Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes

Opioid agonists specific for the , , and opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The opioid agonists U-62,066E and (–) ethylketocyclazocine, but not the agonist [D-Ala²,N-MePhe⁴,Gly⁵-ol]-enkephalin (DAGO) nor the agonist [D-Pen^2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca²⁺ levels, indicating a possible mechanism for agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca²⁺ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca²⁺ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the -opioid specific antagonist ICI 174,864 and the opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that opioid agonists inhibit transmitter release from mossy fiber terminals through both opioid and non- opioid receptor mediated mechanisms.     

Affinity Profiles of Novel δ-Receptor Selective Benzofuran Derivatives of Non-Peptide Opioids

Highly selective heterocyclic opioid ligands with potent -antagonist activity have been developed on the basis of the message-address concept. Using this strategy, benzofuran derivatives corresponding to the non-selective opioid antagonist, naloxone, and to the -opioid receptor selective agonists, oxymorphone and oxycodone, were synthesized. In vitro opioid receptor binding profiles and agonist/antagonist character of these compounds were determined in rat brain membrane preparations with highly selective radioligands. All three benzofuran derivatives displayed high affinities for the -opioid receptor, much less potency toward the -binding site, and were the least effective at the -site. The results indicated that the addition of the bezofuran moiety to these fused ring opioids confers -receptor selectivity. The Na⁺ indices suggested a partial agonist character for oxymorphone- and oxycodone-benzofuran, and an antagonist character for naloxone-benzofuran. These compounds were capable of irreversible inhibition of opioid binding sites in a dosedependent.     

Conformationally constrained opioid peptide analogs with novel activity profiles

Novel conformationally constrained opioid peptide analogs with antagonist, mixed agonist/ antagonist or agonist properties were developed. TIP(P)-related antagonists showed unprecedented antagonist potency and receptor selectivity, and may have potential for use in analgesia in combination with agonists. A definitive model of their receptor-bound conformation was developed. Three prototype mixed agonist/ antagonists were discovered. They represent the only known compounds with this pharmacological profile and, as expected, one of them was shown to be a potent analgesic and to produce no dependence and less tolerance than morphine. Novel dipeptide derivatives turned out to be potent and selective agonists. Because of their low molecular weight and lipophilic character, these compounds may cross the blood-brain barrier and, thus, may have potential as centrally acting analgesics.     

δ and κ Opiate receptors in primary astroglial cultures from rat cerebral cortex

The effects of , , and receptor-agonists on forskolin stimulated cyclic adenosine-3, 5-monophosphate (cAMP) formation were examined in astroglial enriched primary cultures from the cerebral cortex of newborn rats. Intracellular cAMP accumulation was quantified by radioimmunoassay. Morphine was used as a -receptor agonist, D-Ala-D-Leu-Enkephalin (DADLE) as a -receptor agonist and dynorphine 1–13 (Dyn) as a -receptor agonist. Basal cAMP levels were unaffected by either the opiate agonists or the antagonists used. In the presence of the cAMP stimulator forskolin, morphine had no significant effect on the cytoplasmic cAMP levels. DADLE caused a dose related inhibition of the forskolin stimulated cAMP accumulation. The effects of this receptor stimulation was blocked with the selective antagonist ICI 174.864. In the presence of Dyn, the forskolin stimulated cAMP accumulation was inhibited in a dose related manner. This receptor stimulation was blocked with the selective antagonist MR 2266. Co-administration of DADLE and Dyn resulted in a non additive inhibition of the forskolin stimulated accumulation of cAMP. These findings indicate that astroglial enriched cultures from the cerebral cortex of rats express and -receptors co-localized ont he same population of cells, and that these receptors are inhibitory coupled to adenylate cyclase.     

Melatonin enhances Th2 cell mediated immune responses: Lack of sensitivity to reversal by naltrexone or benzodiazepine receptor antagonists

Chronic administration of melatonin for 5 days to antigen-primed mice increased the production of pro-inflammatory cytokine IL10 but decreased the secretion of antiinflammatory cytokine TNF-. These results further confirm that melatonin activates Th2like immune response. Whether melatoninmediated Th2 response is dependent on opioid or central and peripheral benzodiazepine receptors was also examined. Hence, melatonin was administered to antigen-sensitised mice with either naltrexone (a opioid receptor antagonist) or flumazenil (a central benzodiazepine receptor antagonist) or PK11195 (a peripheral benzoidiazepine receptor antagonist). No significant difference in melatonin-induced Th2 cell response was observed by naltrexone, flumazenil or PK11195 treatment. These findings suggest that the Th2 cell response induced by melatonin in antigen sensitised mice neither dependent on endogenous opioid system nor is modulated through the central or peripheral benzodiazepine receptors.     

Effect of anti-regucalcin antibody on neutral phosphatase activity in rat liver cytosol: Involvement of endogenous regucalcin

The effect of anti-regucalcin monoclonal antibody on neutral phoshatase activity in rat liver cytosol was investigated. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate toward phosphatase asssy. Liver cytosolic phosphatase activity with three phosphoaminoacids was significantly increased in the presence of anti-regucalcin antibody (100 and 200 ng/ml) in the enzyme reaction mixture with calcium chloride (0.1 mM) or EGTA (1.0 mM). The effect of anti-regucalcin antibody was completely abolished in the presence of exogenous regucalcin (1.0 M), indicating the involvement of endogenous regucalcin. The anti-regucalcin anti body- increased phosphatase activity was not significantly altered in the presence of trifluoperazine (20 M), an antagonist of calmodulin, or akadaic acid (10 M), an inhibitor of protein phosphatase, although these inihibitors caused a slight decrease in liver cytosolic phosphatase activity. The effect of endogenous regucalcin might be not related to calmodulin, and it was insensitive to okadaic acid. The present findings suggest that endogenous regucalcin is involved in the regulation of protein phasphatase in rat liver cytoplasm.     

Design and bioactivities of melanotropic peptide agonists and antagonists: Design based on a conformationally constrained somatostatin template

-Melanotropin and ACTH, POMC peptides, initiate biological activity by interaction with the classical pigment cell (-MSH receptor, MC1R) and adrenal gland (ACTH receptor, MC2R) melanocortin receptors, respectively. The recently discovered MC3R, MC4R and MC5R receptors provide new targets and new biological functions for POMC peptides. We have developed conformationally constrained -melanotropin peptides that interact with all of these receptors as agonists and antagonists and are examining new approaches to obtain highly selective ligands for each of these melanocortin receptors. Previously, we had converted somatostatin-derived peptides into potent and highly selective analogues that act as antagonists at the opioid receptors. Using the reverse turn template that came out of these studies, we have designed, de novo, agonist and antagonist peptide analogues that interact with melanocortin receptors.     

Coordination dynamics of heme-copper oxidases. The ligand shuttle and the control and coupling of electron transfer and proton translocation

Results are presented which, taken with evidence developed by others, suggest a general mechanism for the entry and binding of exogenous ligands (including O₂) at the binuclear site (Cu_B Fe _a3) of the heme-copper oxidases. The mechanism includes a ligand shuttle wherein the obligatory waystation for incoming ligands is Cu_B and the binding of exogenous ligands at this site triggers the exchange and displacement of endogenous ligands at Fe _a3. It is suggested that these ligand shuttle reactions might be functionally important in providing a coupling mechanism for electron transfer and proton translocation. Scenarios as to how this might happen are delineated.     

The low-light reduction in the quantum yield of photosynthesis: potential errors and biases when calculating the maximum quantum yield

Photosynthesis-irradiance (P-E) curves are widely used to describe photosynthetic efficiency and potential. Contemporary models assume maximal photosynthetic quantum yield () at low irradiances. But P-E observations made with both oxygen evolution and carbon uptake techniques show that this is not always the case. Using new and published data in conjunction with modeling exercises, we demonstrate that regardless of the mechanism there can be reductions in at low irradiances that are not readily observable using conventional P-E analyses. We also show that analytical errors, such as inaccurate estimation of dark oxygen consumption or carbon uptake, can markedly affect the structure of -E curves with negligible effect on P-E curve structure. Whether from respiration `corrections' or other mechanisms, these deviations in at low light levels from the maximum quantum yield of photosynthesis (_max) can lead to significant errors (> 50%) in the estimation of the linear portion of the P-E curve and ultimately _max. Non-linear models of P-E, such as the rectangular hyperbola, quadratic, exponential and hyperbolic tangent that are commonly used to estimate the initial slope () of the P-E curve assume that is maximal at low light levels and therefore can err in the estimation of _max when is reduced at low light levels. Using a diverse data set of 622 P-E curves with a total of 7623 points, we show that although model skills are high (r ² = 0.96 ± 0.05, 0.97 ± 0.04, 0.97 ± 0.04 and 0.97 ± 0.04, respectively), a large fraction of the model-predicted _max differ by greater than 10% from true _max values (91%, 50%, 82% and 46%, respectively). Data from these observations and modeling exercises lead us to suggest that _max be determined by directly estimating the true maximum of a -E curve rather than using the more conventional methodology employing the initial slope of the P-E curve.This revised version was published online in October 2005 with corrections to the Cover Date.     

Retinoic acid receptor isoform RARγ 1: an antagonist of the transactivation of the RARβ RARE in epithelial cell lines and normal human keratinocytes

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR₁ and RAR₂ isoforms, but not by RAR₁. On the contrary, this latter isoform behaved towards the RAR RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR₁ and RAR₂. RAR₁ also behaved as an antagonist of the transactivation produced by cotransfected RXR. The natural RAR gene promoter or RAR RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR₁. It was, however, possible to activate to a certain extent RAR RARE-reporter constructs in NHK by co-transfecting RAR₁, RAR₂ or RXR. The antagonist behavior of RAR₁ towards the RAR RARE may explain why in certain cell types such as keratinocytes, RAR is neither expressed nor induced by RA.Abbreviations DMEM Dulbecco's modified Eagle medium - DMSO dimethyl sulfoxide - FCS fetal calf serum - MEM minimal Eagle medium - NHK normal human keratinocyte - RA retinoic acid - RAR retinoic acid receptor - RARE retinoic acid responsive element - TRE thyroid responsive element - VDRE vitamin D response element - RXR retinoid X receptor     

Transition of outgrowing spores of Bacillus brevis into vegetative cells is not dependent on destruction of gramicidin S

Summary Certain Bacillus brevis strains produce gramicidin S (GS) during sporulation, and germination of such spores is delayed at the stage of outgrowth by endogenous or exogenous GS. Claims have been made that the transition from germinating spores into vegetative cells is dependent on GS destruction. We observed no such destruction of either exogenous or endogenous GS. Thus, in our hands, the recovery of the inhibited germinating spores must be dependent on something other than GS elimination.Offprint requests to: G. Bentzen     

The relationships between the structure of paddy levees and the plant species diversity in cultural landscapes on the west side of Lake Biwa,Shiga, Japan

Paddy levees form networks of narrow linear habitats and play various roles in cultural landscapes. Traditional landscapes on the west side of Lake Biwa consist of paddy field terraces and both stone and soil levees that have been maintained by paddy field management using local resources. Paddy levees in this study site are principally classified into five different types. Our study points out how differences in paddy levee structure as well as in management practices influence the plant species. Seventeen paddy levee transects were split into four habitat types based on their species components by TWINSPAN. Spatial characteristics and physical structures of paddy levees depended on natural conditions and human activities. The species–area curves of each levee type showed a clear distinction: the soil, stone and abandoned curves were steep, while the concrete and consolidated ones were gentle. The vegetation on consolidated levees was utterly different from the vegetation on traditional levee types from the aspect of species richness and species components. Soil type levees contained various woody plant species and included more diverse and indigenous plant species than abandoned type levees.     

Ultrastructural localization of gonadotropin-releasing hormone in the porcine gonadotropic cells

Summary The comparative ultrastructural localization of LH, FSH and GnRH clearly shows that the granules of the FSH/LH cells contain all three hormones. The separate storage of LH and FSH in a significant number of cells, which in the same granules also display GnRH, may suggest that LH-RH is also FSH-RH and may help to explain the non-parallel release of LH and FSH under some functional conditions.     

Reciprocal Innervation between Serotonergic and GABAergic Neurons in Raphe Nuclei of the Rat

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain in order to study serotonergic-GABAergic interaction. The slices were loaded with either [³H] serotonin or [³H]GABA, superfused and the electrically induced efflux of radioactivity was determined. The GABA_A receptor agonist muscimol (3 to 30 M) and the GABA_B receptor agonist baclofen (30 and 100 M) inhibited [³H]serotonin and [³H]GABA release. These effects of muscimol were reversed by the GABA_A antagonists bicuculline (100 M). The GABA_B antagonist phaclofen (100 M) also antagonized the baclofen-induced inhibition of [³H]serotonin and [³H]GABA release. Phaclofen by itself increased [³H]serotonin release but it did not alter [³H]GABA overflow. Muscimol (10 M) and baclofen (100 M) also inhibited [³H]serotonin release after depletion of GABAergic neurons by isoniazid pretreatment. These findings indicate the presence of postsynaptic GABA_A and GABA_B receptors located on serotonergic neurons. The 5-HT_1A receptor agonist 8-OH-DPAT (0.01 to 1 M) and the 5-HT_1B receptor agonist CGS-12066A (0.01 to 1 M) inhibited the electrically stimulated [³H]serotonin and [³H]GABA release. The 5-HT_1A antagonist WAY-100135 (1 M) was without effect on [³H]serotonin and [³H]GABA efflux by itself but it reversed the 8-OH-DPAT-induced transmitter release inhibition. During KCl (22 mM)-induced depolarization, tetrodotoxin (1 M) did not alter the inhibitory effect of CGS-12066A (1 M) on [³H]GABA release, it did blocked, however, the ability of 8-OH-DPAT (1 M) to reduce [³H]GABA efflux. After depletion of raphe serotonin neurons by p-chlorophenylalanine pretreatment, CGS-12066A (1 M) still inhibited [³H]GABA release whereas in serotonin-depleted slices, 8-OH-DPAT (1 M) was without effect on the release. We conclude that reciprocal influence exists between serotonergic projection neurons and the GABAergic interneurons or afferents in the raphe nuclei and these interactions may be mediated by 5-HT_1A/B and GABA_A/B receptors. Both synaptic and non-synaptic neurotransmission may be operative in the 5-HTergic-GABAergic reciprocal interaction which may serve as a local tuning in the neural connection between cerebral cortex and midbrain raphe nuclei.     

Histochemical Discrimination of Endogenous Mammalian β-galactosidase Activity from that Resulting from lac-Z Gene Expression

Minces of several organs from the transgenic mouse ROSA-gal 26 (ROSA-26), which robustly expresses bacterial lac-Z in most tissues, were exposed to 4-bromo, 5-chloro, 3-indoyl, -D-galactopyrosanide (X-gal) at pH ranging from 7.5 to 9.5 to determine the optimal pH for in situ demonstration of bacterial -galactosidase activity (neutral pH optimum) while minimizing detection of potentially confounding endogenous mammalian -galactosidase (acidic pH optimum). Similar studies were performed with organ minces from C57BL/6 mice, Sprague-Dawley rats, New Zealand white rabbits, and macaques to confirm the effect of pH on minimizing detection of endogenous mammalian -galactosidase. In all organs evaluated; heart, liver, spleen, kidney, brain, and skeletal muscle, endogenous -galactosidase activity was rarely detected following incubation at pH greater than 7.5. In contrast, bacterial -galactosidase activity in the ROSA-26 mice was strongly detected in organ minces following incubation at pH 8.0–9.0. These findings are similar to previous observations we have made in lung minces and confirm that a simple alteration of a commonly used histochemical technique for detecting in situ -galactosidase activity, raising the reaction buffer pH to weakly alkaline range, can reliably distinguish between endogenous activity and that resulting from exogenous bacterial gene expression.     

Regulation of biosynthesis of aspartate family amino acids in the photosynthetic bacterium Rhodopseudomonas palustris

The four amino acids of the aspartate family (l-lysine, l-methionine, l-threonine, and l-isoleucine) are produced in bacteria by a branched biosynthetic pathway. Regulation of synthesis of early common intermediates and of carbon flow through distal branches of the pathway requires operation of a number of subtle feedback controls, which are integrated so as to ensure balanced synthesis of the several end products. Earlier studies with nonsulfur purple photosynthetic bacteria were instrumental in revealing the existence of alternative regulatory schemes, and in this communication we report on the control pattern of a representative of this physiological group not previously investigated, Rhodopseudomonas palustris. The results obtained from study of the properties of four key regulatory enzymes of the aspartate family pathway (-aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine deaminase) and of the effects of exogenous amino acids (i. e., the end products) on growth of the bacterium indicate that the control schema in Rps. palustris differs substantially from the schemes described for other Rhodopseudomonas species, but resembles the regulatory pattern observed in Rhodospirillum rubrum.Abbreviations A absorbancy - AK -aspartokinase - ASA aspartate -semialdehyde - DTT dithiothreitol - HS l-homoserine - HSDH homoserine dehydrogenase - HSK homoserine kinase - I l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-methionine - ME -mercaptoethanol - PABA p-aminobenzoic acid - T l-threonine - TD threonine deaminase - RCV synthetic growth medium (see text) - YP agar medium containing 0.3% yeast extract, 0.3% peptone, and 1.5% agar - Y2T synthetic growth medium (see text)     

Regime Shifts in the Sahara and Sahel: Interactions between Ecological and Climatic Systems in Northern Africa

The Sahara and Sahel regions of northern Africa have complex environmental histories punctuated by sudden and dramatic regime shifts in climate and ecological conditions. Here we review the current understanding of the causes and consequences of two environmental regime shifts in the Sahara and Sahel. The first regime shift is the sudden transition from vegetated to desert conditions in the Sahara about 5500 years ago. Geologic data show that wet environmental conditions in this region—giving rise to extensive vegetation, lakes, and wetlands—came to an abrupt end about 5500 years ago. Explanations for climatic changes in northern Africa during the Holocene have suggested that millennial-scale changes in the Earths orbit could have caused the wet conditions that prevailed in the early Holocene and the dry conditions prevalent today. However, the orbital hypothesis, by itself, does not explain the sudden regime shift 5500 years ago. Several modeling studies have proposed that strong, nonlinear feedbacks between vegetation and the atmosphere could amplify the effects of orbital variations and create two alternative stable states (or regimes) in the climate and ecosystems of the Sahara: a green Sahara and a desert Sahara. A recent coupled atmosphere-ocean-land model confirmed that there was a sudden shift from the green Sahara to the desert Sahara regime approximately 5500 years ago. The second regime shift is the onset of a major 30-year drought over the Sahel around 1969. Several lines of evidence have suggested that the interactions between atmosphere and vegetation act to reinforce either a wet Sahel or a dry Sahel climatic regime, which may persist for decades at a time. Recent modeling studies have indicated that the shift from a wet Sahel to a dry Sahel regime was caused by strong feedbacks between the climate and vegetation cover and may have been triggered by slow changes in either land degradation or sea-surface temperatures. Taken together, we conclude that the existence of alternative stable states (or regimes) in the climate and ecosystems of the Sahara and Sahel may be the result of strong, nonlinear interactions between vegetation and the atmosphere. Although the shifts between these regimes occur rapidly, they are made possible by slow, subtle changes in underlying environmental conditions, including slow changes in incoming solar radiation, sea-surface temperatures, or the degree of land degradation.     

Effects of β-alanine treatment on the taurine and DNA content of the rat heart and retina

Experimental evidence has suggested that the high endogenous levels of taurine found in the rat heart and retina are maintained to a large extent by transport processes out of the blood, rather than by endogenous biosynthesis. When these high levels are depleted, dysfunction ensues. In vitro studies have shown that -alanine is a good antagonist of these transport processes. The current studies were done to evaluate the feasibility of depleting heart and retinal taurine levels in vivo through treatment of adult rats either orally or with injections of -alanine. None of the treatments had significant effects on retinal taurine content; ventricular taurine was reduced in some situations, but the effects were not maintained, nor as large as with another transport antagonist. No functional changes were observed. Oral treatment with -alanine had fewer obvious side effects than injections, but all treated rats had body weights less than age-matched controls.     

17 β-Estradiol Enhances the Outgrowth and Survival of Neocortical Neurons in Culture

Results of this investigation demonstrate that exposure to 17 -estradiol differentially and significantly regulates cortical nerve cell outgrowth depending on the cortical region. Parietal and occipital neurons treated with 1 nM 17 -estradiol showed a greater magnitude of neuronal outgrowth whereas outgrowth of temporal cortex neurons was decreased in the presence of 1 nM 17 -estradiol. Frontal cortex neurons showed a consistent enhancement of neuronal outgrowth that did not reach statistical significance. The dose response profile for 17 -estradiol regulation of the macromorphological features exhibited a bimodal dose response relationship whereas the dose response profile for 17 -estradiol regulation of the micromorphological features exhibited a dose response more characteristic of an inverted V-shaped function. An antagonist to the NMDA receptor antagonist, AP5, abolished the growth promoting effect of 17 -estradiol whereas the nuclear estrogen receptor antagonist ICI 182,780 did not. Lastly, neocortical neurons exposed to 17 -estradiol exhibited greater viability and survival than control neurons over a two week period. These data indicate that 17 -estradiol can enhance the growth and viability of select populations of neocortical neurons and that the growth promoting effects of 17 -estradiol can be blocked by an antagonist to the NMDA glutamate receptor and not by an antagonist to the estrogen nuclear receptor.     

Protocol-dependence of equivalent circuit parameters of toad urinary bladder

Summary Determinations of current-voltage relationships are widely employed in the characterization of epithelial sodium transport. In order to determine the protocol dependence of transport parameters in the toad urinary bladder, studies were carried out in the presence and absence of amiloride, an inhibitor of active sodium transport. With symmetric positive and negative perturbations of the transepithelial electrical potential difference (0±100 mV) for 30 sec, the amiloride-sensitive current-voltage (i ^a -) relationship was near linear over the range –75+100 mV, indicating constancy of the conductance ^a and the apparent electromotive force E _Na, lumped parameters of the standard electrical equivalent circuit model of the active transport system. With a reverse protocol (±1000 mV) or 15 min perturbations thei ^a - relationships were highly nonlinear. Nonlinearity reflected voltage dependence of parameters: perturbations that increased active transport decreased E _Na and increased ^a, as evaluated from 10 sec perturbations of ; slowing of active transport produced the converse changes. These effects are usefully analyzed in both quasi-steady states and true steady states by means of a detailed equivalent circuit incorporating the significant ionic currents across each plasma membrane. Precise understanding of the significance of ^a and E _Na will require characterization of the partial ionic conductances on perturbation of .