Changes In Receptor Levels Research Articles

Sympathetic Overdrive and Role of Beta-blockers in Various Forms of Heart Failure: A Consensus Statement from India.

In heart failure, sympathetic overdrive is evidenced by norepinephrine spillover, receptor level changes, etc. Beta-blockers continue to be the cornerstone of treatment in patients with chronic heart failure due to their ability to counteract sympathetic overdrive. Extensive clinical research has demonstrated that long-term beta-blocker treatment with metoprolol succinate, carvedilol, or bisoprolol enhances left ventricular function and reverses left ventricular remodeling, decreases hospitalization risk, and increases survival. The aim of this consensus paper is to identify patient profiles, initiation or up-titration of beta-blockers and benefits of chirally pure beta-blocker in patients with heart failure. This expert consensus will ensure precise implementation of beta-blockers in heart failure as a part of guideline-directed medical therapy.

An Assay to Monitor the Engagement and Modulation of CD6 on T cells as a Clinical Biomarker of Treatment with Itolizumab

Abstract Itolizumab is a novel first-in-class monoclonal antibody that selectively targets the co-stimulatory molecule CD6, a receptor that is highly expressed on CD4 and CD8 T cells and plays an important role in activation and migration. Monitoring target engagement and changes in receptor levels is critically important to interpreting clinical data. To evaluate the pharmacodynamic properties of itolizumab treatment on T cells in patients including those with graft versus host disease (GvHD), Precision for Medicine has developed and validated a 10-color flow cytometry assay to assess the engagement and modulation of cell-surface CD6. The assay was validated using whole blood from healthy donors. Blood was spiked with five concentrations of drug selected based on expected PK in the clinical study. The assay conditions were optimized for sensitivity, signal: noise ratio, detection of free receptor, receptor-bound itolizumab and total surface CD6. Itolizumab was detected using a fluorochrome conjugated α-human IgG1 antibody while total surface CD6 expression was assessed using an antibody to a non-competing site on CD6. For validation, pre-set criteria were used to assess inter-assay, intra-assay, inter-operator precision and post-staining stability. Technical validation was successfully met; and the assay performs within acceptable precision parameters. Measuring cell-based receptor engagement and fate in patients on immuno-modulatory therapies is very challenging. This assay was designed and validated to be both sensitive and selective in the quantification of CD6 receptor occupancy and modulation to facilitate the determination of an optimal therapeutic dose in autoimmune and inflammatory diseases.

Functional Interrogation of a Depression-Related Serotonergic Single Nucleotide Polymorphism, rs6295, Using a Humanized Mouse Model.

The serotonin 1A receptor (5-HT1A) system has been extensively implicated in modulating mood and behavior. Notably, 5-HT1A levels in humans display remarkable variation, and differences in receptor levels have been linked with a variety of psychiatric disorders. Further, reduction of receptor levels by 30-50% in mice suggests that changes in receptor levels that model existing human variation are sufficient to drive behavioral alterations. As a result, genetic mechanisms that modulate human 5-HT1A levels may be important for explaining individual differences in mood and behavior, representing a potential source of psychiatric disease risk. One common genetic variant implicated in differential 5-HT1A levels is the G/C single nucleotide polymorphism (SNP) rs6295, located upstream of the human 5-HT1A gene. This SNP differentially binds the transcription factor, NUDR/Deaf1, leading to cell-type specific effects on transcription in vitro. To investigate the direct effects of this SNP in the heterogeneous cellular context of the brain, we generated humanized transgenic mice using a design that maximized the local transcriptional landscape of the human HTR1A gene while also controlling for effects of genomic insertion location. We integrated a 180 kb human bacteria artificial chromosome (BAC) transgene containing G- and C-alleles of rs6295 flanked by FRT or loxP sites. Subsequent deletion of each allele by Cre- or Flp-recombinase resulted in rs6295G and C alleles in the same genomic location. These alleles were bred onto a 5-HT1A null mouse such that the human BAC was the sole source of 5-HT1A in these mice. We generated three separate lines, two of which had detectable human 5-HT1A levels in the brain, although none displayed expression in the raphe. Of these, one line exhibited rs6295-dependent differences in 5-HT1A levels and differences in behavior, even though the overall levels were considerably lower than native expression levels. The line-dependent effect of rs6295 on protein levels and behavior may depend upon differences in background genetic factors or different insertion sites across each line. This work confirms that relatively subtle differences in 5-HT1A levels can contribute to differences in behavior and highlights the challenges of modeling human noncoding genetic variation in mice.

Differential expression of the receptors for thyroid hormone, thyroid stimulating hormone, vitamin D and retinoic acid and extracellular signal-regulated kinase in uterus of rats under influence of sex-steroids

Sex-steroids play important role in modulating uterine functions. We hypothesized that these hormones affect expression of proteins in the uterus related to thyroid hormone action. Therefore, changes in expression levels of receptors for thyroid hormone (TRα-1 and TRβ-1), thyroid stimulating hormone (TSHR), vitamin D (VDR) and retinoid acid (RAR) as well as extracellular signal-regulated kinase (ERK1/2) in uterus were investigated under sex-steroid influence. MethodsTwo rat models were used: (i) ovariectomised, sex-steroid replaced and (ii) intact, at different phases of oestrous cycle. A day after completion of sex-steroid treatment or following identification of oestrous cycle phases, rats were sacrificed and expression and distribution of these proteins in uterus were identified by Western blotting and immunohistochemistry, respectively. ResultsExpression of TRα-1, TRβ-1, TSHR, VDR, RAR and ERK1/2 in uterus was higher following estradiol (E2) treatment and at estrus phase of oestrous cycle when E2 levels were high. A relatively lower expression was observed following progesterone (P) treatment and at diestrus phases of oestrous cycle when P levels were high. Under E2 influence, TRα, TRβ, TSHR, VDR, RAR and ERK1/2 were distributed in luminal and glandular epithelia while under P influence, TSHR, VDR abn RAR were distributed in the stroma. ConclusionsDifferential expression and distribution of TRα-1, TRβ-1, TSHR, VDR, RAR and ERK1/2 in different uterine compartments could explain differential action of thyroid hormone, TSH, vitamin D, and retinoic acid in uterus under different sex-steroid conditions.

Pharmacological manipulations in animal models of anorexia and binge eating in relation to humans.

Eating disorders, such as anorexia nervosa (AN), bulimia nervosa (BN) and binge eating disorders (BED), are described as abnormal eating habits that usually involve insufficient or excessive food intake. Animal models have been developed that provide insight into certain aspects of eating disorders. Several drugs have been found efficacious in these animal models and some of them have eventually proven useful in the treatment of eating disorders. This review will cover the role of monoaminergic neurotransmitters in eating disorders and their pharmacological manipulations in animal models and humans. Dopamine, 5-HT (serotonin) and noradrenaline in hypothalamic and striatal regions regulate food intake by affecting hunger and satiety and by affecting rewarding and motivational aspects of feeding. Reduced neurotransmission by dopamine, 5-HT and noradrenaline and compensatory changes, at least in dopamine D2 and 5-HT(2C/2A) receptors, have been related to the pathophysiology of AN in humans and animal models. Also, in disorders and animal models of BN and BED, monoaminergic neurotransmission is down-regulated but receptor level changes are different from those seen in AN. A hypofunctional dopamine system or overactive α2-adrenoceptors may contribute to an attenuated response to (palatable) food and result in hedonic binge eating. Evidence for the efficacy of monoaminergic treatments for AN is limited, while more support exists for the treatment of BN or BED with monoaminergic drugs.

A regulatory receptor network directs the range and output of the Wingless signal.

The potent activity of Wnt/Wingless (Wg) signals necessitates sophisticated mechanisms that spatially and temporally regulate their distribution and range of action. The two main receptor components for Wg - Arrow (Arr) and Frizzled 2 (Fz2) - are transcriptionally downregulated by Wg signaling, thus forming gradients that oppose that of Wg. Here, we analyze the relevance of this transcriptional regulation for the formation of the Wg gradient in the Drosophila wing disc by combining in vivo receptor overexpression with an in silico model of Wg receptor interactions. Our experiments show that ubiquitous upregulation of Arr and Fz2 has no significant effects on Wg output, whereas clonal overexpression of these receptors leads to signaling discontinuities that have detrimental phenotypic consequences. These findings are supported by our in silico model for Wg diffusion and signal transduction, which suggests that abrupt changes in receptor levels causes discontinuities in Wg signaling. Furthermore, we identify a 200 bp regulatory element in the arr locus that can account for the Arr gradient, and we show that this is indirectly negatively controlled by Wg activity. Finally, we analyze the role of Frizzled 3 (Fz3) in this system and find that its expression, which is induced by Wg, contributes to the establishment of the Arr and Fz2 gradients through counteracting canonical signaling. Taken together, our results provide a model in which the regulatory network of Wg and the three receptor components account for the range and shape of this prototypical morphogen system.

Age-related loss of the transforming growth factor β receptor ALK5 precedes osteoarthritis development in cartilage

Introduction: Osteoarthritis (OA) is the most common joint disease and (old) age is its main risk factor. One of OA's main hallmarks is degradation of articular cartilage. TGFβ-superfamily signaling plays an important role in cartilage homeostasis and maintenance via induction of Smad phosphorylation (pSmad). pSmad2/3 is a potent inhibitor of chondrocyte terminal differentiation and cell death, whereas pSmad1/5/8 induces these processes. In cartilage, two TGFβ receptors that induce Smad phosphorylation are ALK for Smad1/5/8 and ALK5 for Smad2/3. Previously, we have shown an age-related shift in receptor balance towards dominant Alk1 expression compared to Alk5 expression, and linked elevated Alk1 levels to increased Mmp13 and ColX expression and cartilage damage. However, OA development and ageing are concomitant in mice, and possibly both processes interfere. To uncouple ageing and OA-related processes, we investigated if age-related changes in TGFβ-superfamily signaling occur without interference of the OA process, and if these changes result in an altered cellular response to growth factors. We chose healthy bovine cartilage explants as a model system because bovine samples can be obtained in a wide age range without concurrent OA development. Methods: From the metacarpophalangeal joint of cows (Bos Taurus) (< 3 h post mortem), full thickness cartilage explants were collected. Macroscopically none of the animals showed any signs of OA. Samples were either immediately frozen in liquid nitrogen or, after 24 h equilibration in serum free medium, stimulated with growth factors for 24 h. Subsequently, mRNA was isolated and gene expression was measured by qPCR, using cDNA specific primers and SYBR-green. Results: Gene expression analysis of bovine explants ranging from 0.5 up to 12 years old showed a very significant 128-fold (R = 0.68, p < 0.0001) decrease in Col2a1 expression (fig. 1A) and a 3-fold decrease in Tgfb1 (R = 0.3, p = 0.002) expression with increasing age compared to 4 reference genes; Gapdh, Rpl22, Rps14 and Gusb. Furthermore, Alk5 expression decreased 4-fold (R = 0.3, p = 0.0004) whereas Alk1 expression remained unchanged. These changes in receptor level were reflected in cellular response to growth factors. Over the course of 8 years, expression of the Smad2/3p response gene Serpine1 (Pai1) became 11fold less ( p = 0.01) responsive to 1 ng/ml of the ALK5 ligand TGFβ1 (fig. 1B). In contrast, the response of 8 year old cartilage to the ALK1 ligand BMP9 (5 ng/ml) was not significantly different compared to 1 year old cartilage as measured by expression of the Smad1/5/8 response gene Id-1 (fig. 1C).

P4-01-14: Fulvestrant Regulates Epidermal Growth Factor (EGF) Ligands and Induces EGF Receptor Activation in MCF-7 Breast Cancer Cells.

Abstract Selective Estrogen Receptor Modulators (SERMs) 4 Hydroxy Tamoxifen (4-HT) and fulvestrant (fulv) inhibit estrogen receptor a (ER) positive breast cancer growth. We have observed that treatment (24 to 48 hours) of fulv in MCF-7 cells induced a 190kDa tyrosine phosphorylated band, which was blocked by EGFR and HER-2 tyrosine kinase inhibitors erlotinib and lapatinib. Immunoprecipitation showed EGFR, HER-2, and HER-3 were all phosphorylated after fulv treatment. 4-HT and estradiol did not cause this phosphorylation. No changes in receptor level after fulv treatment were noted. Downstream MAPK signaling was also blocked by erlotinib and lapatinib. Fulv induced activation of EGFR was ER dependent, since fulv treatment in C4-12, an ER negative cell line derived from MCF-7 cells, did not induce EGFR activation. Co-treatment with estradiol and fulv prevented EGFR activation. To explore the possibility that fulv enhanced ligand expression, we collected conditioned media (CM) from MCF-7 cells after 48 hours of treatment. pEGFR was lost when CM was removed, but recurred within 30 minutes. Cycloheximide abolished the ability of fulv to activate EGFR suggesting autorcine production of EGFR ligands was induced by fulv. To detect specific EGFR ligands, we used qPCR to measure various EGFR ligand mRNA levels in MCF-7 and C4-12 cell at 1, 4, 24 and 48 hours after fulv treatment. TGF-α and HB-EGF mRNAs were upregulated over 48 hours, which correlated well with pEGFR activation. 4-HT did not affect mRNA levels of these ligands. In contrast, amphiregulin (AREG) mRNA levels were substantially reduced 48 hours after fulv treatment. A similar trend was seen for AREG mRNA levels in 4HT treated cells but to a much lesser extent. There was no change in any EGFR ligand mRNA levels in ER negative C4-12 cells. These qPCR data suggested differential regulation of EGFR ligands by fulv treatment contributed to EGFR family member activation and was dependent on ER expression. Upon fulv treatment, levels of ER were diminished with decreased detection of S167 and S118 ER phosphorylation sites. Monolayer cell growth analysis showed that while fulv treatment in SFM reduced MCF-7 cell numbers compared to control treatment, erlotinib plus fulv significantly reduced cell numbers below the level of cells maintained in fulv alone. In conclusion, we show that fulv, but not E2 or 4-HT, activates EGFR family members accompanied by upregulation of ligands. Since SERM resistance has been associated with EGFR family member activation, differential control of EGFR ligand gene expression by ER may contribute to the development of fulvestrant resistance in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-14.

Intraperitoneal injection of saline modulates hippocampal brain receptor complex levels but does not impair performance in the Morris Water Maze

The involvement of the hippocampus in pain has been demonstrated but key players, i.e. the major brain receptors have not been shown to be modulated by pain. It was therefore the aim of the study to show the concerted action and pattern of brain receptor complex levels in a non-invasive model of moderate pain. C57BL/6J mice were divided into four groups of 14 animals each: trained injected, trained non-injected, yoked injected and yoked non-injected. Animals were tested in the open field and the elevated plus maze for behavioural evaluation and cognitive functions were tested using the Morris Water Maze. Hippocampi were taken 6 h following sacrification. Membrane proteins were prepared by ultracentrifugation and run on blue native gels to keep the native state, blotted to membranes and western blotting was carried out using the primary antibodies against serotonin receptor 5HT1A, muscarinic acetylcholine receptor M1 (mAChR-M1), nicotinic acetylcholine receptor alpha7 (nAChR-alpha7), glutamate (AMPA) receptor (GluR1) and neurokinin receptor 1 (NK-1). There was no difference between performance in behaviour or in the MWM between groups. Brain receptor level changes involved all receptors given above. Pain affected mAChR-M1, GluR1 and NK-1 complex levels when yoked-injected were compared with yoked non-injected animals. Memory mechanisms affected mAChR-M1 complex levels when trained non-injected animals were compared with yoked non-injected controls. Taken together, the neurochemical basis for testing receptor agonists/antagonists on the role of pain and the hippocampus was generated that may be useful for interpretations of the role of this complex area in moderate pain.

α1‐adrenoceptor activation induces phosphorylation of β2‐adrenoceptors in human prostate tissue

• To test whether β1-adrenoceptor activation leads to phosphorylation of the β2-adrenoceptor in human prostate tissue. • Prostate tissue from patients undergoing radical prostatectomy was stimulated in vitro with the α1-adrenergic agonist phenylephrine (10 µM). • α2-adrenoceptor phosphorylation at serines 345/346 was studied using Western blot analysis with a phospho-specific antibody. • The role of second messenger kinases was assessed by studying the effects of the protein kinase C (PKC) inhibitor Ro 31-8425 and the protein kinase A (PKA) inhibitor H89 on phenylephrine-induced phosphorylation. • The expression of G protein-coupled receptor kinases (GRKs) 2/3 was analysed using quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemistry. • Stimulation of prostate tissue with phenylephrine resulted in phosphorylation of the β2-adrenoceptor (5, 10 and 20 min after stimulation). • This α1-adrenoceptor-induced phosphorylation of β2-adrenoceptors was resistant to inhibition of PKC and PKA. • Changes in phosphorylation levels were not attributable to changes in receptor levels, as these remained constant during stimulation. • RT-PCR and Western blot analysis showed expression of GRK2/3 in human prostate tissues. • Immunohistochemical staining showed that GRK2/3 expression in human prostate tissue is located to stromal and smooth muscle cells. • Activation of α1-adrenoceptors causes phosphorylation of β2-adrenoceptors in the human prostate. This may enhance α1-adrenergic contraction and is possibly mediated by GRK2, which is expressed in prostate smooth muscle. • Mutual regulation between different adrenergic receptors might be involved in the therapeutic effects of α1-blockers in patients with benign prostate hyperplasia.

The Oxytocin-Oxytocin Receptor System and Its Antagonists as Tocolytic Agents

Oxytocin, a hormone involved in numerous physiologic processes, plays a central role in the mechanisms of parturition and lactation. It acts through its receptor, which belongs to the G-protein-coupled receptor superfamily, while Gq/phospholipase C (PLC)/inositol 1,4,5-triphosphate (InsP3) is the main pathway via which it exerts its action in the myometrium. Changes in receptor levels, receptor desensitization, and locally produced oxytocin are factors that influence the effect of oxytocin on uterine contractility in labor. Activation of oxytocin receptor causes myometrial contractions by increasing intracellular Ca+2 and production of prostaglandins. Since oxytocin induces contractions, the inhibition of its action has been a target in the management of preterm labor. Atosiban is today the only oxytocin receptor antagonist that is available as a tocolytic. However, the quest for oxytocin receptor antagonists with a better pharmacological profile has led to the synthesis of peptide and nonpeptide molecules such as barusiban, retosiban, L-368,899, and SSR-126768A. Many of these oxytocin receptor antagonists are used only as pharmacological tools, while others have tocolytic action. In this paper, we summarize the action of oxytocin and its receptor and we present an overview of the clinical and experimental data of oxytocin antagonists and their tocolytic action.

Cholinesterase inhibitors and stress: Effects on brain muscarinic receptor density in mice

Exposure to the reversible cholinesterase inhibitor, pyridostigmine bromide (PB), in conjunction with stress, has been suggested as a possible cause of Gulf War Syndrome. This work explores the hypothesis that PB exposure coupled with stress will alter cholinergic receptor density based on the rationale that prolonged exposure to PB and stress will lead to increased stimulation of cholinergic receptors due to the reduced capacity to degrade acetylcholine, leading to changes in receptor levels. Male C57Bl6 mice were exposed to PB (3 or 10 mg/kg/day) or physostigmine (2.88 mg/kg/day) for 7 days via ALZET mini-osmotic pumps implanted subcutaneously. The mice were stressed by shaking at random intervals (avg of 2 min/30 min) for 1 week, which was sufficient to increase blood cortisol levels. Brain tissue for autoradiographic analysis was collected on day 7 of treatment. While we examined many brain regions, analysis revealed that most of the significant changes ( p < 0.05) were seen in cholinergic nuclei. Stress typically increased muscarinic receptor density, while PB and PHY generally decreased muscarinic receptor density.

Reproductive status regulates expression of sex steroid and GnRH receptors in the olfactory bulb

Neuromodulators including gonadotropin-releasing hormone (GnRH) and sex steroids help integrate an animal's internal physiological state with incoming external cues, and can have profound effects on the processing of behaviorally relevant information, particularly from the olfactory system. While GnRH and steroid receptors are present in olfactory processing regions across vertebrates, little is known about whether their expression levels change with internal physiological state or external social cues. We used qRT-PCR to measure mRNA levels of two GnRH receptors (GnRH-R1, GnRH-R2), five sex steroid receptors (estrogen receptors: ERα, ERβa, ERβb; androgen receptors: ARα, ARβ), and aromatase in the olfactory bulb of the highly social African cichlid fish Astatotilapia burtoni. We asked whether these receptor levels changed with reproductive condition in females, or with social status, which regulates reproductive capacity in males. Our results reveal that mRNA levels of multiple sex steroid, GnRH receptor subtypes, and aromatase in the olfactory bulb vary with sex, social status in males, and reproductive condition in females, which highlights the potential importance of changing receptor levels in fine-tuning the olfactory system during the reproductive cycle. Further, steroid receptor mRNA levels were positively correlated with circulating steroid levels in males, but negatively correlated in females, suggesting different regulatory control between sexes. These results provide support for the hypothesis that the first-order olfactory relay station is a substrate for both GnRH and sex steroid modulation, and suggest that changes in receptor levels could be an important mechanism for regulating reproductive, social, and seasonal plasticity in olfactory perception observed across vertebrates.

A correlation of haloperidol-induced cognitive deficit with dysfunctional dopamine receptor activity in nonhuman primate

Haloperidol is an antipsychotic drug that acts through blockage of dopamine D 2 receptors. Chronic administration of this antipsychotic drug in nonhuman primates induces a pronounced cognitive deficit. However, receptor subtypes that are responsible for this cogni - tive dysfunction remain unknown. Therefore, brains of chronic haloperidol-treated young and aged monkeys were used to analyze the intricate relation of receptor activity, cognitive dysfunction, and haloperidol-mediated actions in the production of harmful effects. Tak- ing into account the significant cognitive loss observed after haloperidol treatment, it was predicted that changes in the cognitive status that correlate with the receptor activity in the prefrontal cortex and striatum, areas implicated in the processing of haloperidol-mediated effects in brain, should be common in both young and aged animals. Based on this concept, we observed that in the prefrontal cortex, dopamine D 1 and D 2 receptors showed changes in receptor levels that were common in both age groups. However, this relationship was absent in GABA A , serotonin 5HT2 and muscaranic receptors. In contrast to the prefrontal cortex, in striatum, this change was restricted to the dopamine D 2 receptors only. Therefore, from our results, it seems that apart from the downregulation of D 1 receptor activity in the prefrontal cortex, an upregulation of D 2 receptors could also contribute to the generation of the cognitive loss observed in haloperidol-treated monkeys. Additionally, reduced excitatory input due to hampered cortico-striatal D 1 dopaminergic activity and stronger inhibition at the synapse of excitatory input site by upregulated striatal D 2 receptor activity could promote the side effects

Drastic decrease in dopamine receptor levels in the striatum of acetylcholinesterase knock-out mouse

The acetylcholinesterase knock-out mouse lives to adulthood despite 60-fold elevated acetylcholine concentrations in the brain that are lethal to wild-type animals. Part of its mechanism of survival is a 50% decrease in muscarinic and nicotinic receptors and a 50% decrease in adrenoceptor levels. The hypothesis was tested that the dopaminergic neuronal system had also adapted. Radioligand binding assays measured dopamine receptor level and binding affinity in the striatum. Immunohistochemistry of brain sections with specific antibodies visualized dopamine transporter. Effects on the intracellular compartment were measured as cAMP content, PI-phospholipase C activity. Dopamine receptor levels were decreased 28-fold for the D(1)-like, and more than 37-fold for the D(2)-like receptors, though binding affinity was normal. Despite these huge changes in receptor levels, dopamine transporter levels were not affected. The intracellular compartment had normal levels of cAMP and PI-phospholipase C activity. Survival of the acetylcholinesterase knock-out mouse could be linked to adaptation of many neuronal systems during development including the cholinergic, adrenergic and dopaminergic. These adaptations balance the overstimulation of cholinergic receptors caused by high acetylcholine concentrations and thus maintain homeostasis inside the cell, allowing the animal to live.

Delivery of Recombinant Adeno-Associated Virus-Mediated Human Tissue Kallikrein for Therapy of Chronic Renal Failure in Rats

The tissue kallikrein-kinin system is important in regulating cardiovascular and renal function, and dysregulation of the system has been implicated in heart and kidney pathologies. These findings suggest that if balance can be restored to the kallikrein-kinin axis, then associated disease progression may be attenuated. To test this hypothesis, recombinant adeno-associated virus (rAAV)-mediated human tissue kallikrein (HK) expression was induced in a rodent model of chronic renal failure involving 5/6 nephrectomy (nephrectomy plus 70% reduction of remaining kidney). rAAV-HK treatment attenuated the rise in blood pressure, glomerular sclerosis, and tubulointerstitial injury observed in this model. rAAV-HK treatment also attenuated renal function decline as measured by urinary microalbumin, osmolarity, and cGMP levels. Reverse transcriptase-polymerase chain reaction analysis showed that rAAV-HK-treated rats had higher levels of bradykinin receptor-2 (B(2)R) and dopamine receptor-1 mRNAs. In contrast, angiotensin II receptor-1, endothelin receptor-A, and vasopressin receptor-2 mRNAs were markedly downregulated in kidneys from HK-treated rats. Bradykinin induced similar changes in receptor levels and prevented transforming growth factor-beta(1)-induced tubulointerstitial fibrosis. The effects of bradykinin could be reversed with the B(2)R antagonist HOE-140. Together, these findings suggest that restoration of the kallikrein-kinin system reduces kidney injury and protects renal function in 5/6-nephrectomized rats via changes in the expression and activation of G protein-coupled receptors including B(2)R.

Nicotinic α5 subunit deletion locally reduces high‐affinity agonist activation without altering nicotinic receptor numbers

Neuronal nicotinic acetylcholine receptor subunit alpha5 mRNA is widely expressed in the CNS. An alpha5 gene polymorphism has been implicated in behavioral differences between mouse strains, and alpha5-null mutation induces profound changes in mouse acute responses to nicotine. In this study, we have examined the distribution and prevalence of alpha5* nicotinic acetylcholine receptor in mouse brain, and quantified the effects of alpha5-null mutation on pre-synaptic nicotinic acetylcholine receptor function (measured using synaptosomal (86)Rb(+) efflux) and overall [(125)I]epibatidine binding site expression. alpha5* nicotinic acetylcholine receptor expression was found in nine of fifteen regions examined, although < 20% of the total nicotinic acetylcholine receptor population in any region contained alpha5. Deletion of the alpha5 subunit gene resulted in localized loss of function (thalamus, striatum), which was itself confined to the DHbetaE-sensitive receptor population. No changes in receptor expression were seen. Consequently, functional changes must occur as a result of altered function per unit of receptor. The selective depletion of high agonist activation affinity sites results in overall nicotinic function being reduced, and increases the overall agonist activation affinity. Together, these results describe the receptor-level changes underlying altered behavioral responses to nicotine in nicotinic acetylcholine receptor alpha5 subunit-null mutants.

The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis

In our laboratory we have developed a quantitative-polymerase chain reaction (Q-PCR) strategy to examine the differential expression of adenosine receptor (ADOR), A 1, A 2A, A 2B and A 3, and estrogen receptors (ER) α and β. Brain and uterine mRNA were first used to optimise specific amplification conditions prior to SYBR Green I real time analysis of receptor subtype expression. SYBR Green I provided a convenient and sensitive means of examining specific PCR amplification product in real time, and allowed the generation of standard curves from which relative receptor abundance could be determined. Real time Q-PCR analysis was then performed, to examine changes in receptor expression levels in brains of adult female Wistar rats 3-month post ovariectomy. Comparison with sham-operated age-matched control rats demonstrated both comparative and absolute-copy number changes in receptor levels. Evaluation of both analytical methods investigated 18S rRNA as an internal reference for comparative gene expression analysis in the brain. The results of this study revealed preferential repression of ADORA 2A (>4-fold down) and consistent (>2-fold) down-regulation of ADORA 1, ADORA 3, and ER-β, following ovariectomy. No change was found in ADORA 2B or ER-α. Analysis of absolute copy number in this study revealed a correlation between receptor expression in response to ovariectomy, and relative receptor subtype abundance in the brain.

The relationships of animal age and caloric intake to cellular replication in vivo and in vitro: a review.

This brief review examines aging at the cellular level as expressed by cell replication rates in vivo, clone size limits in vitro, and cell function in several tissues and organs. Studies are presented in which in vivo and in vitro cell replication measurements were made for several cell types and organs in relation to animal age, diet, life span, and specific age-related pathologies. Among the events examined that affect cell replication and cell survival in vitro and in vivo over a lifetime are oxidative damage, telomere shortening, and hormone and hormone receptor level changes. Long-term caloric restriction (CR) is favorable or protective for all of these events when measured in later life and comparisons are made to ad libitum (AL)-fed animals, and it is accompanied by more youthful rates of cell replication. It is proposed that in vivo and in vitro measures of cellular replication constitute biomarkers of aging when applied to comparisons of CR and AL diet rodents, where they correlate with the delay of disease and extension of life span. Longitudinal studies are needed to confirm this. The occurrence of certain age-related pathophysiologic states, such as immune (T cell) insufficiency, cataract, and senile osteopenia/osteoporosis, are accompanied by major diminishments of replication rates, numbers, and functions of the essential cell types in the organs and tissues involved. However, direct evidence is lacking that diminished cell replication in specific organs contributes to the limitation of life span.

AT1 and AT2 angiotensin receptor gene expression in human heart failure.

The availability of selective antagonists for angiotensin II receptors has focused interest on the gene expression of angiotensin II-receptor subtypes in the human heart. We analyzed expression of the AT1 and AT2 subtypes of the angiotensin II receptor in ventricular myocardium taken from 9 donor hearts before implantation and from 12 patients with heart failure (6 with dilated cardiomyopathy and 6 with ischemic heart disease). Competitive reverse transcription-polymerase chain reaction with synthetic RNA internal standards was used to detect mRNA for both subtypes and to quantify relative differences in levels between failing and non-failing ventricular myocardium. AT1- and AT2-receptor mRNA could be detected in all samples. AT1-receptor gene expression was 2.5-fold greater in nonfailing hearts than in patients with failing hearts (P = .015). There was no significant difference in AT2-receptor mRNA expression in failing and nonfailing hearts. The level of expression of the angiotensin AT1 receptor appears to decrease in the failing human ventricle whereas the level of AT2 expression is unaffected. These changes parallel the changes found in human ventricular myocardium at the receptor level, suggesting that the changes in receptor level may result from changes in gene expression or mRNA stability.