THE EFFECT OF RELAXIN ON BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS OF RAT MYOMETRIAL CELLS IN CULTURE (CYCLIC-AMP, CELL SHAPE CHANGE)

Relaxin is able to inhibit spontaneous, oxytocin-and prostaglandin-driven uterine contractions. The intracellular mechanism of action of relaxin on uterine relaxation had previously been studied using isometrically suspended uterine strips. Since uterine strips contain stroma as well as myometrium, the changes in biochemical parameters induced by relaxin treatment may not occur in the same cell types responsible for the physical changes. In these studies, cultures of enriched populations of rat myometrial cells were used to investigate the effect of relaxin on biochemical and morphological parameters which are related to relaxation.^ Under optimal culture conditions (initial plating density 1 - 1.5 x 10('6)cells/ml, 3 ml/35 mm dish, 2 days culture), enzymatically isolated rat myometrial cells were able to respond to relaxin with cAMP elevation. Relaxin elevated cAMP levels in the presence but not the absence of 0.1 mM methylisobutylxanthine or 0.4 um forskolin in a time- and concentration-dependent manner. In contrast, isoproterenol was able to elevate cAMP levels in the presence and absence of 0.1 mM methylisobutylxanthine.^ Oxytocin treatment caused a decrease in mean cell length and area of myometrial cells in culture which could be considered analogous to contraction. Under optimal culture conditions, relaxin increased myometrial cell length and area (i.e. analogous to relaxation) of oxytocin-treated cells in a time- and concentration-dependent manner. Other relaxants such as isoproterenol and dibutyryl cAMP also increased cell length and area of oxytocin - treated myometrial cells in culture.^ Under optimal culture conditions, relaxin decreased myosin light chain kinase activity in a time-and concentration-dependent manner by increasing the K(,50) of the enzyme for calmodulin (CaM), i.e. decreasing the affinity of the enzyme for CaM. The decrease in the affinity of myosin light chain kinase for CaM may be due to the phosphorylation of the enzyme by cAMP-dependent protein kinase. Relaxin also decreased the Ca('2+)(.)CaM-independent myosin light chain kinase activity to a lesser extent than that of the Ca('2+)(.)CaM-dependent enzyme activity. This was not attributable to a decrease in the affinity of the enzyme for myosin in myometrial cells in culture, in contrast to the finding of such a change following relaxin treatment of uterine strips. Further studies are required to clarify this point.^ There was a temporal association between the effects of relaxin on elevation of cAMP levels in the presence of 0.4 uM forskolin, increase in cell length and decrease in myosin light chain kinase activity. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

REGULATION OF BRAIN NORADRENERGIC-RECEPTOR FUNCTION BY ADRENOCORTICOTROPHIN AND ANTIDEPRESSANT DRUGS (ADRENERGIC RECEPTOR, CYCLIC-AMP, ADENYLATE CYCLASE, IMIPRAMINE, STRESS)

Human behavior appears to be regulated in part by noradrenergic transmission since antidepressant drugs modify the number and function of (beta)-adrenergic receptors in the central nervous system. Affective illness is also known to be associated with the endocrine system, particularly the hypothalamic-pituitary-adrenal axis. The aim of the present study was to determine whether hormones, in particular adrencorticotrophin (ACTH) and corticosterone, may influence behavior by regulating brain noradrenergic receptor function.^ Chronic treatment with ACTH accelerated the increase or decrease in rat brain (beta)-adrenergic receptor number induced by a lesion of the dorsal noradrenergic bundle or treatment with the antidepressant imipramine. Chronic administration of ACTH alone had no effect on (beta)-receptor number although it reduced norepinephrine stimulated cyclic AMP accumulation in brain slices. Treatment with imipramine also reduced the cyclic AMP response to norepinephrine but was accompanied by a decrease in (beta)-adrenergic receptor number. Both the imipramine and ACTH treatments reduced the affinity of (beta)-adrenergic receptors for norepinephrine, but only the antidepressant modified the potency of the neurotransmitter to stimulate second messenger production. Neither ACTH nor imipramine treatment altered Gpp(NH)p- or fluoride-stimulated adenylate cyclase, cyclic AMP, cyclic GMP, or cyclic GMP-stimulated cyclic AMP phosphodiesterase, or the activity of the guanine nucleotide binding protein (Gs). These findings suggested that post-receptor components of the cyclic nucleotide generating system are not influenced by the hormone or antidepressant. This conclusion was verified by the finding that neither treatment altered adenosine-stimulated cyclic AMP accumulation in brain tissue.^ A detailed examination of the (alpha)- and (beta)-adrenergic receptor components of norepinephrine-stimulated cyclic AMP production revealed that ACTH, but not imipramine, administration reduced the contribution of the (alpha)-receptor mediated response. Like ACTH treatment, corticosterone diminished the (alpha)-adrenergic component indicating that adrenal steroids probably mediate the neurochemical responses to ACTH administration. The data indicate that adrenal steroids and antidepressants decrease noradrenergic receptor function by selectively modifying the (alpha)- and (beta)-receptor components. The functional similarity in the action of the steroid and antidepressants suggests that adrenal hormones normally contribute to the maintenance of receptor systems which regulate affective behavior in man. ^

Environmental analyses of cyclic Middle Eocene sediments and palynomorph records of ODP Hole 189-1172A

The influence of orbital precession on early Paleogene climate and ocean circulation patterns in the southeast Pacific region is investigated by combining environmental analyses of cyclic Middle Eocene sediments and palynomorph records recovered from ODP Hole 1172A on the East Tasman Plateau with climate model simulations. Integration of results indicates that in the marine realm, direct effects of precessional forcing are not pronounced, although increased precipitation/runoff could have enhanced dinoflagellate cyst production. On the southeast Australian continent, the most pronounced effects of precessional forcing were fluctuations in summer precipitation and temperature on the Antarctic Margin. These fluctuations resulted in vegetational changes, most notably in the distribution of Nothofagus (subgenus Brassospora). The climate model results suggest significant fluctuations in sea ice in the Ross Sea, notably during Austral summers. This is consistent with the influx of Antarctic heterotrophic dinoflagellates in the early part of the studied record. The data demonstrate a strong precessionally driven climate variability and thus support the concept that precessional forcing could have played a role in early Antarctic glaciation via changes in runoff and/or precipitation.

Cyclic behaviour of saturated sands subject to previous horizontal shear stresses

The dynamic behaviour of saturated sands has been studied from different perspectives. However, most experimental research on this field does not take into account the shear stress conditions existing prior to the application of dynamic loads; i.e., a null initial static shear stress (τo = 0) is assumed. The main objective of this work is to report on the influence that static shear stresses (τo) have on the behaviour of saturated sands under cyclic shear loads. This article presents the results and analysis of part of a wider experimental programme involving 30 monotonic and 26 cyclic simple shear tests for different combinations of static shear stress (τo) and cyclic shear stress (τc) (all undrained), besides identification and classification tests. The tested samples have been taken from the area of the North Entrance Mouth at the Port of Barcelona (Spain).

Dynamic & Cyclic behaviour of ballast in the long term as determined in Cedex's track box

The 6 cylinder servo-hydraulic loading system of CEDEX's track box (250 kN, 50 Hz) has been recently implemented with a new piezoelectric loading system (±20 kN, 300 Hz) allowing the incorporation of low amplitude high frequency dynamic load time histories to the high amplitude low frequency quasi-static load time histories used so far in the CEDEX's track box to assess the inelastic long term behavior of ballast under mixed traffic in conventional and high- speed lines. This presentation will discuss the results obtained in the first long-duration test performed at CEDEX's track box using simultaneously both loading systems, to simulate the pass-by of 6000 freight vehicles (1M of 225 kN axle loads) travelling at a speed of 120 km/h over a line with vertical irregularities corresponding to a medium quality lin3e level. The superstructure of the track tested at full scale consisted of E 60 rails, stiff rail pads (mayor que 450 kN/mm), B90.2 sleepers with USP 0.10 N/mm and a 0.35 m thick ballast layer of ADIF first class. A shear wave velocity of 250 m/s can be assumed for the different layers of the track sub-base. The ballast long-term settlements will be compared with those obtained in a previous long-duration quasi- static test performed in the same track, for the RIVAS [EU co-funded] project, in which no dynamic loads where considered. Also, the results provided by a high diameter cyclic triaxial cell with ballast tested in full size will be commented. Finally, the progress made at CEDEX's Geotechnical Laboratory to reproduce numerically the long term behavior of ballast will be discussed.

Health monitoring of web plastifying dampers subjected to cyclic loading through vibration tests

This article investigates experimentally the application of health monitoring techniques to assess the damage on a particular kind of hysteretic (metallic) damper called web plastifying dampers, which are subjected to cyclic loading. In general terms, hysteretic dampers are increasingly used as passive control systems in advanced earthquake-resistant structures. Nonparametric statistical processing of the signals obtained from simple vibration tests of the web plastifying damper is used here to propose an area index damage. This area index damage is compared with an alternative energy-based index of damage proposed in past research that is based on the decomposition of the load?displacement curve experienced by the damper. Index of damage has been proven to accurately predict the level of damage and the proximity to failure of web plastifying damper, but obtaining the load?displacement curve for its direct calculation requires the use of costly instrumentation. For this reason, the aim of this study is to estimate index of damage indirectly from simple vibration tests, calling for much simpler and cheaper instrumentation, through an auxiliary index called area index damage. Web plastifying damper is a particular type of hysteretic damper that uses the out-of-plane plastic deformation of the web of I-section steel segments as a source of energy dissipation. Four I-section steel segments with similar geometry were subjected to the same pattern of cyclic loading, and the damage was evaluated with the index of damage and area index damage indexes at several stages of the loading process. A good correlation was found between area index damage and index of damage. Based on this correlation, simple formulae are proposed to estimate index of damage from the area index damage.

Evidence of a role for cyclic ADP-ribose in long-term synaptic depression in hippocampus

Ca2+ released from presynaptic and postsynaptic intracellular stores plays important roles in activity-dependent synaptic plasticity, including long-term depression (LTD) of synaptic strength. At Schaffer collateral–CA1 synapses in the hippocampus, presynaptic ryanodine receptor-gated stores appear to mobilize some of the Ca2+ necessary to induce LTD. Cyclic ADP-ribose (cADPR) has recently been proposed as an endogenous activator of ryanodine receptors in sea urchin eggs and several mammalian cell types. Here, we provide evidence that cADPR-mediated signaling pathways play a key role in inducing LTD. We show that biochemical production of cGMP increases cADPR concentration in hippocampal slices in vitro, and that blockade of cGMP-dependent protein kinase, cADPR receptors, or ryanodine-sensitive Ca2+ stores each prevent the induction of LTD at Schaffer collateral–CA1 synapses. A lack of effect of postsynaptic infusion of either cADPR antagonist indicates a probable presynaptic site of action.

An isoform of the rod photoreceptor cyclic nucleotide-gated channel β subunit expressed in olfactory neurons

Sensory transduction in olfactory neurons involves the activation of a cyclic nucleotide-gated (CNG) channel by cAMP. Previous studies identified a CNG channel α subunit (CNG2) and a β subunit (CNG5), which when heterologously expressed form a channel with properties similar but not identical to those of native olfactory neurons. We have cloned a new type of CNG channel β subunit (CNG4.3) from rat olfactory epithelium. CNG4.3 derives from the same gene as the rod photoreceptor β subunit (CNG4.1) but lacks the long, glutamic acid-rich domain found in the N terminus of CNG4.1. Northern blot and in situ hybridization revealed that CNG4.3 is expressed specifically in olfactory neurons. Expression of CNG4.3 in human embryonic kidney 293 cells did not lead to detectable currents. Coexpression of CNG4.3 with CNG2 induced a current with significantly increased sensitivity for cAMP whereas cGMP affinity was not altered. Additionally, CNG4.3 weakened the outward rectification of the current in the presence of extracellular Ca2+, decreased the relative permeability for Ca2+, and enhanced the sensitivity for l-cis diltiazem. Upon coexpression of CNG2, CNG4.3, and CNG5, a conductance with a cAMP sensitivity greater than that of either the CNG2/CNG4.3 or the CNG2/CNG5 channel and near that of native olfactory channel was observed. Our data suggest that CNG4.3 forms a subunit of the native olfactory CNG channel. The expression of various CNG4 isoforms in retina and olfactory epithelium indicates that the CNG4 subunit may be necessary for normal function of both photoreceptor and olfactory CNG channels.

Cyclic AMP regulates potassium channel expression in C6 glioma by destabilizing Kv1.1 mRNA

The tissue distributions and physiological properties of a variety of cloned voltage-gated potassium channel genes have been characterized extensively, yet relatively little is known about the mechanisms controlling expression of these genes. Here, we report studies on the regulation of Kv1.1 expressed endogenously in the C6 glioma cell line. We demonstrate that elevation of intracellular cAMP leads to the accelerated degradation of Kv1.1 RNA. The cAMP-induced decrease in Kv1.1 RNA is followed by a decrease in Kv1.1 protein and a decrease in the whole cell sustained K+ current amplitude. Dendrotoxin-I, a relatively specific blocker of Kv1.1, blocks 96% of the sustained K+ current in glioma cells, causing a shift in the resting membrane potential from −40 mV to −7 mV. These data suggest that expression of Kv1.1 contributes to setting the resting membrane potential in undifferentiated glioma cells. We therefore suggest that receptor-mediated elevation of cAMP reduces outward K+ current density by acting at the translational level to destabilize Kv1.1 RNA, an additional mechanism for regulating potassium channel gene expression.

Molecular determinants of the modulation of cyclic nucleotide-activated channels by calmodulin

The action of calmodulin (CaM) on target proteins is important for a variety of cellular functions. We demonstrate here, however, that the presence of a CaM-binding site on a protein does not necessarily imply a functional effect. The α-subunit of the cGMP-gated cation channel of human retinal cones has a CaM-binding site on its cytoplasmic N-terminal region, but the homomeric channel that it forms is not functionally modulated by CaM. Mutational analysis based on comparison to the highly homologous olfactory cyclic nucleotide-gated channel α-subunit, which does form a CaM-modulated channel, indicates that residues downstream of the CaM-binding domain on these channels are also important for CaM to have an effect. These findings suggest that a CaM-binding site and complementary structural features in a protein probably evolve independently, and an effect caused by CaM occurs only in the presence of both elements. More generally, the same may be true for other recognized binding sites on proteins for modulators or activators, so that a demonstrated physical interaction does not necessarily imply functional consequence.

Production of cyclic peptides and proteins in vivo

Combinatorial libraries of synthetic and natural products are an important source of molecular information for the interrogation of biological targets. Methods for the intracellular production of libraries of small, stable molecules would be a valuable addition to existing library technologies by combining the discovery potential inherent in small molecules with the large library sizes that can be realized by intracellular methods. We have explored the use of split inteins (internal proteins) for the intracellular catalysis of peptide backbone cyclization as a method for generating proteins and small peptides that are stabilized against cellular catabolism. The DnaE split intein from Synechocystis sp. PCC6803 was used to cyclize the Escherichia coli enzyme dihydrofolate reductase and to produce the cyclic, eight-amino acid tyrosinase inhibitor pseudostellarin F in bacteria. Cyclic dihydrofolate reductase displayed improved in vitro thermostability, and pseudostellarin F production was readily apparent in vivo through its inhibition of melanin production catalyzed by recombinant Streptomyces antibioticus tyrosinase. The ability to generate and screen for backbone cyclic products in vivo is an important milestone toward the goal of generating intracellular cyclic peptide and protein libraries.

Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels

Native cylic nucleotide-gated (CNG) channels are composed of α and β subunits. Olfactory CNG channels were expressed from rat cDNA clones in Xenopus oocytes and studied in inside-out patches. Using tandem dimers composed of linked subunits, we investigated the stoichiometry and arrangement of the α and β subunits. Dimers contained three subunit types: αwt, βwt, and αm. The αm subunit lacks an amino-terminal domain that greatly influences gating, decreasing the apparent affinity of the channel for ligand by 9-fold, making it a reporter for inclusion in the tetramer. Homomeric channels from injection of αwtαwt dimers and from αwt monomers were indistinguishable. Channels from injection of αwtαm dimers had apparent affinities 3-fold lower than αwt homomultimers, suggesting a channel with two αwt and two αm subunits. Channels from coinjection of αwtαwt and ββ dimers were indistinguishable from those composed of α and β monomers and shared all of the characteristics of the α+β phenotype of heteromeric channels. Coinjection of αwtαm and ββ dimers yielded channels also of the α+β phenotype but with an apparent affinity 3-fold lower, indicating the presence of αm in the tetramer and that α+β channels have adjacent α-subunits. To distinguish between an α-α-α-β and an α-α-β-β arrangement, we compared apparent affinities for channels from coinjection of αwtαwt and βαwt or αwtαwt and βαm dimers. These channels were indistinguishable. To further argue against an α-α-α-β arrangement, we quantitatively compared dose–response data for channels from coinjection of αwtαm and ββ dimers to those from α and β monomers. Taken together, our results are most consistent with an α-α-β-β arrangement for the heteromeric olfactory CNG channel.

Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to Eag and cyclic nucleotide-gated channels

We have isolated a novel cDNA, that appears to represent a new class of ion channels, by using the yeast two-hybrid system and the SH3 domain of the neural form of Src (N-src) as a bait. The encoded polypeptide, BCNG-1, is distantly related to cyclic nucleotide-gated channels and the voltage-gated channels, Eag and H-erg. BCNG-1 is expressed exclusively in the brain, as a glycosylated protein of ≈132 kDa. Immunohistochemical analysis indicates that BCNG-1 is preferentially expressed in specific subsets of neurons in the neocortex, hippocampus, and cerebellum, in particular pyramidal neurons and basket cells. Within individual neurons, the BCNG-1 protein is localized to either the dendrites or the axon terminals depending on the cell type. Southern blot analysis shows that several other BCNG-related sequences are present in the mouse genome, indicating the emergence of an entire subfamily of ion channel coding genes. These findings suggest the existence of a new type of ion channel, which is potentially able to modulate membrane excitability in the brain and could respond to regulation by cyclic nucleotides.

A Novel Ras-interacting Protein Required for Chemotaxis and Cyclic Adenosine Monophosphate Signal Relay in Dictyostelium

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced ∼60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.