Changes in the brain kappa-opioid receptor levels of rats in withdrawal from physical dependence upon butorphanol

Changes in kappa-opioid receptor levels have been implicated in the development of physical dependence upon and withdrawal from the mixed agonist-antagonist opioid, butorphanol. Immunoblotting analysis was performed to determine the levels of kappa- and mu-opioid receptors in brain regions of rats in withdrawal from dependence upon butorphanol or morphine. Physical dependence was induced by a 72 h i.c.v. infusion with either butorphanol or morphine (26 nmol/microl/h). Withdrawal was subsequently precipitated by i.c.v. challenge with naloxone (48 nmol/5 microl/rat), administered 2 h following cessation of butorphanol or morphine infusion. Immunoblotting analysis of kappa-opioid receptors from butorphanol-withdrawal rats showed significant increases in 11 of 21 brain regions examined, including the nucleus accumbens, amygdala, dorsomedial hypothalamus, hypothalamus, paraventricular thalamus, thalamus, presubiculum, and locus coeruleus, when compared with saline treated, non-dependent controls. In addition, significant reductions were found in the hippocampus and in cortical brain regions, including the parietal cortex and temporal cortex from butorphanol-withdrawal rats. These findings contrasted with those from morphine-withdrawal rats, in which the only changes noted were increases in the thalamus and paraventricular thalamus. Changes in the levels of the mu-opioid receptor protein were observed in 11 of 21 brain regions examined in morphine-withdrawal rats, but only in three of 21 in butorphanol-withdrawal rats. These results implicate a substantive and largely unique role for kappa-opioid receptors in mediation of the development of physical dependence upon, and the expression of withdrawal from, butorphanol, as opposed to the prototypical opioid analgesic, morphine.     

Nonopioid receptor-mediated effects of U-50,488H on [Ca(2+)]i and extracellular dopamine in PC12 cells

The present studies were carried out to determine the effects of a kappa-opioid receptor agonist on cytosolic Ca(2+) concentration, [Ca(2+)](i), and extracellular dopamine in undifferentiated PC12 cells. The kappa-opioid receptor agonist U-50,488H caused concentration-dependent increases in [Ca(2+)](i) and extracellular dopamine. Neither effect was blocked by the selective kappa-opioid receptor antagonist nor-binaltorphimine. Increases in extracellular dopamine content and [Ca(2+)](i) caused by U-50,488H were correlated positively in the presence of extracellular Ca(2+); however, reduction of extracellular Ca(2+) abolished the increase in [Ca(2+)](i), but not that in dopamine. The latter observation suggests that stimulation of exocytotic release is not the primary mechanism involved in the increase in extracellular dopamine caused by U-50,488H. Effects on dopamine synthesis or catabolism also seem unlikely because the enhancement of extracellular dopamine occurred rapidly, and the amount of a major metabolite of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), was not affected. In any event, neither the increase in [Ca(2+)](i) nor the increase in extracellular dopamine caused by U-50,488H is mediated by the kappa-opioid receptor.     

The effects of wild ginger (Costus speciosus (Koen) Smith) rhizome extract and diosgenin on rat uterine contractions

The aim of this study was to investigate the effects of wild ginger (Costus speciosus (Koen) Smith, Costaceae) rhizome extract on uterine contractility. We particularly examined the effects on spontaneous phasic contractions and the mechanisms whereby it exerts its effects. Wild ginger rhizomes were ethanolic extracted and their constituents analyzed. Isometric force was measured in strips of longitudinal myometrium and the effects of the extract studied. The extract (10 mg/100 mL) increased spontaneous contractions. The amplitude and frequency of the phasic contraction were significantly increased along with basal tension. Force produced in the presence of the extract was abolished by inhibition of l-type calcium channels or myosin light chain kinase (MLCK). The actions of the extract were not blocked by the estrogen receptor blocker, fulvestrant. Although significant amounts of diosgenin were present in the extract, we found that, depending upon its concentration, diosgenin had either no effect or was inhibitory on force. Interestingly, the extract induced significant amounts of force in the absence of extracellular calcium, which could be blocked by inhibition of the sarcoplasmic reticulum calcium-ATPase (SERCA), but not fulvestrant. We conclude that wild ginger rhizome extract stimulates phasic activity in rat uterus. Our data suggest that the uterotonic effect is due to nonestrogenic effects and not those of diosgenin. Wild ginger was able to increase contraction via calcium entry on l-type calcium channels and sarcoplasmic reticulum (SR) calcium release. We suggest that wild ginger rhizome extract may be a useful uterine stimulant.     

Mechanism of the inhibitory action of capsaicin on energy metabolism by rat liver mitochondria

Capsaicin was found to inhibit the oxidation of exogenous NADH by digitonin particles and hypotonic-treated rat liver mitochondria without impairing the oxidation of succinate by these mitochondrial preparations. The action of methyl capsaicin, the non-phenolic derivative of capsaicin, on mitochondrial oxidative phosphorylation and NADH oxidation was found identical to that of capsaicin. However, on the basis of I₅₀, values, methyl capsaicin was approximately three times more potent. The inhibition of NADH oxidation produced by capsaicin and its methylated derivative was completely relieved by addition of menadione, an electron acceptor at the NADH dehydrogenase segment of mitochondrial electron transport chain. From these observations, it is concluded that (a) both capsaicin and methyl capsaicin inhibit mitochondrial energy metabolism by retarding electrons flow from NADH to coenzyme Q; (b) the location of this action is beyond the site at which menadione accepts electrons from the NADH dehydrogenase system; and (c) the phenolic group in capsaicin molecule is not essential for the above mitochondrial action of this compound.     

Effects of piperine on bioenergetic functions of isolated rat liver mitochondria

The in vitro effects of piperine on three bioenergetic reactions namely, oxidative phosphorylation, ATPase activity and calcium transport by isolated rat liver mitochondria have been investigated. Piperine was found to inhibit state 3 and DNP-stimulated respiration by mitochondria respiring with glutamate plus malate or succinate as substrates. The I50 values of piperine on oxidative phosphorylation in the presence of glutamate plus malate and succinate were 22 and 12 micrograms/mg mitochondrial protein respectively. With HTM preparations, the oxidation of added NADH and succinate was depressed by piperine while ascorbate plus TMPD oxidation was slightly affected. Piperine did not inhibit the mitochondrial ATPase activity induced by DNP, but by itself exerted stimulating activity on this enzyme. Piperine was also found to diminish calcium uptake and to facilitate the release of accumulated calcium by the mitochondria incubated with succinate or ATP. These results suggest that piperine inhibits mitochondrial oxidative phosphorylation at the level of respiratory chain, and the inhibitory site(s) is in the segment(s) ahead of cytochrome C. The mechanism of the piperine-induced ATPase activity is not known; but the effect of piperine on calcium transport is likely to be consequential to the effects of this compound on the mitochondrial respiratory chain and ATPase activity.     

Lupinifolin from <Emphasis Type="Italic">Derris reticulata</Emphasis> possesses bactericidal activity on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> by disrupting bacterial cell membrane

In this study, lupinifolin, a prenylated flavonoid, was isolated from Derris reticulata stem, identified by NMR spectra and confirmed with mass spectrometry. Lupinifolin was freshly prepared by solubilizing in 0.1 N NaOH and immediately diluted in Müller–Hinton broth for antibacterial testing. The data showed that Gram-positive bacteria were more susceptible to lupinifolin than Gram-negative bacteria. Of four strains of Gram-positive bacteria tested, Staphylococcus aureus was the most susceptible. Using the two-fold microdilution method, it was found that lupinifolin possessed antimicrobial activity against S. aureus with minimum inhibitory concentration and minimum bactericidal concentration of 8 and 16 µg/ml, respectively, which is less potent than ampicillin. However, from the time–effect relationship, it was shown that lupinifolin had faster onset than ampicillin. The faster onset of lupinifolin was confirmed by scanning electron microscopy. To investigate the mechanism of action of lupinifolin, transmission electron microscopy (TEM) was performed to observe the ultrastructure of S. aureus. The TEM images showed that lupinifolin ruptured the bacterial cell membrane and cell wall. Due to its fast onset, it is suggested that the action of lupinifolin is likely to be the direct disruption of the cell membrane. This hypothesis was substantiated by the data from flow cytometry using DiOC₂ as an indicator. The result showed that the red/green ratio which indicated bacterial membrane integrity was significantly decreased, similar to the known protonophore carbonyl cyanide 3-chlorophenylhydrazone. It is concluded that lupinifolin inhibits the growth of S. aureus by damaging the bacterial cytoplasmic membrane.     

Proteomic analysis of phosphotyrosyl proteins in the rat brain: effect of butorphanol dependence

Butorphanol (17-cyclobutylmethyl-3,14-dihydroxymorphinan) tartrate (Stadol) is a mixed agonist-antagonist opioid analgesic agent that is about five to seven times as potent as morphine in analgesic effects. The chronic use of butorphanol produces physical dependence in humans and animals. Phosphorylation plays a very important role in developing butorphanol dependence; however, global phosphorylation events induced by chronic butorphanol administration have not been reported. The aim of this study is to determine the alteration of tyrosine phosphorylation of brain frontal cortical proteins in butorphanol-dependent rats using a proteomic approach. Dependence was produced by continuous intracerebroventricular (i.c.v.) infusion of butorphanol (26 nmol/microl/hr) for 72 hr via osmotic minipump in rats. Similar patterns of protein expression were detected by two-dimensional electrophoresis (2-DE) in brain frontal cortex of butorphanol-dependent and saline-treated control rats. All 65 phosphotyrosyl (p-Tyr) protein spots detected in pH 3-10 phosphotyrosine 2-DE of control rat brains were detected in butorphanol-dependent rat brains. The densities of most p-Tyr protein spots were increased in butorphanol-dependent rat brains compared to saline-treated control samples. Eighteen additional p-Tyr protein spots were detected in pH 3-10 2-DE images of butorphanol-dependent rat brains. Immobilized pH strips with three different narrow pH ranges were examined to improve the resolution of p-Tyr proteins in 2-DE gels. Fifty-three p-Tyr protein spots were identified as known proteins involved in cell cytoskeleton, cell metabolism, and cell signaling. This proteomic approach can provide useful information for understanding the complex mechanism of butorphanol dependence in vivo.     

Effects of 2,4,3′,5′-tetrahydroxystilbene on oxidative phosphorylation by rat liver mitochondria

The effects of an antifungal compound 2,4,3′,5′-tetrahydroxystilbene (THS)2 on oxidative phosphorylation by rat liver mitochondria were studied. THS at low concentrations was found to inhibit state 3 respiration (I₅₀ = 44 nmoles/mg protein) but had no effect on uncoupled respiration induced by 2,4-dinitrophenol. The inhibitory effect of THS on state 3 respiration was observed with either glutamate or succinate as respiratory substrates. However when rat liver mitochondria were respiring with glutamate as substrate and in the presence of calcium chloride and inorganic phosphate, THS produced a strong inhibition of 2,4-dinitrophenol-stimulated respiration. This inhibition was over-come by adding succinate. The tetramethoxy derivative of THS had no effect on the mitochondrial reactions mentioned above suggesting that the phenolic groups are essential for the action of THS. The sites at which THS inhibits mitochondrial oxidative phosphorylation are proposed.     

Studies on the effect of capsaicin on metabolic reactions of isolated rat liver mitochondria.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) was found to inhibit the respiratory response of rat liver mitochondria, respiring in the presence of glutamate as substrate, to the additions of ADP(+P_i), 2,4-dinitrophenol(DNP), and CaCl₂(+P_i). The inhibitory effect of capsaicin on mitochondrial oxidative phosphorylation can also be observed with other NAD-linked substrates such as β-hydroxybutyrate and malate plus pyruvate and was partially reversed by adding bovine serum albumin to the reaction mixtures. When the mitochondria were respiring with succinate as substrate, capsaicin was less effective in inhibiting state 3 and DNP-stimulated respiration but exerted an uncoupling action on mitochondrial respiration. This uncoupling effect was evident at the doses of capsaicin much higher than that required to depress oxidative phosphorylation when NAD-linked substrates were used. Capsaicin was also found to depress the DNP-activated adenosine triphosphatase (ATPase) activity of rat liver mitochondria. These results indicate that capsaicin has profound effect on the energy-linked functions of isolated mitochondria.     

Streptomyces chiangmaiensis SSUT88A mediated green synthesis of silver nanoparticles: characterization and evaluation of antibacterial action against clinical drug-resistant strains

This study involved the characterization of AgNPs synthesized from soil isolate Streptomyces sp. SSUT88A and their antimicrobial activities. The strain SSUT88A revealed 98.8% similarity of the 16s rRNA gene to Streptomyces chiangmaiensis TA4-1^T. The AgNPs were synthesized by mixing either intracellular or extracellular cell-free supernatant of strain SSUT88A with AgNO₃. The synthesized AgNPs from intracellular cell-free supernatant and extracellular cell-free supernatant were designated as IS-AgNPs and ES-AgNPs, respectively. The IS-AgNPs showed maximum absorbance of UV-vis spectra at 418 nm, while ES-AgNPs revealed maximum absorbance at 422 nm. The TEM observation of synthesized AgNPs revealed a spherical shape with an average diameter of 13.57 nm for IS-AgNPs and 30.47 nm for ES-AgNPs. The XRD and XANES spectrum profile of both synthesized AgNPs exhibited similar spectrum energy, which corresponded to AgNPs. The IS-AgNPs revealed antimicrobial activity against clinical isolate drug-resistant bacteria (Acinetobacter baumannii, Escherichia coli 8465, Klebsiella pneumoniae 1617, and Pseudomonas aeruginosa N90PS), while ES-AgNPs had no antimicrobial activity. When compared to commercial AgNPs, IS-AgNPs exhibited antibacterial efficacy against all clinical isolate bacteria including A. baumannii, one of the most threatening multi-drug resistant strains, while commercial AgNPs did not. Thus, IS-AgNPs has potential to be further developed as an antimicrobial agent against drug-resistant bacteria.

The first synthesis of AgNPs by Streptomyces chiangmaiensis which had superior antibacterial action against serious drug-resistant pathogens than commercial AgNPs.