Receptor Message Research Articles

Endothelin blockade prevents the long-term cardiovascular and renal sequelae of acute kidney injury in mice.

Acute kidney injury (AKI) is common and associated with increased risks of cardiovascular and chronic kidney disease. Causative molecular/physiological pathways are poorly defined. There are no therapies to improve long-term outcomes. An activated endothelin system promotes cardiovascular and kidney disease progression. We hypothesized a causal role for this in the transition of AKI to chronic disease. Plasma endothelin-1 was threefold higher; urine endothelin-1 was twofold higher; and kidney preproendothelin-1, endothelin-A, and endothelin-B receptor message up-regulated in patients with AKI. To show causality, AKI was induced in mice by prolonged ischemia with a 4-week follow-up. Ischemic injury resulted in hypertension, endothelium-dependent and endothelium-independent macrovascular and microvascular dysfunction, and an increase in circulating inflammatory Ly6Chigh monocytes. In the kidney, we observed fibrosis, microvascular rarefaction, and inflammation. Administration of endothelin-A antagonist, but not dual endothelin-A/B antagonist, normalized blood pressure, improved macrovascular and microvascular function, and prevented the transition of AKI to CKD. Endothelin-A blockade reduced circulating and renal proinflammatory Ly6Chigh monocytes and B cells, and promoted recruitment of anti-inflammatory Ly6Clow monocytes to the kidney. Blood pressure reduction alone provided no benefits; blood pressure reduction alongside blockade of the endothelin system was as effective as endothelin-A antagonism in mitigating the long-term sequelae of AKI in mice. Our studies suggest up-regulation of the endothelin system in patients with AKI and show in mice that existing drugs that block the endothelin system, particularly those coupling vascular support and anti-inflammatory action, can prevent the transition of AKI to chronic kidney and cardiovascular disease.

CHANGE IN ENVIRONMENTAL GLUCOSE CONCENTRATION ALTERS ASTROCYTIC INSULIN AND INSULIN RECEPTOR LEVELS

BACKGROUNDThe actions of insulin in the brain include effects on glucose homeostasis, cell proliferation, cell differentiation, cognition, and memory. Cellular constituents of the neurovascular unit, including the astrocyte, contribute to insulin responses in the brain. Astrocytes are an ideal target cell type for investigation because of their dynamic character, multitude of functions, and their important role in brain homeostasis. We hypothesize that astrocytes respond to environmental glucose changes and could be a source of insulin supply to the microenvironment.OBJECTIVETo investigate astrocyte insulin and insulin receptor response to environmental glucose changes and to determine insulin secretion levels.METHODSNeonatal rat astrocytes in culture were exposed to changes in environmental glucose levels (LOW‐ 0mM, NORMAL‐5.5mM, and HIGH‐25mM) over three passages. Astrocyte whole cell lysates were used to determine message (qPCR) and protein levels (Western blot) for insulin and insulin receptor. Insulin Ultrasensitive ELISA was used to measure NORMAL astrocytic insulin secretion in response to glucose (25mM) and potassium (30mM) stimuli.RESULTSAstrocytes grown in LOW, NORMAL, and HIGH glucose levels resulted in equal cell counts and morphological appearances. Increasing glucose level from LOW, to NORMAL, or to HIGH in astrocytic culture media significantly increased insulin‐1 (exon 1–2) and insulin‐2 (exon 1–2) mRNA levels (One‐way ANOVA, n=4, p<0.05). Increased glucose level also resulted in a positive correlation with increased protein levels of insulin and insulin receptor in astrocytic cell lysate (Pearson Correlation, r=>0.85, p<0.01, n=3, R2=>0.73). Astrocytes secreted measurable levels of insulin (0.02–0.05μg/L) but did not increase significantly with glucose or potassium stimuli.CONCLUSIONSAstrocytes respond to changes in environmental glucose levels with increased insulin and insulin receptor message and protein levels. Astrocytes secrete measurable amounts of insulin but do not respond to stimuli as other insulin secreting cells. We speculate that such insulin responses to glucose levels from the astrocyte may be adaptive for its own homeostasis rather than to affect the microenvironment.Support or Funding InformationFunding from NIH (NHLBI) R01 HL033833 and HL105997 and the Veterans Administration Research Career Scientist Award

IL-21 receptor expression in human tendinopathy.

The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β) in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy.

5-hydroxytryptamine 2C receptors tonically augment synaptic currents in the nucleus tractus solitarii.

The nucleus tractus solitarii (nTS) is the primary termination and integration point for visceral afferents in the brain stem. Afferent glutamate release and its efficacy on postsynaptic activity within this nucleus are modulated by additional neuromodulators and transmitters, including serotonin (5-HT) acting through its receptors. The 5-HT(2) receptors in the medulla modulate the cardiorespiratory system and autonomic reflexes, but the distribution of the 5-HT(2C) receptor and the role of these receptors during synaptic transmission in the nTS remain largely unknown. In the present study, we examined the distribution of 5-HT(2C) receptors in the nTS and their role in modulating excitatory postsynaptic currents (EPSCs) in monosynaptic nTS neurons in the horizontal brain stem slice. Real-time RT-PCR and immunohistochemistry identified 5-HT(2C) receptor message and protein in the nTS and suggested postsynaptic localization. In nTS neurons innervated by general visceral afferents, 5-HT(2C) receptor activation increased solitary tract (TS)-EPSC amplitude and input resistance and depolarized membrane potential. Conversely, 5-HT(2C) receptor blockade reduced TS-EPSC and miniature EPSC amplitude, as well as input resistance, and hyperpolarized membrane potential. Synaptic parameters in nTS neurons that receive sensory input from carotid body chemoafferents were also attenuated by 5-HT(2C) receptor blockade. Taken together, these data suggest that 5-HT(2C) receptors in the nTS are located postsynaptically and augment excitatory neurotransmission.

Cospeptin, a novel reproductive peptide

Using a bioinformatics screen, our group recently discovered the novel neuropeptide cospeptin. We detected highest levels of cospeptin in the hypothalamus, including the periventricular nucleus, suggesting that cospeptin may influence pituitary hormone secretion. Cospeptin treatment alone failed to alter secretion of anterior pituitary (AP) hormones from dispersed primary rat pituitary cells; however, we found that cospeptin pre‐treatment potentiated release of luteinizing hormone in response to gonadotrophin‐releasing hormone (GnRH) stimulation. To determine if cospeptin plays a role in regulation of reproductive function, we compromised production of endogenous cospeptin by administration of siRNA directed against cospeptin into the lateral cerebroventricle of adult female rats, and used vaginal cytology to track the integrity of the estrous cycle. Treatment with cospeptin siRNA caused a delay in the appearance of the next estrus, and led to a decrease in GnRH receptor message in AP, suggesting cospeptin sensitizes gonadotrophs by upregulating the transcription of the GnRH receptor. These and other results suggest that cospeptin is an important reproductive hormone that modulates the sensitivity of pituitary gonadotrophs to GnRH through regulation of the expression of GnRH receptor.

Enhanced actions of adenosine in medial entorhinal cortex layer II stellate neurons in temporal lobe epilepsy are mediated via A1‐receptor activation

The adenosinergic system is known to exert an inhibitory affect in the brain, and as such adenosine has been considered an endogenous anticonvulsant. Entorhinal cortex (EC) layer II neurons, which serve as the primary input to the hippocampus, are spared in temporal lobe epilepsy (TLE) and become hyperexcitable. Because these neurons also express adenosine receptors, the activity of these neurons may be controlled by adenosine, specifically during seizure activity when adenosine levels are thought to rise. In light of this, we determined if the actions of adenosine on medial EC (mEC) layer II stellate neurons are augmented in TLE and by which receptor subtype. Horizontal brain slices were prepared from rats exhibiting spontaneous seizures (TLE) induced by electrical stimulation and compared with age-matched control rats. mEC layer II stellate neurons were visually identified, and action potentials (APs) were evoked either by a series of depolarizing current injection steps or via presynaptic stimulation of mEC deep layers. The effects of adenosine were compared with actions of adenosine A(1) and A(2A) receptor-specific agonists (CPA and CGS-21680) and antagonists (DPCPX and ZM-241385), respectively. Immunohistochemical and qPCR techniques were also employed to assess relative adenosine A(1)-receptor message and expression. mEC layer II stellate neurons were hyperexcitable in TLE, evoking a higher frequency of APs when depolarized and generating bursts of APs when synaptically stimulated. Adenosine reduced AP frequency and synaptically evoked APs in a dose-dependent manner (500 nM-100 μM); however, in TLE, the inhibitory actions of adenosine occurred at concentrations that were without affect in control neurons. In both cases, the inhibitory actions of adenosine were mediated via activation of the A(1)- and not the A(2A)-receptor subtype. Quantitative polymerase chain reaction (qPCR) and immunohistochemical experiments revealed an upregulation of the adenosine A(1) mRNA and an increase in A(1)-receptor staining in TLE neurons compared to control. Our data indicate that the actions of adenosine on mEC layer II stellate neurons is accentuated in TLE due to an upregulation of adenosine A(1)-receptors. Because adenosine levels are thought to rise during seizure activity, activation of adenosine A(1)-receptors could provide a possible endogenous mechanism to suppress seizure activity and spread within the temporal lobe.

Afferent arteriolar vasodilator effect of adenosine predominantly involves adenosine A2B receptor activation

Adenosine is an important paracrine agent regulating renal vascular tone via adenosine A(1) and A(2) receptors. While A(2B) receptor message and protein have been localized to preglomerular vessels, functional evidence on the role of A(2B) receptors in mediating the vasodilator action of adenosine on afferent arterioles is not available. The present study determined the role of A(2B) receptors in mediating the afferent arteriolar dilation and compared the effects of A(2B) and A(2A) receptor blockade on afferent arterioles. We used the rat in vitro blood-perfused juxtamedullary nephron technique combined with videomicroscopy. Single afferent arterioles of Sprague-Dawley rats were visualized and superfused with solutions containing adenosine or adenosine A(2) receptor agonist (CV-1808) along with adenosine A(2B) and A(2A) receptor blockers. Adenosine (10 micromol/l) caused modest constriction and subsequent superfusion with SCH-58261 (SCH), an A(2A) receptor blocker, at concentrations up 10 micromol/l elicited only slight additional decreases in afferent arteriolar diameter with maximum effect at a concentration of 1 micromol/l (-11.0 +/- 2.5%, n = 6, P < 0.05). However, superfusion of adenosine-treated vessels with MRS-1754 (MRS), an A(2B) receptor blocker, elicited greater decreases in afferent arteriolar diameter (-26.0 +/- 4.7%, n = 5, P < 0.01). SCH did not significantly augment the adenosine-mediated afferent constriction elicited by MRS; however, adding MRS after SCH caused further significant vasoconstriction. Superfusion with CV-1808 dilated afferent arterioles (17.2 +/- 2.4%, n = 6, P < 0.01). This effect was markedly attenuated by MRS (-22.6 +/- 2.0%, n = 5, P < 0.01) but only slightly reduced by SCH (-9.0 +/- 1.1%, n = 5, P < 0.05) and completely prevented by adding MRS after SCH (-24.7 +/- 1.8%, n = 5, P < 0.01). These results indicate that, while both A(2A) and A(2B) receptors are functionally expressed in juxtamedullary afferent arterioles, the powerful vasodilating action of adenosine predominantly involves A(2B) receptor activation, which counteracts A(1) receptor-mediated vasoconstriction.

Expression of leptin receptor gene in developing and adult zebrafish

Interactions of leptin and leptin receptors play crucial roles during animal development and regulation of appetite and energy balance. In this study we analyzed expression pattern of a zebrafish leptin receptor gene in both developing and adult zebrafish using in situ hybridization and Q-PCR methods. Zebrafish leptin receptor message ( lepr) was detected in all embryonic and larval stages examined, and in adult zebrafish. In embryonic zebrafish, lepr was mainly expressed in the notochord. As development proceeded, lepr expression in the notochord decreased, while its expression in several other tissues, including the trunk muscles and gut, became evident. In both larval and adult brains, large lepr expressing cells were detected in similar regions of the hindbrain. In adult zebrafish, lepr expression was also observed in several other brain regions including the hypothalamic lateral tuberal nucleus, the fish homolog of the arcuate nucleus. Q-PCR experiments confirmed lepr expression in the adult fish brain, and also showed lepr expression in several adult tissues including liver, muscle and gonads. Our results showed that lepr expression was both spatially and temporally regulated.

Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells

To develop a method of labeling and micro-dissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection (LCM). Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model. mRNA extracted from the microdissected cell population displayed marked increases in colony-stimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer cells derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant (2.5-fold, q value < 10) change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury. The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.

Hypothalamic and pituitary expression of ghrelin receptor message is increased during lactation

In the lactating rat there is a dramatic increase in food intake that peaks at around day 15 postpartum, a time when pups are near weaning age, yet still fully dependant on maternal nourishment. We examined whether the orexigenic hormone ghrelin plays a role in increasing food intake during lactation. To do this, we compared plasma levels ghrelin, as well as brain and pituitary expression of the growth hormone secretagogue receptor (GHS-R 1a) rats in one of three groups: (1) dams whose litters were removed the day after giving birth (non-lactating); (2) dams whose litters were removed on day 13 postpartum (litter removed), and dams allowed keeping their litters (lactating). On day 15 postpartum, all dams were decapitated and trunk blood collected for plasma analysis of active ghrelin levels. Also, brain and pituitaries were collected and snap frozen using liquid nitrogen and stored at -80 degrees C before mRNA extraction and RT-PCR analysis. Results show no differences in ghrelin concentrations between lactating and non-lactating rats. Hypothalamic and pituitary expression of GHS-R 1a, however, was significantly increased in lactating animals compared to non-lactating animals. Interestingly, litter removed dams had higher levels of plasma ghrelin concentrations than either lactating or non-lactating females. Furthermore, GHS-R mRNA expression in these animals remained elevated in the pituitary but not the hypothalamus. These data suggest that the hypothalamus and pituitary of lactating rats are more sensitive to the effects of ghrelin, and that hypothalamic sensitivity to ghrelin depends on the presence of a suckling litter.

ADP Stimulates Human Endothelial Cell Migration via P2Y 1 Nucleotide Receptor–Mediated Mitogen-Activated Protein Kinase Pathways

Extensive research on the role of ADP in platelet activation led to the design of new anti-thrombotic drugs, such as clopidogrel (Plavix; sanofi-aventis); however, very little is known about the ADP-preferring nucleotide receptors (P2Y1, P2Y12, and P2Y13) in endothelium. Here, we show that ADP stimulates migration of cultured human umbilical vein endothelial cells (HUVECs) in both Boyden chamber and in vitro wound repair assays. This promigratory effect was mimicked by 2-MeSADP, but not by AMP, and was inhibited by MRS2179 (P2Y1 receptor antagonist) but not by AR-C69931MX (P2Y12/13 receptor antagonist). RT-PCR revealed abundant P2Y1, barely detectable P2Y12, and absent P2Y13 receptor message in these cells. In addition, both ADP and 2-MeSADP, but not AMP, activated the mitogen-activated protein kinase pathways as evidenced by increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), and p38 kinase. ADP also stimulated phosphorylation of p90RSK, a downstream substrate of phosphorylated ERK1/2, and induced phosphorylation of such transcription factors downstream of the JNK and p38 pathways as c-Jun and activating transcription factor-2. These signaling events were inhibited by MRS2179 but not by AR-C69931MX. Furthermore, blockade of the ERK or JNK pathways by U0126 and SP600125, respectively, abolished ADP- and 2-MeSADP-stimulated HUVEC migration. However, inhibition of the p38 pathway by SB203580 partially suppressed ADP- and 2-MeSADP-induced HUVEC migration. We conclude that ADP promotes human endothelial cell migration by activating P2Y1 receptor-mediated MAPK pathways, possibly contributing to reendothelialization and angiogenesis after vascular injury.

Increased P2X7 Receptor Expression and Function in Thyroid Papillary Cancer: A New Potential Marker of the Disease?

Nucleotides are increasingly recognized as nonredundant extracellular signals for chemotaxis, cell growth, and cytokine release. Effects of extracellular nucleotides are mediated by P2 receptors, among which the P2X(7) subtype is attracting increasing attention for its involvement in apoptosis, cell growth, and cytokine release. Recent studies showed that P2X(7) is overexpressed in chronic lymphocytic leukemia and breast and prostate cancer. The aim of the present study was to better understand the clinical significance of P2X(7) receptor expression in normal and cancer human thyroid tissues. P2X(7) receptor message and protein expression and functional activity were tested in two cell lines (FB1 and FB2) established from either anaplastic or papillary primary thyroid cancer and in several histological samples of human papillary cancer. We show here that human thyroid papillary carcinoma, whether of the classical or follicular variant, expresses the P2X(7) receptor (P2X(7)R) to a much higher level than normal thyroid tissue. The P2X(7)R was similarly up-regulated in FB1 and FB2 cell lines. In contrast to normal thyroid cells, both cell lines responded to extracellular nucleotide stimulation with a large increase in intracellular Ca(2+) and secretion of IL-6. Ca(2+) increase was attenuated and release of IL-6 was fully blocked by P2X(7)R inhibitors. Finally, the thyroid carcinoma cell lines had at least a 3-fold higher intracellular ATP concentration and maintained at least a 3-fold higher extracellular ATP level, compared with control cells. These data suggest that an enhanced P2X(7)R function might be a feature of human thyroid cancer.

Human thrombopoietin reduces myocardial infarct size, apoptosis, and stunning following ischaemia/reperfusion in rats

Thrombopoietin (Tpo) is known for its ability to stimulate platelet production. However, it is currently unknown whether Tpo plays a physiological function in the heart. We assessed the potential protective role of Tpo in vitro and in vivo in two rat models of myocardial ischaemia/reperfusion. Tpo receptor (c-mpl) message was detected in the heart using RT-PCR, and the Tpo receptor protein was detected using western blotting and immunohistochemistry. Tpo treatment immediately before ischaemia reduced myocardial necrosis, apoptosis, and decline in ventricular function following ischaemia/reperfusion in the rat in a concentration- and dose-dependent manner with an optimal concentration of 1.0 ng/mL in vitro and an optimal dose of 0.05 microg/kg iv in vivo. Tpo also reduced infarct size when given after the onset of ischaemia or at reperfusion. Tpo activated JAK-2 (Janus kinase-2) and p44 MAPK (mitogen-activated protein kinase) during reperfusion but not prior to ischaemia. Inhibition of JAK-2 (AG-490), p42/44 MAPK (PD98059), mitochondrial K(ATP) channels (5-HD), and sarcolemmal K(ATP) channels (HMR 1098) abolished Tpo-induced resistance to injury from myocardial ischaemia/reperfusion. AG-490, PD98059, 5-HD, and HMR1098 alone had no effect on cardioprotection. Treatment with a single dose of Tpo (0.05 or 1.0 microg/kg iv) did not result in the elevation of platelet count or haematocrit over a 16-day period. A single treatment of Tpo confers cardioprotection through JAK-2, p42/44 MAPK, and K(ATP) channels, suggesting a potential therapeutic role of Tpo in the treatment of injury resulting from myocardial ischaemia and reperfusion.

ADP Stimulates Human Endothelial Cell Migration via P2Y1 Receptor‐mediated MAPK Pathways

Extensive research on the role of ADP in platelet activation led to the design of new anti-thrombotic drugs, such as Plavix; however, very little is known about the ADP-preferring nucleotide receptors (P2Y1, P2Y12, P2Y13) in endothelium. Here, we show that ADP stimulated migration of cultured human umbilical vein endothelial cells (HUVEC) in both Boyden chamber and in vitro wound repair assays. This pro-migratory effect was mimicked by 2-MsADP, but not by AMP, and was inhibited by MRS2179 (P2Y1 antagonist), but not by AR-C69931MX (P2Y12/13 antagonist), suggesting a role for the P2Y1 receptor. Consistent with this interpretation, RT-PCR revealed abundant P2Y1, barely detectable P2Y12, and absent P2Y13 receptor message in these cells. In addition, both ADP and 2-MsADP, but not AMP and adenosine, activated the MAPK pathways as evidenced by increased phosphorylation of ERK1/2, JNK and P38 kinases. ADP also stimulated phosphorylation of p90RSK, a downstream substrate of phosphorylated ERK1/2, and induced phosphorylation of several transcription factors downstream of the JNK and P38 pathways, including ATF-2 and c-JUN. These signaling events were inhibited by MRS2179, but not by AR-C69931MX. Furthermore, blockade of the P2Y1 receptor (MRS2179), or the MAPK pathways, including ERK (U0126), JNK (SP600125) or P38 (SB203580), abolished or decreased ADP- and 2-MsADP-induced HUVEC migration. We conclude that ADP promotes human endothelial cell migration by activating P2Y1 receptor-mediated MAPK pathways, possibly contributing to re-endothelialization and angiogenesis after vascular injury. Support: NIH HL29582 and RR00080.

Cloning of the Chicken Pituitary Receptor for Growth Hormone-Releasing Hormone

Details of the regulation of GH in birds are unclear. In this report, a receptor was cloned from chicken pituitary cDNA with 61% amino acid sequence identity to the human pituitary GHRH receptor. Phylogenies inferred from sequence alignments support that this is the chicken counterpart of the GHRH receptor known in mammals. Northern blotting shows that this receptor message is expressed in chicken pituitary, with lesser amounts seen in hypothalamus and brain but not in liver. The recombinant chicken receptor binds human GHRH with high affinity and specificity and signals cAMP accumulation. Surprisingly, available peptides synthesized to the published sequence for chicken GHRH-like peptide (cGHRH-LP) were inactive at this receptor. To address this we recloned the cDNA for this cGHRH-LP from chicken hypothalami. The revised sequence encodes lysine at position 21, which is consistent with all reported GHRH sequences from other species but different from the originally published chicken sequence. When this revised cGHRH-LP sequence was synthesized, it had improved but still weak potency at the cloned receptor. Consistent with the activity at the cloned receptor, human GHRH was potent when assayed in live chickens or on chicken pituitary membranes, but cGHRH-LP was not. We conclude that we have cloned a putative GHRH receptor that is homologous to mammalian GHRH receptors and functionally expressed in chicken pituitary, but that the identity of the endogenous ligand remains unclear. The chicken GHRH receptor cloned in this study can serve as a tool to identify its ligand and to clarify the evolutionary development of the regulation of GH.

Forebrain dopamine receptors in cognitive, memory and learning processes

Neurons that utilize dopamine (DA) as a neurotransmitter have attracted great interest because of their involvement in the behavioral, endocrine and descending control of major brain functions. DA is known to exert its physiological effects on target neurons through D1-like (D1 and D5) and D2-like (D2, D3 and D4) receptor subtypes. To better understand the DA modulation of brain functions, the distribution and cellular localization of D1 and D2 DA receptors in the rat forebrain is reviewed, and their possible role in cognitive aspects of behavior is discussed. In general, DA receptors are differentially expressed and mostly distributed in different target fields of DA neurons. Both D1 and D2 receptor gene products are found in the cerebral cortex, basal ganglia and hippocampal formation, albeit in different cell groups or neuronal subpopulations, whereas D3 receptors are mainly located in limbic regions. In the cortex, mRNA signals are seen in all the areas and cortical layers except layer I. In the striatum, the most intense signal is found in the caudate-putamen, nucleus accumbens and olfactory tubercle where a large number of cells are strongly labeled for D1 and D2. In the globus pallidus only scattered D2 mRNA-containing cells are present. In contrast, no D1 or D2 messages can be seen in the ventral pallidum. In the basal forebrain, mRNA encoding the D1 receptor is detected in the islands of Calleja. The medial and lateral septal nuclei show a low D2 signal. In the amygdaloid nuclear complex, the strongest D1 receptor message is observed in the basomedial and basolateral nuclei. Conversely, the highest density of D2 mRNA-expressing cells is revealed in the central nucleus. Moderately labeled for D1 and D2 cells are scattered throughout the anterior and posterior subdivisions of the bed nucleus of stria terminalis, and within all subfields of the hippocampal formation and dentate granule cell layer. Differential regional and cellular distribution of DA receptors in the forebrain provides anatomical evidence for an area-specific regulation of the DAergic neurotransmission. It can be inferred that DA facilitates learning, memory and cognition processes via activation of both the D1 and D2 receptors. Biomedical Reviews 2005; 16: 59-75.

KGF Prevents Oxygen-Mediated Damage in ARPE-19 Cells

Oxidative stress has been implicated in a variety of diseases of the eye. In several other tissues, keratinocyte growth factor (KGF) has been shown to prevent negative cellular changes associated with oxidative insult, such as permeability increases and nuclear DNA damage. In this study, we looked at whether KGF provided these same protective effects to cultured human retinal pigmented epithelial (RPE) cells (ARPE-19). Reverse transcriptase-polymerase chain reaction (RT-PCR) using a published primer pair sequence followed by restriction endonuclease digestion with AvaI and HincII was used to look for the KGF receptor message in ARPE-19 cells. Cellular response to KGF was verified through proliferation assays and Western blot analysis for mitogen-activated protein kinase (MAPK). Single-cell gel electrophoresis was used to assess DNA damage, Western blot analysis was used to assay actin cytoskeletal changes, and electrical resistance and tracer experiments with Transwell tissue plates were used to assess permeability changes. Immunostaining was used to verify the existence of the tight junction protein occludin. It was verified through RT-PCR that the ARPE-19 cell line exhibited the message for FGFR2-IIIb, otherwise known as KGFR. KGF was also shown to increase cellular proliferation and activated the MAPK p44/p42 cascade. KGF ameliorated nuclear DNA damage and cytoskeletal rearrangement caused by oxidative stress through the addition of exogenous hydrogen peroxide but was unable to prevent permeability changes. KGF was shown to significantly reduce DNA damage and cytoskeletal rearrangement caused by oxidative stress in cultured ARPE-19 cells. This result may be useful in targeting future therapies to combat a multitude of diseases of the eye that result from increases in reactive oxygen species.

Silencing of the mutant SCAP allele accounts for restoration of a normal phenotype in CT60 cells selected for NPC1 expression

The sterol regulatory element binding protein (SREBP)/SREBP cleavage-activating protein (SCAP) complex regulates the transcription of numerous genes involved in cellular cholesterol metabolism. The CHO mutant, CT60, and its parental cell line, 25RA, possess a gain-of-function mutation in one allele of the SCAP gene that renders the cells resistant to sterol-mediated suppression of cholesterol synthesis and uptake. In addition, CT60 cells do not express a functional Niemann-Pick type C1 (NPC1) protein, which leads to lysosomal accumulation of free cholesterol. Correction of the NPC1 defect by expression of a yeast artificial chromosome (YAC) containing the NPC1 genetic interval restored normal mobilization of cholesterol from the lysosomal compartment. Unexpectedly, the YAC-containing cell lines have overall cellular cholesterol concentrations that are comparable to wild-type levels, despite the assumed presence of the SCAP mutation. This phenotypic change results from a reduction in endogenous sterol synthesis, LDL receptor message, and HMG-CoA reductase message. Genetic analysis of the SCAP gene revealed that the YAC-expressing CT60 cells have normal regulation of these sentinel cholesterogenic genes as a result of selective silencing of the mutant SCAP allele, which appears to be independent of functional NPC1 expression.

Selective Upregulation of the A3 Adenosine Receptor in Eyes with Pseudoexfoliation Syndrome and Glaucoma

Adenosine is increasingly released in metabolic stress conditions, like hypoxia or ischemia, and regulates many physiologic processes, such as aqueous humor secretion and intraocular pressure, via activation of four adenosine receptors. In the current study, the role of the adenosine system in the pathophysiology of pseudoexfoliation (PEX) syndrome, which is typically associated with anterior chamber hypoxia and elevated intraocular pressure, was examined. RT-PCR, Northern hybridization, in situ hybridization, and immunohistochemistry were applied to analyze the mRNA and protein expression of the adenosine receptor subtypes A1, A2A, A2B, and A3 in anterior segment tissues of PEX eyes, without and with glaucoma, in comparison to eyes with primary open-angle or angle-closure glaucoma and normal control eyes. Real-time PCR was used to study the effect of hypoxia and oxidative stress on adenosine receptor expression by nonpigmented ciliary epithelial cells in vitro. Levels of adenosine and its catabolites inosine, hypoxanthine, and xanthine were measured in cell culture supernatants and aqueous humor samples by HPLC. All four adenosine receptor subtypes (A2A > A1 > A2B > A3) were coexpressed but differently distributed in the ciliary epithelium of control eyes, with the A3 receptor being localized to the basolateral membrane infoldings of the nonpigmented epithelial cells. A selective, approximately 10-fold upregulation of A3 receptor mRNA and protein was consistently found in the nonpigmented ciliary epithelium of all PEX eyes, with and without glaucoma, compared with the normal and glaucomatous control eyes. Significant upregulation of A3 receptor message in nonpigmented epithelial cells was induced by both hypoxia and oxidative stress in vitro, together with increased levels of inosine, hypoxanthine, and xanthine in the supernatants. Levels of adenosine and its catabolites, however, were not significantly elevated in the aqueous humor of patients with PEX. Considering the known role of the A3 adenosine receptor in modulating aqueous humor secretion, its selective, probably hypoxia-induced upregulation in the ciliary epithelium may not only confer cytoprotection but also influence aqueous humor dynamics and may be accessible to therapeutic intervention in patients with PEX.

MHC Class I, β2 microglobulin, and the INF‐γ receptor are upregulated in aged motoneurons

During aging, spinal cord motoneurons show characteristic changes including the loss of afferent boutons, a selective process that associates with gliosis and behavioral motor impairment. Evidence suggests that the major histocompatibility complex Class I (MHC I) system may be involved in synaptic plasticity of neurons during development and regeneration. In search of a mechanism governing senescent changes in synaptic connectivity, we report evidence for increased expression of MHC I and beta2 microglobulin (beta2M) in motoneurons and glial-like profiles of 30-month-old rats. The regulatory signal(s) for MHC I expression in normal neurons remains unresolved but among tentative molecules are cytokines such as interferon-gamma (INF-gamma) and tumor necrosis factor alpha (TNF-alpha). Interestingly, aged motoneurons, overlapping with those showing increased levels of MHC I, contained increased levels of INF-gamma receptor message. INF-gamma mRNA was detected at low levels in most (8/9) of the aged spinal cords but only infrequently (2/9) in young adult spinal cords; however, the cellular localization of INF-gamma mRNA could not be determined. Our data also indicates that TNF-alpha is upregulated in the senescent spinal cord but that TNF-alpha immunoreactive protein does not associate with motoneurons. We report evidence for an increased expression of MHC I and beta2M in senescent spinal motoneurons and discuss the possibility that this regulation associates with INF-gamma or changes in neurotrophin signaling and neuron activity in senescence.