that chronic dopamine receptor blockade with haloperidol increased striatal concentrations of both Met‐enkephalin and Tyr‐Gly‐Gly.