Receptor-mediated Changes Research Articles

Characterization of Tachykinin‐related Peptides from Different Insect Species on Drosophila Tachykinin Receptor‐expressing Cell Line

Drosophila tachykinin receptor, a neurokinin receptor cloned from the fruit fly Drosophila melanogaster, is a G-protein-coupled receptor, which upon activation by a peptide agonist induces a transient increase in the concentration of intracellular calcium. The functional assay based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca(2+) levels was used to examine and compare the effect of tachykinin-related peptides from different species. Among the endogenous Drosophila peptides, Drm-TK I induced the strongest calcium response. The most potent tachykinin-related peptides from Leucophaea maderae, Locusta migratoria, and Calliphora vomitoria, were partial agonists at the Drosophila tachykinin receptor.

D-1-Nal 4]endomorphin-2 is a potent μ-opioid receptor antagonist in the aequorin luminescence-based calcium assay

A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca 2+ levels, was used to examine relative potency and efficacy of the μ-opioid receptor antagonists. A series of position 3- and 4-substituted endomorphin-2 (Tyr–Pro–Phe–Phe–NH 2) analogues containing d-3-(1-naphthyl)-alanine ( d-1-Nal) or d-3-(2-naphthyl)-alanine ( d-2-Nal), which were previously shown to reverse antinociception induced by endomorphin-2 in the in vivo hot-plate test in mice, was tested in the aequorin luminescence-based calcium assay to examine their μ-opioid antagonist potency in vitro. A recombinant mammalian cell line expressing the μ-opioid receptor together with a luminescent reporter protein, apoaequorin, was used in the study. The results obtained in this functional assay indicated that analogues with d-1-Nal or d-2-Nal substitutions in position 4 of endomorphin-2 are strong μ-opioid receptor antagonists, while those substituted in position 3 are partial agonists. Exceptional antagonist potency in the calcium assay was observed for [ d-1-Nal 4]endomorphin-2. The pA 2 value for this analogue was 7.95, compared to the value of 8.68 obtained for the universal, non-selective opioid antagonist of the alkaloid structure, naloxone. The obtained results were compared with the data from the hot-plate test in mice. In that in vivo assay [ d-1-Nal 4]endomorphin-2 was also the most potent analogue of the series.

Functional Characterization of Opioid Receptor Ligands by Aequorin Luminescence-Based Calcium Assay

A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in intracellular calcium levels, was used to examine relative potency and efficacy of the mu-opioid agonists endomorphin-1, endomorphin-2, morphiceptin, and their position 3-substituted analogs, as well as the delta-agonist deltorphin-II. The results of the aequorin assay, performed on recombinant cell lines, were compared with those obtained in the functional assay on isolated tissue preparations (guinea pig ileum and mouse vas deferens). A range of nine opioid peptide ligands produced a similar rank order of potency for the mu- and delta-opioid receptor agonists in both functional assays. The highest potency at the mu-receptor was observed for endomorphin-1, endomorphin-2, and [D-1-Nal3]morphiceptin, whereas deltorphin-II was the most potent delta-receptor agonist. In the aequorin assay, the mu- and delta-agonist-triggered luminescence was inhibited by the opioid antagonists naloxone and naltrindole, respectively. We can conclude that the use of the aequorin assay for new mu- and delta-receptor-selective opioid analogs gives pharmacologically relevant data and allows high-throughput compound screening, which does not involve radioactivity or animal tissues. This is the first study that validates the application of this assay in the screening of opioid analogs.

TGFβ2-Induced Changes in Human Trabecular Meshwork: Implications for Intraocular Pressure

Transforming growth factor (TGF)-beta2 levels are elevated in glaucomatous human aqueous humor. TGFbeta is a cytokine that alters extracellular matrix (ECM) metabolism, and excess ECM has been proposed to increase aqueous outflow resistance in the trabecular meshwork (TM) of glaucomatous eyes. This study was undertaken to investigate effects of TGFbeta2 on secretion of fibronectin and the protease inhibitor plasminogen activator inhibitor (PAI)-1 from human TM cell cultures and perfused human ocular anterior segments. Total RNA was isolated from pooled human TM cell monolayers and used for a gene microarray expression analysis. Supernatants from treated human TM cells were analyzed by ELISA for fibronectin or PAI-1 content. TGFbeta2 effects on intraocular pressure (IOP) were evaluated in a perfused organ culture model using human anterior segments, and eluates were analyzed for fibronectin and PAI-1 content. Overnight treatment of TM cells with TGFbeta2 upregulated multiple ECM-related genes, such as PAI-1. TGFbeta2 also increased secretion of both fibronectin and PAI-1 from TM cells. TGFbeta2 effects on TM cells were blocked by inhibitors of the TGFbeta type I receptor. In perfused human anterior segments, TGFbeta2 treatment elevated IOP and increased eluate fibronectin and PAI-1 content. TGFbeta2 effects on IOP may be transduced by TGFbeta type-I receptor-mediated changes in TM secretion of ECM-related factors such as fibronectin and PAI-1. Modulation of TGFbeta2-induced changes in the ECM may provide a novel and viable approach to the management of glaucoma.

Hyaluronan‐based polymer scaffold modulates the expression of inflammatory and degradative factors in mesenchymal stem cells: Involvement of Cd44 and Cd54

Hyaluronan (HA), in the bone marrow stroma, is the major non-protein glycosaminoglycan component of extracellular matrix (ECM) involved in cell positioning, proliferation, differentiation as well as in receptor-mediated changes in gene expression. Repair of bone and regeneration of bone marrow is dependent on ECM, inflammatory factors, like chemokines and degradative factors, like metalloproteinases. We analyzed the interaction between human mesenchymal stem cells (h-MSCs) and a three-dimensional (3-D) HA-based scaffold in vitro. The expression of CXC chemokines/receptors, CXCL8 (IL-8)/CXCR1-2, CXCL10 (IP-10)/CXCR3, CXCL12 (SDF-1)/CXCR4, and CXCL13 (BCA-1)/CXCR5, and metalloproteinases/inhibitors MMP-1, MMP-3, MMP-13/TIMP-1 were evaluated in h-MSCs grown on plastic or on HA-based scaffold by Real-time PCR, ELISA, and immunocytochemical techniques. Moreover, the expression of two HA receptors, CD44 and CD54, was analyzed. We found both at mRNA and protein levels that HA-based scaffold induced the expression of CXCR4, CXCL13, and MMP-3 and downmodulated the expression of CXCL12, CXCR5, MMP-13, and TIMP-1 while HA-based scaffold induced CD54 expression but not CD44. We found that these two HA receptors were directly involved in the modulation of CXCL12, CXCL13, and CXCR5. This study demonstrates a direct action of a 3-D HA-based scaffold, widely used for cartilage and bone repair, in modulating both h-MSCs inflammatory and degradative factors directly involved in the engraftment of specific cell types in a damaged area. Our data clearly demonstrate that HA in this 3-D conformation acts as a signaling molecule for h-MSCs.

Rapid modulation of TRH-like peptides in rat brain by thyroid hormones

Recent identification of membrane receptors for T 4, T 3, 3,5-T 2, and 3-iodothyronamine that mediate rapid physiologic effects of thyroid hormones suggested that such receptors may supplement the regulation of TRH and TRH-like peptides by nuclear T 3 receptors. For this reason 200 g male Sprague–Dawley rats received daily i.p. injections of PTU or T 4. Levels of TRH and TRH-like peptides were measured 0, 2 h or 1, 2, 3, or 4 days later. Rapid increases or decreases in TRH and TRH-like peptide levels were observed in response to PTU and T 4 treatments in various brain regions involved in mood regulation. Significant effects were measured within 2 h of T 4 injection. Nuclear T 3 receptor-mediated changes in gene expression altering translation, post-translational processing and constitutive release of peptides require more than 2 h. We conclude that non-genomic mechanisms may contribute to the psychiatric effects of thyroid disease and thyroid hormone adjuvant treatment for major depression.

Sex-Specific Effects of Prenatal Low-Protein and Carbenoxolone Exposure on Renal Angiotensin Receptor Expression in Rats

Experimental models have shown the developing cardiovascular and renal systems to be sensitive to mild shifts in maternal nutrition, leading to altered function and risk of disease in adult life. The offspring of Wistar rats fed a low-protein diet during pregnancy exhibit a reduced nephron number and hypertension in postnatal life, providing a useful tool to examine the mechanistic basis of programming. Evidence indicates that upregulation of the renin-angiotensin system plays an important role, in particular through receptor-mediated changes in angiotensin II activity. However, although programmed hypertension has proven dependent on maternal glucocorticoids, there appear to be conflicting effects of prenatal low-protein and glucocorticoid exposure on postnatal angiotensin receptor expression. This study aimed to resolve this issue by comparing the effects of low-protein and glucocorticoid exposures on postnatal nephron number and angiotensin receptor expression. In addition, this study examined the modulation of prenatal treatment effects by postnatal inhibition of type 1 angiotensin receptor. The data demonstrates that whereas prenatal low-protein and glucocorticoid exposure have a similar effect in reducing nephron number, there are age- and gender-related differences in their effects on postnatal angiotensin receptor expression. In addition, this study provides novel evidence of a substantial upregulation of type 2 angiotensin receptor expression in low-protein- and glucocorticoid-exposed female offspring at 20 weeks of age, with implications for subsequent renal remodeling and function. Despite being targeted to the postnephrogenic period, inhibition of type 1 angiotensin receptor had an inhibitory effect on renal and somatic growth, additionally indicating its unsuitability during early life.

Comparison of antagonist activity of spantide family at human neurokinin receptors measured by aequorin luminescence-based functional calcium assay

Neurokinin receptors (NK 1, NK 2, NK 3) are G-protein-coupled receptors, which upon activation by a peptide agonist induce a transient increase in the concentration of intracellular calcium. The functional assay based on aequorin-derived luminescence triggered by receptor-mediated changes in Ca 2+ levels was used to compare the effect of spantides I–III on SP-, NKA- and NKB-stimulated NK 1, NK 2 and NK 3 receptors, respectively. Recombinant cell lines expressing neurokinin receptors and apoaequorin were used in the study. The obtained results indicate that all three spantides acted as competitive antagonists at the NK 1 and NK 2 receptors and inhibited agonist-induced calcium responses. The rank order of antagonism at the NK 1 receptor was spantide II > spantide III > spantide I and at the NK 2 receptor was spantide III > spantide II > spantide I. All three spantides failed to antagonize NKB-induced calcium responses at the NK 3 receptor.

Serotonin Excites Neurons in the Human Submucous Plexus via 5-HT 3 Receptors

Serotonin (5-hydroxytryptamine [5-HT]) is a key signaling molecule in the gut. Recently, the neural 5-HT3 receptor received a lot of attention as a possible target in functional bowel diseases. Yet, the 5-HT3 receptor-mediated changes in properties of human enteric neurons is unknown. We used a fast imaging technique in combination with the potentiometric dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine to monitor directly the membrane potential changes in neurons of human submucous plexus from surgical specimens of 21 patients. An Ussing chamber technique was used to study 5-HT3 receptor involvement in chloride secretion. Local microejection of 5-HT directly onto ganglion cells resulted in a transient excitation of enteric neurons characterized by increased spike discharge. This response was mimicked by the 5-HT3 receptor agonist, 2-methyl-5-HT, and blocked by the 5-HT3 receptor antagonist, tropisetron. The proportions of 5-HT-responsive nerve cells per ganglion ranged from 25.5% +/- 18.4% in the duodenum to 54.2% +/- 46.9% in the colon. Interestingly, 2-methyl-5-HT did not evoke chloride secretion in the human intestine but it did in the guinea-pig intestine. Specific 5-HT3A and 5-HT3B receptor subunit immunoreactivity as well as 5-HT3A and 5-HT3B receptor-specific messenger RNA were detected in the tissue samples. Based on co-labeling with the pan-neuronal marker HuC/D we conclude that submucous nerve cells potentially express heteromeric 5-HT3A/B receptors. We show that 5-HT excited human enteric neurons via 5-HT3 receptors, which may comprise both 5-HT3A and 5-HT3B receptor subunits.

Differential expression of striatal synaptotagmin mRNA isoforms in hemiparkinsonian rats

Synaptotagmins (Syts) constitute a multi-gene family of 15 putative membrane trafficking proteins. The expression of some of the Syts in the brain might be dopaminergically controlled and thus affected by dopamine depletion in Parkinson’s disease. We used hemiparkinsonian rats to investigate the effects of chronic striatal dopamine depletion and the acute effects of antiparkinsonic drug l-DOPA or D1 agonist (±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrobromide (SKF82958) on the levels of striatal Syt I, II, IV, VI, VII, X, XI mRNA isoforms. On the 6-hydroxydopamine (6-OHDA)-lesioned side we observed a nearly total loss of tyrosine hydroxylase (TH), synaptotagmin I, Syt IV, Syt VII and Syt XI mRNA levels in the substantia nigra compacta (SNc). In dopamine-depleted striatum we also found a significant down-regulation Syt II and up-regulation of Syt X mRNA levels that could not be reversed by the acute treatment either with l-DOPA or SKF82958. By contrast, these two drugs induced an increase of Syt IV and Syt VII mRNA levels. A time-course study revealed the highest levels of Syt IV and VII mRNAs to occur at two hours and 12 hours after the treatment with SKF82958, respectively. D1 antagonist (±)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390) but not D2 antagonist haloperidol prevented the l-DOPA-driven increase of Syt IV and VII mRNAs. These results imply that synaptic plasticity in response to chronic striatal dopamine depletion involves a complex pattern of changes in striatal Syt mRNA expression. The l-DOPA treatment does not reverse the changes in Syt II and Syt X gene expression, but recruits additional, D1 receptor-mediated changes in Syt IV and Syt VII gene expression. Whether these D1 receptor-mediated changes play a role in the alterations of synaptic transmission that results in the unwanted side effects of chronic l-DOPA treatment in Parkinson’s disease remains to be determined.

Changes in Plasma Membrane Properties and Phosphatidylcholine Subspecies of Insect Sf9 Cells Due to Expression of Scavenger Receptor Class B, Type I, and CD36

In mammalian cells scavenger receptor class B, type I (SR-BI), mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester into hepatic and steroidogenic cells. In addition, SR-BI has a variety of effects on plasma membrane properties including stimulation of the bidirectional flux of free cholesterol (FC) between cells and HDL and changes in the organization of plasma membrane FC as indicated by increased susceptibility to exogenous cholesterol oxidase. Recent studies in SR-BI-deficient mice and in SR-BI-expressing Sf9 insect cells showed that SR-BI has significant effects on plasma membrane ultrastructure. The present study was designed to test the range of SR-BI effects in Sf9 insect cells that typically have very low cholesterol content and a different phospholipid profile compared with mammalian cells. The results showed that, as in mammalian cells, SR-BI expression increased HDL cholesteryl ester selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These activities were diminished or absent upon expression of the related scavenger receptor CD36. Thus, SR-BI has fundamental effects on cholesterol flux and membrane properties that occur in cells of evolutionarily divergent origins. Profiling of phospholipid species by electrospray ionization mass spectrometry showed that scavenger receptor expression led to the accumulation of phosphatidylcholine species with longer mono- or polyunsaturated acyl chains. These changes would be expected to decrease phosphatidylcholine/cholesterol interactions and thereby enhance cholesterol desorption from the membrane. Scavenger receptor-mediated changes in membrane phosphatidylcholine may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.

Tyrosine-phosphorylated and Nonphosphorylated Sodium Channel β1 Subunits Are Differentially Localized in Cardiac Myocytes

Voltage-gated sodium channel alpha and beta subunits expressed in mammalian heart are differentially localized to t-tubules and intercalated disks. Sodium channel beta subunits are multifunctional molecules that participate in channel modulation and cell adhesion. Reversible, receptor-mediated changes in beta1 tyrosine phosphorylation modulate its ability to recruit and associate with ankyrin. The purpose of the present study was to test our hypothesis that tyrosine-phosphorylated beta1 (pYbeta1) and nonphosphorylated beta1 subunits may be differentially localized in heart and thus interact with different cytoskeletal and signaling proteins. We developed an antibody that specifically recognizes pYbeta1 and investigated the differential subcellular localization of beta1 and pYbeta1 in mouse ventricular myocytes. We found that pYbeta1 colocalized with connexin-43, N-cadherin, and Nav1.5 at intercalated disks but was not detected at the t-tubules. Anti-pYbeta1 immunoprecipitates N-cadherin from heart membranes and from cells transfected with beta1 and N-cadherin in the absence of other sodium channel subunits. pYbeta1 does not associate with ankyrinB in heart membranes. N-cadherin and connexin-43 associate with Nav1.5 in heart membranes as assessed by co-immunoprecipitation assays. We propose that sodium channel complexes at intercalated disks of ventricular myocytes are composed of Nav1.5 and pYbeta1 and that these complexes are in close association with both N-cadherin and connexin-43. beta1 phosphorylation appears to regulate its localization to differential subcellular domains.

Integrating rapid responses to 1,25-dihydroxyvitamin D 3 with transcriptional changes in osteoblasts: Ca 2+ regulated pathways to the nucleus

It is well established that 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) treatment of target cells including osteoblasts activates both membrane-initiated rapid Ca 2+ responses linked to influx through voltage sensitive Ca 2+ channels (VSCCs) and longer term nuclear receptor-mediated changes in gene expression. We recently reported use of a cDNA microarray strategy to identify transcriptional changes after 3 and 24 h of treatment with 1,25(OH) 2D 3 and with an analog of 1,25(OH) 2D 3 (25(OH)-16ene-23yne-D 3 [AT]) that activates Ca 2+ influx without binding to the nuclear receptor. Among 5000 different clones on the array filters, we identified families of genes in osteoblasts that were altered two-fold or greater following treatment with 1,25(OH) 2D 3 or analog AT for 3 h. Cluster analysis further revealed complex patterns of changes in gene expression, indicative of multiple pathways to the nucleus. Evidenced by changes in target gene expression, activation of a Ca 2+/CaMK/CREB/CRE pathway clearly occurs and modulates expression of a variety of genes associated with changes in protein secretion including those involved in paracrine regulation of bone resorption, RANKL and osteoprotegerin (OPG). The changes in gene expression can be inhibited by L-type VSCC channel blockers, confirming the role of Ca 2+ entry in pathway activation. These findings provide clear evidence of rapid changes in gene expression associated with Ca 2+ influx after treatment with 1,25(OH) 2D 3, and open the door to novel nuclear receptor-independent signaling pathways that affect gene transcription.

Effect of omeprazole on gastric adenosine A1 and A2A receptor gene expression and function.

Adenosine has been shown to inhibit immunoreactive gastrin (IRG) release and to stimulate somatostatin-like immunoreactivity (SLI) release by activating adenosine A(1) and A(2A) receptors, respectively. Since the synthesis and release of gastrin and somatostatin are regulated by the acid secretory state of the stomach, the effect of achlorhydria on A(1) and A(2A) receptor gene expression and function was examined. Omeprazole-induced achlorhydria was shown to suppress A(1) and A(2A) receptor gene expression in the antrum and corporeal mucosa, but not in the corporeal muscle. Omeprazole treatment produced reciprocal changes in A(1) receptor and gastrin gene expression, and parallel changes in A(2A) receptor and somatostatin gene expression. The localization of A(1) and A(2A) receptors on gastrinsecreting G-cells and somatostatin-secreting D-cells, respectively, suggests that changes in adenosine receptor expression may modulate the synthesis and release of gastrin and somatostatin. Thus, the effect of omeprazole on adenosine receptor-mediated changes in IRG and SLI release was also examined in the vascularly perfused rat stomach. After omeprazole treatment, the A(1) receptor-mediated inhibition of IRG and SLI release induced by N(6)-cyclopentyladenosine (A(1) receptor-selective agonist) was not altered, but the A(2A) receptor-mediated augmentation of SLI release induced by 2-p-(2-carboxyethyl-)phenethylamino-5'-N-ethylcarboxamidoadenosine (A(2A)-selective agonist) was significantly attenuated. These findings agree well with the corresponding omeprazole-induced decrease in antral A(2A) receptor mRNA expression. Overall, the present study suggests that adenosine receptor gene expression and function may be altered by omeprazole treatment. Acid-dependent changes in adenosine receptor expression may represent a novel purinergic regulatory feedback mechanism in controlling gastric acid secretion.

Characterization of NMDA induced depression in rat hippocampus: involvement of AMPA and NMDA receptors

The involvement of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) vs. N-methyl- d-aspartate (NMDA) receptor mediated changes in NMDA-induced long-term depression (LTD) was assessed by monitoring isolated AMPA, isolated NMDA and composite field excitatory postsynaptic potentials (EPSP) in the CA1 area of acute rat hippocampal slices. Application of NMDA (20–50 μM) for 3–5 min led to LTD of both AMPA and NMDA receptor mediated EPSPs with near equal changes of the responses. However, AMPA EPSPs displayed a faster initial recovery than NMDA EPSPs. In addition, during the first 15–25 min after NMDA application, there was a superimposed potentiation of the later, but not early, part of AMPA EPSPs, implying a prolongation of waveform. In contrast, the NMDA EPSP waveform remained unaltered throughout the experiments. While it has been maintained that NMDA-induced depression is equivalent to stimulus-induced LTD, our results reveal additional complexity, suggesting a multitude of changes, most likely at the postsynaptic receptor level.

The different effects of endocrine-disrupting chemicals on estrogen receptor-mediated transcription through interaction with coactivator TRAP220 in uterine tissue.

An endocrine-disrupting chemical (EDC) can alter endocrine functions through a variety of mechanisms, including nuclear receptor-mediated changes in protein synthesis, interference with membrane receptor binding, steroidogenesis or synthesis of other hormones. Although major chemicals have been shown to disrupt estrogenic actions mainly through their binding to estrogen receptor (ER) or androgen receptor, it is not clear how EDCs affect endocrine functions in vivo. We present evidence that the EDCs bisphenol A and phthalate activate ER-mediated transcription through interaction with TRAP220. Moreover, bisphenol A had positive effects on the interaction between ER-beta and TRAP220 and on the expression of ER-beta and TRAP220 compared with phthalate and estradiol in uterine tIssue. These data suggested that some EDCs might alter endocrine function through the change of the receptor and coactivator levels in uterine tIssue and through the different effect on the interaction between ERs and coactivator TRAP220.

Growth inhibition by the muscarinic M(3) acetylcholine receptor: evidence for p21(Cip1/Waf1) involvement in G(1) arrest.

We have assessed the growth response of Chinese-hamster ovary (CHO) cells to activation of recombinantly expressed G-protein-coupled muscarinic M(2) or M(3) acetylcholine receptors (AChRs). We show that activation of these receptors leads to divergent growth responses: M(2) AChR activation causes an increase in DNA synthesis, whereas M(3) AChR activation causes a dramatic decrease in DNA synthesis. We have characterized the M(3) AChR-mediated growth inhibition and show that it involves a G(1) phase cell-cycle arrest. Further analysis of this arrest indicates that it involves an increase in expression of the cyclin-dependent kinase (CDK) inhibitor, p21(Cip1/Waf1) (where Cip1 is CDK-interacting protein 1 and Waf1 is wild-type p53-associated fragment 1), in response to M(3) AChR activation. This increase in protein expression leads to an increase in p21(Cip1/Waf1) association with CDK2, a decrease in CDK2 activity and an accumulation of hypophosphorylated retinoblastoma protein. The increased p21(Cip1/Waf1) expression is due, at least in part, to an increase in p21(Cip1/Waf1) mRNA, and receptor-mediated changes in phosphorylation of c-Jun provide a mechanism to account for this p21(Cip1/Waf1) transcriptional regulation. Evaluation of the extracellular signal-regulated protein kinase and c-Jun N-terminal kinase activities has shown striking differences in the profiles of activation of these mitogen-activated protein kinases by the M(2) and M(3) AChRs, and their potential involvement in mediating growth arrest by the M(3) AChR is discussed.

In vitro networks: cortical mechanisms of anaesthetic action

In vitro networks: cortical mechanisms of anaesthetic action

Microarray detection of gene expression changes induced by 1,25(OH)(2)D(3) and a Ca(2+) influx-activating analog in osteoblastic ROS 17/2.8 cells.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) treatment of osteoblastic ROS 17/2.8 cells initiates membrane-initiated rapid responses through activation of Ca(2+) influx and longer-term nuclear receptor-mediated changes in gene expression. Ca(2+) influx triggers a change in the phosphorylation state of the bone matrix protein, osteopontin (OPN), detectable at 3 h and prior to nuclear receptor-mediated events. This study aimed to determine if Ca(2+) influx induced by 1,25(OH)(2)D(3) would produce nuclear receptor-independent changes in gene expression. We employed a rat cDNA microarray strategy to screen the transcriptional changes at 3 h of treatment with 1,25(OH)(2)D(3) and with an analog of 1,25(OH)(2)D(3) (25(OH)-16ene-23yne-D(3) [AT]) that we previously showed to activate Ca(2+) influx without binding to the nuclear receptor. Arrays also were screened with cDNA from ROS 17/2.8 cells treated for 24 h, when nuclear receptor-mediated transcriptional events would occur. Rat gene filters (GeneFilter, Research Genetics) were hybridized with labeled cDNA probes from treatment groups. Among 5000 different clones on the array filters, we identified a family of genes which were altered 2-fold or greater following treatment with 1,25(OH)(2)D(3) or analog AT for 3 h. Cluster analysis also revealed genes whose expression was significantly up-regulated at 24 h, including OPN. Analysis of rapid changes in gene expression revealed changes affecting a diverse range of cellular pathways and functions, including protein kinases and phosphatases, Ca(2+) signaling, cell adhesion and secretion. These findings provide clear evidence of rapid changes in gene expression associated with Ca(2+) influx mediated by 1,25(OH)(2)D(3), and shed light on the nuclear-receptor independent signaling pathway affecting OPN phosphorylation.

Purinergic Regulation of Sound Transduction and Auditory Neurotransmission

In the cochlea, extracellular ATP influences the endocochlear potential, micromechanics, and neurotransmission via P2 receptors. Evidence for this arises from studies demonstrating widespread expression of ATP-gated ion channels (assembled from P2X receptor subunits) and G protein-coupled receptors (P2Y receptors). P2X₂ receptor subunits are localized to the luminal membranes of epithelial cells and hair cells lining scala media. These ion channels provide a shunt pathway for K⁺ ion egress. Thus, when noise exposure elevates ATP levels in this cochlear compartment, the K⁺ conductance through P2X receptors reduces the endocochlear potential. ATP-mediated K⁺ efflux from scala media is complemented by a P2Y receptor G protein-coupled pathway that provides coincident reduction of K⁺ transport into scala media from the stria vascularis when autocrine or paracrine ATP signalling is invoked. This purinergic signalling likely provides a basis for a reactive homoeostatic regulatory mechanism limiting cochlear sensitivity under stressor conditions. Elevation of ATP in the perilymphatic compartment under such conditions is also likely to invoke purinergic receptor-mediated changes in supporting cell micromechanics, mediated by Ca²⁺ influx and gating of Ca²⁺ stores. Independent of these humoral actions, ATP can be classified as a putative auditory neurotransmitter based on the localization of P2X receptors at the spiral ganglion neuron-hair cell synapse, and functional verification of ATP-gated currents in spiral ganglion neurons in situ. Expression of P2X receptors by type II spiral ganglion neurons supports a role for ATP as a transmitter encoding the dynamic state of the cochlear amplifier.