Antinociceptive potencies of beta-casomorphin analogs as compared to their affinities towards mu and delta opiate receptor sites in brain and periphery

beta-Casomorphins and their analogs were tested for their opioid activities in the myenteric plexus longitudinal muscle preparation of the guinea pig ileum (GPI), the isolated mouse vas deferens (MVD), and for their affinities to mu- delta- and kappa- binding sites in rat brain membranes. C-terminal amidation of beta-casomorphin-4 and (-5) increased opioid potency in both organ preparations (GPI, MVD) and affinity to mu-binding sites in brain whereas binding to delta-sites was diminished. These beta-casomorphin-amides displayed a 2-3 times greater naloxone reversible antinociceptive effect than natural beta-casomorphins. Introduction of D-alanine at position 2 in the beta-casomorphin-amides increased potency in the GPI whereas activity in the MVD was only slightly changed. These compounds, however, showed a remarkable increase in binding to delta-sites in brain with an unaffected or slightly increased binding to mu-sites and decreased binding to kappa-sites. D-Ala2-beta-casomorphin-4 and (-5) amides were 10 times more potent antinociceptive agents than corresponding beta-casomorphin-amides. These results suggest firstly, that peripheral delta-receptors in the MVD are not as closely related to delta-binding sites at rat brain membranes as is the case with mu-receptors in the GPI and mu-binding sites, and secondly, in addition to mu-receptors, delta-receptors may be of importance in mediating antinociception.     

The Presence of Endogenous Morphine Signaling in Animals

Recent empirical findings have contributed valuable mechanistic information in support of a regulated de novo biosynthetic pathway for chemically authentic morphine and related morphinan alkaloids within animal cells. Importantly, we and others have established that endogenously expressed morphine represents a key regulatory molecule effecting local circuit autocrine/paracrine cellular signaling via a novel μ₃ opiate receptor coupled to constitutive nitric oxide production and release. The present report provides an integrated review of the biochemical, pharmacological, and molecular demonstration of μ₃ opiate receptors in historical linkage to the elucidation of mechanisms of endogenous morphine production by animal cells and organ systems. Ongoing research in this exciting area provides a rare window of opportunity to firmly establish essential biochemical linkages between dopamine, a morphine precursor, and animal biosynthetic pathways involved in morphine biosynthesis that have been conserved throughout evolution. Special issue article in honor of Dr. Ji-Sheng Han.     

Peptide E and other proenkephalin-derived peptides are potent kappa opiate receptor agonists

Various proenkephalin-derived peptides such as peptide E and the bovine adrenal medulla peptides BAM-12P and BAM-22P are potent competitors on mu and kappa binding sites in guinea pig brain sections. Moreover, they are all potent agonists in the rabbit vas deferens, a specific kappa opiate receptor bioassay. As described before, dynorphin and some of its fragments are also potent kappa agonists. Our results suggest that not only prodynorphin-derived peptides could act as endogenous kappa ligands but also some proenkephalin-derived peptides such as peptide E.     

Cocaine Treatment Causes Early and Long-Lasting Changes in Muscarinic and Dopaminergic Receptors

1. The study of changes that persist after drug discontinuation could be fundamental to understand the mechanisms involved in craving and relapse. 2. In this work the changes occurring in muscarinic, D1- and D2-like receptors after 30 min (immediate), 1 day (early), 5 and 30 days (late) withdrawal periods were studied, in the striatum of rats treated once a day for 7 days with cocaine 20 and 30 mg/kg, i.p. 3. Binding assays were performed in 10% homogenates and ligands used were [3H]-N-methylscopolamine, [3H]-SCH 23390, and [3H]-spiroperidol for muscarinic (M1 + M2-like), D1-, and D2-like receptors, respectively. 4. Muscarinic receptors presented a downregulation at all doses and discontinuation times, while the dissociation constant (Kd) for this receptor decreased after 30 min, 5 and 30 days abstinence times. In relation to D1-like receptors we found an antagonistic effect with 100% increase in receptor number 30 min after the last cocaine injection, but after 1-day withdrawal a downregulation was observed with both doses that persisted up to 30 days, only with the higher dose. The dissociation constant value (Kd) for this receptor showed a decrease only with 5 and 30 days withdrawal. An increase occurred with D2-like receptors at all doses and withdrawal periods studied, while Kd increased in 30-min, 5, and 30 days withdrawal. 5. In this work we found that the subchronic cocaine treatment produces early and long-lasting modifications in cholinergic muscarinic as well in dopaminergic receptors that persist up to 30 days of cocaine withdrawal.     

Effect of oral nalmefene, an opiate-receptor antagonist, on meal-stimulated gastric acid secretion and serum gastrin concentration in man

We evaluated whether nalmefene, an orally administered opiate-receptor antagonist, would inhibit gastric acid secretion in response to a meal in healthy humans. On separate days either 50 mg nalmefene or a placebo tablet was administered by mouth 90 min before a blenderized steak meal was infused into the stomach through a nasogastric tube. Compared to placebo, nalmefene inhibited meal-stimulated acid secretion in each of 6 subjects studied (P less than 0.05). During the second and third hours after the meal, nalmefene inhibited mean acid secretion by 16%. Nalmefene also resulted in significantly higher meal-stimulated serum gastrin concentrations than placebo (P less than 0.05) even though intragastric pH was kept constant at 5.0 in both experiments. These studies indicate that an orally administered opiate-receptor antagonist can inhibit gastric acid secretion in response to a meal in humans, yet increase meal-stimulated serum gastrin concentrations.     

Protein Connectivity and Protein Complexity Promotes Human Gene Duplicability in a Mutually Exclusive Manner

It has previously been reported that protein complexity (i.e. number of subunits in a protein complex) is negatively correlated to gene duplicability in yeast as well as in humans. However, unlike in yeast, protein connectivity in a protein–protein interaction network has a positive correlation with gene duplicability in human genes. In the present study, we have analyzed 1732 human and 1269 yeast proteins that are present both in a protein–protein interaction network as well as in a protein complex network. In the human case, we observed that both protein connectivity and protein complexity complement each other in a mutually exclusive manner over gene duplicability in a positive direction. Analysis of human haploinsufficient proteins and large protein complexes (complex size >10) shows that when protein connectivity does not have any direct association with gene duplicability, there exists a positive correlation between gene duplicability and protein complexity. The same trend, however, is not found in case of yeast, where both protein connectivity and protein complexity independently guide gene duplicability in the negative direction. We conclude that the higher rate of duplication of human genes may be attributed to organismal complexity either by increasing connectivity in the protein–protein interaction network or by increasing protein complexity.     

Evolution of structure,order and complexity in biological systems: Part III of a general theory

In this, Part III of a general theory, the large-scale features of evolution of structure, order, and complexity are considered as characteristic features of the biological state of matter. This starts with a rigorous formal definition of structure, classes of structural order, complexity, measures of complexity, and how these arise through evolution by a cumulative process of storing information in memory systems. Three such memory systems have evolved: the genetic memory, the immune memory, and the memories of the nervous system. The evolution, characteristic parameters and the limitations of these memory systems are explored. From these considerations emerge the large-scale features of the evolutionary pathways of biological structure, function, and complexity.     

人防御素分子生物学研究进展及其应用

人防御素(human defensins)是一类内源性阳离子抗微生物肽,在天然免疫防御中起重要作用,也能增强继承性免疫应答.它具有强烈的广谱抗微生物活性,并且不易对微生物产生耐药性.对人防御素的分子生物学特性方面的研究进展作一综述,重点在于比较和探讨其结构的异同,并对其应用和发展的前景进行展望.     

Stimulation of Distinct D2 Dopaminergic and α2-Adrenergic Receptors Induces Light-Adaptive Pigment Dispersion in Teleost Retinal Pigment Epithelium

In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules aggregate and disperse in response to changes in light conditions. Pigment granules aggregate into the RPE cell body in the dark and disperse into the long apical projections in the light. Pigment granule movement retains its light sensitivity in vitro only if RPE is explanted together with neural retina. In the absence of retina, RPE pigment granules no longer move in response to light onset or offset. Using a preparation of mechanically isolated fragments of RPE from green sunfish, Lepomis cyanellus, we investigated the effects of catecholamines on pigment migration. We report here that 3,4-dihydoxyphenylethylamine (dopamine) and clonidine each mimic the effect of light in vivo by inducing pigment granule dispersion. Dopamine had a half-maximal effect at approximately 2 nM; clonidine, at 1 microM. Dopamine-induced dispersion was inhibited by the D2 dopaminergic antagonist sulpiride but not by D1 or alpha-adrenergic antagonists. Furthermore, a D2 dopaminergic agonist (LY 171555) but not a D1 dopaminergic agonist (SKF 38393) mimicked the effect of dopamine. Clonidine-induced dispersion was inhibited by the alpha 2-adrenergic antagonist yohimbine but not by sulpiride. These results suggest that teleost RPE cells possess distinct D2 dopaminergic and alpha 2-adrenergic receptors, and that stimulation of either receptor type is sufficient to induce pigment granule dispersion. In addition, forskolin, an activator of adenylate cyclase, induced pigment granule movement in the opposite direction, i.e., dark-adaptive pigment aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exploiting Oxidative Stress and Signaling in Chemotherapy of Resistant Neoplasms

Neural crest tumors of childhood are particularly resistant to apoptosis induction by chemotherapeutic agents. Mechanisms of resistance include altered glutathione handling that accompanies up-regulation of Bcl-2 and its relatives. We have designed and tested in preclinical model systems approaches to this problem. These approaches include adjunctive use of oxygen radical-generating neurotransmitter analogs taken up by these neural crest tumor cells with scavenging (i.e., "rescue") agents that are selective for normal neural crest and the use of reduction-dependent prodrugs of apoptosis-inducing agents. Promising prototypes for these conceptual approaches include, respectively, adjunctive use of the oxygen radical generator, 6-hydroxydopamine, with the normal cell-selective antioxidant, Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), and use of the reduction-dependent chemotherapeutic prodrug neocarzinostatin.     

Endogenous Dopamine Functionally Activates D-1 and D-2 Receptors in Striatum

Rat striatal slices incubated with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine at 1 mM were exposed to different concentrations (1-100 microM) of the catecholamine-releasing drug amphetamine. This produced both a concentration-dependent release of endogenous dopamine and accumulation of cyclic AMP in the slices. The cyclic AMP accumulation due to amphetamine was greatly increased when slices were coincubated with the selective dopamine D-2 antagonist (-)-sulpiride (30 microM), but the amphetamine-induced release of dopamine from the slices was the same in the presence or absence of (-)-sulpiride. Pretreatment of animals with reserpine (5 mg/kg s.c., 18 h before death) and in vitro incubation with alpha-methyl-p-tyrosine (50 microM for 90 min), respectively, reduced the ability of amphetamine (1-100 microM) [in the presence of 30 microM (-)-sulpiride] to induce release of dopamine and to elevate cyclic AMP accumulation in striatal slices. A similar reduction in amphetamine-induced dopamine release and cyclic AMP accumulation in striatal slices was observed 7 days following unilateral 6-OHDA lesions of the medial forebrain bundle of rats. These results suggest that amphetamine induces release of endogenous dopamine from the terminals of nigrostriatal dopamine neurones. Released dopamine is then able functionally and concomitantly to activate D-1 and D-2 receptors, seen as stimulation and inhibition of cyclic AMP accumulation, respectively.     

Bombesin-like peptides and receptors in human tumor cell lines

Human cancer cell lines were assayed for bombesin-like peptides and receptors. Acid extracts derived from small cell lung cancer, but not other types of cancer had high levels of immunoreactive bombesin. Regardless of patient treatment, site of tumor origin (bone marrow, lymph node, or pleural effusion) or culture conditions, small cell lung cancer cell lines had high levels of bombesin-like peptides. Thus, bombesin levels in small cell lung, but not other types of human cancer, are routinely elevated. Also, small cell lung cancer lines in contrast to other cell lines have a high density of binding sites for a radiolabeled bombesin analogue. The presence of high concentrations of bombesin-like peptides and receptors suggests that bombesin may function as an important regulatory agent in human small cell lung cancer.     

Atrial natriuretic peptide in heart and specific binding in organs from fetal and newborn rats

To assess the possibility that atrial natriuretic peptide plays a role in salt and water balance during early mammalian development, we examined hearts from fetal and neonatal rates for the presence of this peptide and presumed target tissues for their ability to bind the hormone. Immunohistochemistry was used to localize and radioimmunoassay to quantify this peptide in heart. Immunoreactive atrial natriuretic peptide was visualized in the fetal heart on day 17.5 post-conception. It was distributed throughout the atrial appendages and free wall and, in ventricle, in the trabeculae carnae and chordae tendineae. The concentrations of immunoreactive atrial natriuretic peptide in atria of rats on day 19.5 post-conception were one-tenth of those in the adult. Levels of this peptide in fetal ventricle were low and virtually absent from the adult tissue. Specific binding of radiolabelled atrial natriuretic peptide measured by whole organ counting occurred in several organs from 19.5-day fetal and neonatal rats. A number of these tissues, including the kidney, ileum, adrenal, lung and liver, are targets for and/or bind the peptide in adult rats. Specific binding in these tissues was localized using autoradiography at anatomical sites similar to those in adult organs. Specific binding was also seen in fetal but not neonatal skin. In the kidney, binding was associated with immature as well as mature glomeruli. These findings support the proposition that atrial natriuretic peptide may function in the perinatal rat as it does in the adult and, in addition, may play a unique role during fetal life.     

Identification and expression of natriuretic peptide receptor type-A and -B mRNA in freshwater and seawater rainbow trout

Natriuretic peptide receptors mediate the physiological response of natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.     

Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades

Mitogen-activated protein kinase (MAPK) cascades can operate as bistable switches residing in either of two different stable states. MAPK cascades are often embedded in positive feedback loops, which are considered to be a prerequisite for bistable behavior. Here we demonstrate that in the absence of any imposed feedback regulation, bistability and hysteresis can arise solely from a distributive kinetic mechanism of the two-site MAPK phosphorylation and dephosphorylation. Importantly, the reported kinetic properties of the kinase (MEK) and phosphatase (MKP3) of extracellular signal-regulated kinase (ERK) fulfill the essential requirements for generating a bistable switch at a single MAPK cascade level. Likewise, a cycle where multisite phosphorylations are performed by different kinases, but dephosphorylation reactions are catalyzed by the same phosphatase, can also exhibit bistability and hysteresis. Hence, bistability induced by multisite covalent modification may be a widespread mechanism of the control of protein activity.     

Further Identification of Neurokinin Receptor Types and Mechanisms of Calcium Signaling Evoked by Neurokinins in the Murine Neuroblastoma C1300 Cell Line

Abstract: It has been suggested that murine neuroblastoma C1300 cells express endogenous neurokinin NK₂ receptors with features that differ from those of NK₂ receptors characterized in other systems. In this study, we have further characterized the neurokinin receptor types present in this cell line. RNA blots showed that mRNAs of NK₂ and NK₃ receptors, but not of NK₁ receptors, were expressed in C1300 cells. The increase in the cytosolic calcium concentration ([Ca²⁺]_i) induced by 0.33 µM neurokinin A was completely inhibited by SR 48968, an NK₂ receptor antagonist, whereas the partial response to 0.33 µM neurokinin B was unaffected, and the response was completely inhibited by SR 142801, an NK₃ receptor antagonist. In addition, the [Ca²⁺]_i increase by 0.33 µM senktide, an NK₃ receptor agonist, was inhibited by SR 142801 but not by SR 48968. These findings indicated that C1300 cells endogenously express functional NK₂ and NK₃ receptors. It was also demonstrated that NK₂ and NK₃ receptors can be activated independently by 3.3 µM neurokinin A in the presence of 1.0 µM SR 142801 or 1.0 µM senktide, respectively. Therefore, the mechanisms of Ca²⁺ signaling mediated by endogenous NK₂ and NK₃ receptors were investigated. The independent activation of NK₂ or NK₃ receptors induced not only the [Ca²⁺]_i increase, but also stimulated the formation of inositol trisphosphates; both these responses were inhibited by U73122, a phospholipase C (PLC) inhibitor. In addition, NK₂ and NK₃ receptor-mediated [Ca²⁺]_i increase was partially attenuated in the absence of extracellular Ca²⁺ or in the presence of nickel, an inorganic Ca²⁺ influx blocker, but was unaffected by nifedipine and ω-conotoxin, L- and N-type voltage-dependent Ca²⁺ channel blockers, respectively. Furthermore, the depolarization by 60 mM K⁺ did not affect the [Ca²⁺]_i. These findings suggested that the NK₂ and NK₃ receptor-mediated [Ca²⁺]_i increase was due to the activation of PLC and was dependent on the mobilization of internal Ca²⁺ and the entry of extracellular Ca²⁺ through voltage-independent channels. This study showed that the C1300 cell line is a useful system with which to investigate pharmacological functions and signaling pathways of endogenous NK₂ and NK₃ receptors.     

Reviews in Molecular Biology and Biotechnology: Transmembrane Signaling by G Protein-Coupled Receptors

As the most diverse type of cell surface receptor, the importance heptahelical G protein-coupled receptors (GPCRs) to clinical medicine cannot be overestimated. Visual, olfactory and gustatory sensation, intermediary metabolism, cell growth and differentiation are all influenced by GPCR signals. The basic receptor-G protein-effector mechanism of GPCR signaling is tuned by a complex interplay of positive and negative regulatory events that amplify the effect of a hormone binding the receptor or that dampen cellular responsiveness. The association of heptahelical receptors with a variety of intracellular partners other than G proteins has led to the discovery of potential mechanisms of GPCR signaling that extend beyond the classical paradigms. While the physiologic relevance of many of these novel mechanisms of GPCR signaling remains to be established, their existence suggests that the mechanisms of GPCR signaling are even more diverse than previously imagined.     

Differential Effects of Cocaine-Induced Seizures and Lethality on M1-Like Muscarinic and Dopaminergic D1- and D2-Like Binding Receptors in Mice Brain

Summary This work was designed to study the changes produced by cocaine-induced seizures and lethality on dopaminergic D₁- and D₂-like receptors, muscarinic M₁-like binding sites, as well as acetylcholinesterase activity in mice prefrontal cortex (PFC) and striatum (ST). Binding assays were performed in brain homogenates from the PFC and ST and ligands used were [³H]-N-methylscopolamine, [³H]-NMS (in the presence of carbachol), [³H]-SCH 23390 and [³H]-spiroperidol (in presence of mianserin), for muscarinic (M₁-like), D₁- and D₂-like receptors, respectively. Brain acetylcholinesterase (AChE) activity was also determined in these brain areas. Cocaine-induced SE decreased [³H]-SCH 23390 binding in both ST and PFC areas. A decrease in [³H]-NMS binding and an increase in [³H]-spiroperidol binding in PFC was also observed. Cocaine-induced lethality increased [³H]-spiroperidol binding in both areas and decreased [³H]-NMS binding only in PFC, while no difference was seen in [³H]-SCH 23390 binding. Neither SE, nor lethality altered [³H]-NMS binding in ST. AChE activity increased after SE in ST while after death the increase occurred in both PFC and ST. In conclusion, cocaine-induced SE and lethality produces differential changes in brain cholinergic and dopaminergic receptors, depending on the brain area studied suggesting an extensive and complex involvement of these with cocaine toxicity in central nervous system.     

A radioassay for nonoxidized methionine in peptides. A method for identifying (after isoelectric focusing) and for estimating biologically active forms of corticotropin and other hormones

A radioassay for nonoxidized methionine in peptides is described; it has advantages over other methods currently used because of its simplicity, sensitivity, accuracy, and applicability to individual peptide components in mixtures and to many samples at a time. Methionyl residues were S-carboxymethylated with iodo[2-14C]acetic acid; iodo[2-3H]acetic acid did not provide a stable radioactive tracer. The labeled peptide was isolated by carboxymethylcellulose chromatography or by isoelectric focusing (IEF) or electrophoresis in polyacrylamide gel, and its radioactivity measured. The assay was applied to corticotropins, alpha-melanotropin, bombesin, glucagon, substance P, parathormone, and calcitonin. Twenty-four to thirty samples were conveniently analyzed at a time with a lower detection limit of less than 1 nmol of methionine per sample. The accuracy of the assay, assessed also by reverse-phase high-performance liquid chromatography, is a consequence of its precision, the specificity of the reaction with iodoacetic acid, and the use of an appropriate standard of the peptide being assayed. Methionine was identified, and could be estimated, in individual peptide components of a mixture by using IEF to separate simultaneously the labeled peptide from iodo[2-14C]acetic acid and from other peptide and protein components. This was facilitated by a convenient method for detecting and quantifying these peptides after IEF. The assay is particularly useful for several peptide hormones whose biological activity depends on their sole methionine residue being in a nonoxidized state. It can be used for monitoring their isolation or synthesis and their stability during processing and storage, as well as for evaluating differences in biological potency between preparations and analogues.