Role of α1-adrenoceptor subtypes on corneal epithelial thickness and cell proliferation in mice.

Adrenergic stimuli are important for corneal epithelial structure and healing. The purpose of the present study was to examine the hypothesis that the lack of a single α1-adrenoceptor (α1-AR) subtype affects corneal epithelial thickness and cell proliferation. Expression levels of α1-AR mRNA were determined in mouse cornea using real-time PCR. In mice devoid of one of the three α1-AR subtypes (α1A-AR-/-, α1B-AR-/-, α1D-AR-/-) and in wild-type controls, thickness of individual corneal layers, the number of epithelial cell layers, and average epithelial cell size were determined in cryosections. Endothelial cell density and morphology were calculated in corneal explants, and epithelial cell proliferation rate was determined with immunofluorescence microscopy. Moreover, the ultrastructure of the corneal epithelium was examined by transmission electron microscopy. Messenger RNA for all three α1-AR subtypes was expressed in whole cornea and in corneal epithelium from wild-type mice with a rank order of abundance of α1A ≥ α1B > α1D. In contrast, no α1-AR mRNA was detected in the stroma, and only α1B-AR mRNA was found in the Descemet endothelial complex. Remarkably, corneal epithelial thickness and mean epithelial cell size were reduced in α1A-AR-/- mice. Our findings suggest that the α1A-AR exerts growth effects in mouse corneal epithelial cells.

Contribution of nitric oxide synthase isoforms to cholinergic vasodilation in murine retinal arterioles.

Nitric oxide synthases (NOSs) are critically involved in regulation of ocular perfusion. However, the contribution of the individual NOS isoforms to vascular responses is unknown in the retina. Because some previous findings suggested an involvement of inducible nitric oxide synthase (iNOS) in the regulation of retinal vascular tone, a major goal of the present study was to examine the hypothesis that iNOS is involved in mediating cholinergic vasodilation responses of murine retinal arterioles. Another subject of this study was to test the contribution of the other two NOS isoforms, neuronal (nNOS) and endothelial NOS (eNOS), to cholinergic retinal arteriole responses. Expression of individual NOS isoforms was determined in murine retinal arterioles using real-time PCR. All three NOS isoforms were expressed in retinal arterioles. However, eNOS mRNA was found to be most, and iNOS mRNA least abundant. To examine the functional relevance of iNOS for mediating vascular responses, retinal vascular preparations from gene-targeted iNOS-deficient mice (iNOS-/-) and wild-type mice were studied in vitro. Changes in luminal vessel diameter in response to the thromboxane mimetic 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619), the endothelium-dependent vasodilator acetylcholine, and the nitric oxide donor nitroprusside were measured by video microscopy. To determine the contribution of individual NOS isoforms to cholinergic vasodilation responses, retinas from iNOS-/- and wild-type mice were incubated with Nω-nitro-l-arginine methyl ester (l-NAME), a non-isoform-selective inhibitor of NOS, 7-nitroindazole, a selective nNOS blocker and aminoguanidine, a selective iNOS inhibitor. U-46619 evoked concentration-dependent vasoconstriction that was similar in retinal arterioles from iNOS-/- and wild-type mice. In retinal arterioles preconstricted with U-46619, acetylcholine and nitroprusside produced dose-dependent dilation that did not differ between iNOS-/- and wild-type mice. Remarkably, in both genotypes, vasodilation to acetylcholine was negligible after incubation with l-NAME. In contrast, pharmacological inhibition of nNOS and iNOS had no effect on acetylcholine-induced vasodilation. These findings suggest that dilation of murine retinal arterioles to acetylcholine is mediated predominantly by eNOS.

Expression of fourteen novel obesity-related genes in Zucker diabetic fatty rats.

BACKGROUND: Genome-wide association studies (GWAS) are useful to reveal an association between single nucleotide polymorphisms and different measures of obesity. A multitude of new loci has recently been reported, but the exact function of most of the according genes is not known. The aim of our study was to start elucidating the function of some of these genes. METHODS: We performed an expression analysis of fourteen genes, namely BDNF, ETV5, FAIM2, FTO, GNPDA2, KCTD15, LYPLAL1, MCR4, MTCH2, NEGR1, NRXN3, TMEM18, SEC16B and TFAP2B, via real-time RT-PCR in adipose tissue of the kidney capsule, the mesenterium and subcutaneum as well as the hypothalamus of obese Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats at an age of 22 weeks. RESULTS: All of our target genes except for SEC16B showed the highest expression in the hypothalamus. This suggests a critical role of these obesity-related genes in the central regulation of energy balance. Interestingly, the expression pattern in the hypothalamus showed no differences between obese ZDF and lean ZL rats. However, LYPLAL1, TFAP2B, SEC16B and FAIM2 were significantly lower expressed in the kidney fat of ZDF than ZL rats. NEGR1 was even lower expressed in subcutaneous and mesenterial fat, while MTCH2 was higher expressed in the subcutaneous and mesenterial fat of ZDF rats. CONCLUSION: The expression pattern of the investigated obesity genes implies for most of them a role in the central regulation of energy balance, but for some also a role in the adipose tissue itself. For the development of the ZDF phenotype peripheral rather than central mechanisms of the investigated genes seem to be relevant.

Bronchoepithelial expression of CXCR1 and CXCR2 does not facilitate transepithelial migration of neutrophils.

BACKGROUND: Neutrophilic airway inflammation is one of the key features of chronic obstructive pulmonary disease (COPD). The chemokine receptors 1 (CXCR1) and 2 (CXCR2) are expressed in the bronchial mucosa during chronic inflammation and might be of importance for transepithelial migration of neutrophils. OBJECTIVES: This study addressed the role of bronchoepithelial CXCR1 and CXCR2 expression with respect to transepithelial migration of neutrophils. METHODS: Primary bronchial epithelial cells (PBECs) derived from COPD patients and healthy controls as well as transiently CXCR1- and CXCR2-transfected Calu-6 cells were used for transepithelial migration assays of neutrophils under various conditions. Epithelial CXCR1 and CXCR2 expression was verified by means of flow cytometry. RESULTS: Transepithelial migration of neutrophils was significantly increased following lipopolysaccharide pretreatment of epithelial cells. Transient transfection of CXCR1 and CXCR2 neither augmented the transepithelial migration of neutrophils, nor did the selective blockade of CXCR1 and CXCR2 have any significant effect on neutrophilic transepithelial migration. In addition, no differences were found in PBECs and neutrophils derived from healthy controls and COPD patients. CONCLUSIONS: The data of the present study do not support the hypothesis that bronchoepithelial expression of CXCR1 and/or CXCR2 facilitate transepithelial migration of neutrophils.

Assessment of Physiological Rat Kidney Ageing-Implications for the Evaluation of Allograft Quality Prior to Renal Transplantation.

Due to organ shortage and rising life expectancy the age of organ donors and recipients is increasing. Reliable biomarkers of organ quality that predict successful long-term transplantation outcomes are poorly defined. The aim of this study was the identification of age-related markers of kidney function that might accurately reflect donor organ quality. Histomorphometric, biochemical and molecular parameters were measured in young (3-month-old) and old (24-month-old) male Sprague Dawley rats. In addition to conventional methods, we used urine metabolomics by NMR spectroscopy and gene expression analysis by quantitative RT-PCR to identify markers of ageing relevant to allograft survival. Beside known markers of kidney ageing like albuminuria, changes in the concentration of urine metabolites such as trimethylamine-N-oxide, trigonelline, 2-oxoglutarate, citrate, hippurate, glutamine, acetoacetate, valine and 1-methyl-histidine were identified in association with ageing. In addition, expression of several genes of the toll-like receptor (TLR) pathway, known for their implication in inflammaging, were upregulated in the kidneys of old rats. This study led to the identification of age-related markers of biological allograft age potentially relevant for allograft survival in the future. Among those, urine metabolites and markers of immunity and inflammation, which are highly relevant to immunosuppression in transplant recipients, are promising and deserve further investigation in humans.

Endothelin type A and B receptors in the control of afferent and efferent arterioles in mice.

BACKGROUND: Endothelin 1 contributes to renal blood flow control and pathogenesis of kidney diseases. The differential effects, however, of endothelin 1 (ET-1) on afferent (AA) and efferent arterioles (EA) remain to be established. METHODS: We investigated endothelin type A and B receptor (ETA-R, ETB-R) functions in the control of AA and EA. Arterioles of ETB-R deficient, rescued mice [ETB(-/-)] and wild types [ETB(+/+)] were microperfused. RESULTS: ET-1 constricted AA stronger than EA in ETB(-/-) and ETB(+/+) mice. Results in AA: ET-1 induced similar constrictions in ETB(-/-) and ETB(+/+) mice. BQ-123 (ETA-R antagonist) inhibited this response in both groups. ALA-ET-1 and IRL1620 (ETB-R agonists) had no effect on arteriolar diameter. L-NAME did neither affect basal diameters nor ET-1 responses. Results in EA: ET-1 constricted EA stronger in ETB(+/+) compared to ETB(-/-). BQ-123 inhibited the constriction completely only in ETB(-/-). ALA-ET-1 and IRL1620 constricted only arterioles of ETB(+/+) mice. L-NAME decreased basal diameter in ETB(+/+), but not in ETB(-/-) mice and increased the ET-1 response similarly in both groups. The L-NAME actions indicate a contribution of ETB-R in basal nitric oxide (NO) release in EA and suggest dilatory action of ETA-R in EA. CONCLUSIONS: ETA-R mediates vasoconstriction in AA and contributes to vasoconstriction in EA in this mouse model. ETB-R has no effect in AA but mediates basal NO release and constriction in EA. The stronger effect of ET-1 on AA supports observations of decreased glomerular filtration rate to ET-1 and indicates a potential contribution of ET-1 to the pathogenesis of kidney diseases.

Dissecting the action of an evolutionary conserved non-coding region on renin promoter activity.

Elucidating the mechanisms of the human transcriptional regulatory network is a major challenge of the post-genomic era. One important aspect is the identification and functional analysis of regulatory elements in non-coding DNA. Genomic sequence comparisons between related species can guide the discovery of cis-regulatory sequences. Using this technique, we identify a conserved region CNSmd of approximately 775 bp in size, approximately 14 kb upstream of the renin gene. Renin plays a pivotal role for mammalian blood pressure regulation and electrolyte balance. To analyse the cis-regulatory role of this region in detail, we perform 132 combinatorial reporter gene assays in an in vitro Calu-6 cell line model. To dissect the role of individual subregions, we fit several mathematical models to the experimental data. We show that a multiplicative switch model fits best the experimental data and that one subregion has a dominant effect on promoter activity. Mapping of the sub-sequences on phylogenetic conservation data reveals that the dominant regulatory region is the one with the highest multi-species conservation score.

Wilms' tumor protein (-KTS) modulates renin gene transcription.

Renin plays a crucial role in the control of various physiological processes such as blood pressure and body fluid homeostasis. Here, we show that a splice variant of the Wilms' tumor protein lacking three amino acids WT1(-KTS) suppresses renin gene transcription. Using bioinformatics tools, we initially predicted that a WT1-binding site exists in a regulatory region about 12 kb upstream of the renin promoter; this was confirmed by reporter gene assays and gel shift experiments in heterologous cells. Co-expression of Wt1 and renin proteins was found in rat kidney sections, mouse kidney blood vessels, and a cell line derived from the juxtaglomerular apparatus that produces renin. Knockdown of WT1 protein by siRNA significantly increased the cellular renin mRNA content, while overexpression of WT1(-KTS) reduced renin gene expression in stable and transiently transfected cells. A mutant WT1(-KTS) protein found in Wilms' tumors failed to suppress renin gene reporter activity and endogenous renin expression. Our findings show that renin gene transcription is regulated by the WT1(-KTS) protein and this may explain findings in patients with WT1 gene mutations of increased plasma renin and hypertension.

Adenosine restores angiotensin II-induced contractions by receptor-independent enhancement of calcium sensitivity in renal arterioles.

Adenosine is coupled to energy metabolism and regulates tissue blood flow by modulating vascular resistance. In this study, we investigated isolated, perfused afferent arterioles of mice, which were subjected to desensitization during repeated applications of angiotensin II. Exogenously applied adenosine restores angiotensin II-induced contractions by increasing calcium sensitivity of the arterioles, along with augmented phosphorylation of the regulatory unit of the myosin light chain. Adenosine restores angiotensin II-induced contractions via intracellular action, because inhibition of adenosine receptors do not prevent restoration, but inhibition of NBTI sensitive adenosine transporters does. Restoration was prevented by inhibition of Rho-kinase, protein kinase C, and the p38 mitogen-activated protein kinase, which modulate myosin light chain phosphorylation and thus calcium sensitivity in the smooth muscle. Furthermore, adenosine application increased the intracellular ATP concentration in LuciHEK cells. The results of the study suggest that restoration of the angiotensin II-induced contraction by adenosine is attributable to the increase of the calcium sensitivity by phosphorylation of the myosin light chain. This can be an important component of vascular control during ischemic and hypoxic conditions. Additionally, this mechanism may contribute to the mediation of the tubuloglomerular feedback by adenosine in the juxtaglomerular apparatus of the kidney.

CCR7 Is Important for Mesangial Cell Physiology and Repair.

The homeostatic chemokine receptor CCR7 serves as key molecule in lymphocyte homing into secondary lymphoid tissues. Previous experiments from our group identified CCR7 also to be expressed by human mesangial cells. Exposing cultured human mesangial cells to the receptor ligand CCL21 revealed a positive effect on these cells regarding proliferation, migration, and survival. In the present study, we localized CCR7 and CCL21 during murine nephrogenesis. Analyzing wild-type and CCR7 deficient (CCR7-/-) mice, we observed a retarded glomerulogenesis during renal development and a significantly decreased mesangial cellularity in adult CCR7-/- mice, as a consequence of less mesangial cell proliferation between embryonic day E17.5 and week 5 postpartum. Cell proliferation assays and cell-wounding experiments confirmed reduced proliferative and migratory properties of mesangial cells cultured from CCR7-/- kidneys. To further emphasize the role of CCR7 as important factor for mesangial biology, we examined the chemokine receptor expression in rats after induction of a mesangioproliferative glomerulonephritis. Here, we demonstrated for the first time that extra- and intraglomerular mesangial cells that were CCR7-negative in control rats exhibited a strong CCR7 expression during the phase of mesangial repopulation and proliferation.

Role of nucleolin in posttranscriptional control of MMP-9 expression.

Matrix-metalloproteinases (MMPs), which are able to degrade extra cellular matrix (ECM) components, are crucial in ECM-remodeling, under physiological (e.g., embryogenesis, wound healing, angiogenesis) or pathophysiological conditions (e.g., arthritis, cancer progression and metastasis, fibrosis). Treating HT1080 cells, a human fibrosarcoma cell line, with the iron chelator 2,2-Dipyridyl, which mimics certain aspects of hypoxia, leads to a 3-fold elevated Matrix-metalloproteinase-9 (MMP-9) protein level. This elevation occurs within 3 h, without any change of mRNA-concentration. The rapid increase in MMP-9 expression is caused by an enhancement of translational efficiency characterized by a recruitment of translationally inactive MMP-9 mRNP-complexes into the rough endoplasmatic reticulum (rER). Reporter gene assays, which depend on the untranslated regions (UTR) of MMP-9 mRNA, reveal that the posttranscriptional regulation is mainly attributed to the 3'UTR. RNA/protein interaction studies indicate that the elevated binding of nucleolin ( approximately 64 kDa form) to the 3'UTR may be of major importance for the increased efficiency of MMP-9 translation. The results show that MMP-9 expression can be regulated posttranscriptionally, affecting the efficiency of translation and localization of the mRNA.

Effect of the M1 Muscarinic Acetylcholine Receptor on Retinal Neuron Number Studied with Gene-Targeted Mice.

Pharmacological activation of the M1 muscarinic receptor subtype was suggested to promote the survival of retinal neurons. We examined the hypothesis that the M1 receptor is crucial for retinal neuron survival in vivo by using mice devoid of the M1 receptor gene. Muscarinic receptor gene expression was determined in the retina using real-time PCR. The amount of neurons in the retinal ganglion cell layer and of axons in the optic nerve was determined in retinal wholemounts stained with cresyl blue and in optic nerve cross-sections stained with toluidine blue, respectively. mRNA of all five muscarinic receptor subtypes (M1-M5) was detected in the retina from wild-type mice. Remarkably, M2 and M3 receptor mRNA were most abundant. In retinas from M1 receptor-deficient mice, M4 receptor mRNA expression was increased compared to that of wild-type mice, while no marked changes in the mRNA expression levels of the other muscarinic receptor subtypes were observed. The amount of cells in the retinal ganglion cell layer and the amount of axons in the optic nerve did not differ between M1 receptor-deficient and wild-type mice. The present findings suggest that the M1 receptor is not essential for the survival of retinal neurons in vivo.

The α1B -adrenoceptor subtype mediates adrenergic vasoconstriction in mouse retinal arterioles with damaged endothelium.

BACKGROUND AND PURPOSE: The α1-adrenoceptor family plays a critical role in regulating ocular perfusion by mediating responses to catecholamines. The purpose of the present study was to determine the contribution of individual α1-adrenoceptor subtypes to adrenergic vasoconstriction of retinal arterioles using gene-targeted mice deficient in one of the three adrenoceptor subtypes (α1A-AR(-/-), α1B-AR(-/-) and α1D-AR(-/-) respectively). EXPERIMENTAL APPROACH: Using real-time PCR, mRNA expression for individual α1-adrenoceptor subtypes was determined in murine retinal arterioles. To assess the functional relevance of the three α1-adrenoceptor subtypes for mediating vascular responses, retinal vascular preparations from wild-type mice and mice deficient in individual α1-adrenoceptor subtypes were studied in vitro using video microscopy. KEY RESULTS: Retinal arterioles expressed mRNA for all three α1-adrenoceptor subtypes. In functional studies, arterioles from wild-type mice with intact endothelium responded only negligibly to the α1-adrenoceptor agonist phenylephrine. In endothelium-damaged arterioles from wild-type mice, phenylephrine evoked concentration-dependent constriction that was attenuated by the α1-adrenoceptor blocker prazosin. Strikingly, phenylephrine only minimally constricted endothelium-damaged retinal arterioles from α1B-AR(-/-) mice, whereas arterioles from α1A -AR(-/-) and α1D-AR(-/-) mice constricted similarly to arterioles from wild-type mice. Constriction to U46619 was similar in endothelium-damaged retinal arterioles from all four mouse genotypes. CONCLUSIONS AND IMPLICATIONS: The present study is the first to demonstrate that α1-adrenoceptor-mediated vasoconstriction in murine retinal arterioles is buffered by the endothelium. When the endothelium is damaged, a vasoconstricting role of the α1B-adrenoceptor subtype is unveiled. Hence, the α1B-adrenoceptor may represent a target to selectively modulate retinal blood flow in ocular diseases associated with endothelial dysfunction.

Role of the M3 muscarinic acetylcholine receptor subtype in murine ophthalmic arteries after endothelial removal.

PURPOSE: We tested the hypothesis that the M3 muscarinic acetylcholine receptor subtype mediates cholinergic responses in murine ophthalmic arteries after endothelial removal. METHODS: Muscarinic receptor gene expression was determined in ophthalmic arteries with intact and with removed endothelium using real-time PCR. To examine the role of the M3 receptor in mediating vascular responses, ophthalmic arteries from M3 receptor-deficient mice (M3R(-/-)) and respective wild-type controls were studied in vitro. Functional studies were performed in nonpreconstricted arteries with either intact or removed endothelium using video microscopy. RESULTS: In endothelium-intact ophthalmic arteries, mRNA for all five muscarinic receptor subtypes was detected, but M3 receptor mRNA was most abundant. In endothelium-removed ophthalmic arteries, M1, M2, and M3 receptors displayed similar mRNA expression levels, which were higher than those for M4 and M5 receptors. In functional studies, acetylcholine evoked vasoconstriction in endothelium-removed arteries from wild-type mice that was virtually abolished after incubation with the muscarinic receptor blocker atropine, indicative of the involvement of muscarinic receptors. In concentration-response experiments, acetylcholine and carbachol concentration-dependently constricted endothelium-removed ophthalmic arteries from wild-type mice, but produced only negligible responses in arteries from M3R(-/-) mice. In contrast, acetylcholine concentration-dependently dilated ophthalmic arteries with intact endothelium from wild-type mice, but not from M3R(-/-) mice. Responses to the nitric oxide donor nitroprusside and to KCl did not differ between ophthalmic arteries from wild-type and M3R(-/-) mice, neither in endothelium-intact nor in endothelium-removed arteries. CONCLUSIONS: These findings provide evidence that in murine ophthalmic arteries the muscarinic M3 receptor subtype mediates cholinergic endothelium-dependent vasodilation and endothelium-independent vasoconstriction.

Role of α1-adrenoceptor subtypes in pupil dilation studied with gene-targeted mice.

PURPOSE: The α1A-adrenoceptor (α1A-AR) subtype was suggested to mediate contraction and trophic effects in the iris dilator muscle, and thus its pharmacological blockade may be involved in intraoperative floppy iris syndrome. We tested the hypothesis that the α1A-AR mediates pupil dilation and trophic effects in the mouse iris. METHODS: The α1-AR subtype mRNA expression was quantified in iris tissue by real-time PCR. To assess the role of individual α1-ARs for mediating pupil dilation, the α1-AR agonist phenylephrine was topically applied to the ocular surface of mice deficient in one of the three α1-AR subtypes (α1A-AR(-/-), α1B-AR(-/-), α1D-AR(-/-), respectively) and wild-type controls. Changes in pupil diameter were measured under a microscope in restrained mice. Moreover, iris and iris muscle thickness were determined in cryosections. RESULTS: Messenger RNA for all three α1-AR subtypes was detected the iris of wild-type mice with a rank order of abundance of α1A ≥ α1B > > α1D. The lack of a single α1-AR gene did not affect mRNA expression of the remaining two receptor subtypes. Phenylephrine induced pupil dilation in wild-type mice that was reduced in extent and duration in α1A-AR(-/-) and, less so, in α1B-AR(-/-) but not in α1D-AR(-/-) mice. The lack of a single α1-AR subtype had no effect on iris or iris muscle thickness. CONCLUSIONS: The α1-AR-induced mydriasis in mice is mediated mainly by the α1A-AR, with a smaller contribution of the α1B-AR, matching the relative abundance of these subtypes at the mRNA level. The lack of a single α1-AR subtype does not appear to cause atrophy in the mouse iris.

Comprehensive morphometric analysis of mononuclear cell infiltration during experimental renal allograft rejection.

The role of specific subtypes of infiltrating cells in acute kidney allograft rejection is still not clear and was so far not examined by different analyzing methods under standardized conditions of an experimental kidney transplantation model. Immunohistochemical staining of CD3, CD20 and CD68 was performed in rat allografts, in syngeneically transplanted rats and in control rats with a test duration of 6 and 28 days. The detailed expression and localization of infiltrating cells were analyzed manually in different kidney compartments under light microscope and by the two different morphometric software programs. Data were correlated with the corresponding kidney function as well as with histopathological classification. The information provided by the morphometric software programs on the infiltration of the specific cell types after renal transplantation was in accordance with the manual analysis. Morphometric methods were solid to analyze reliably the induction of cellular infiltrates after renal transplantation. By manual analysis we could clearly demonstrate the detailed localization of the specific cell infiltrates in the different kidney compartments. Besides infiltration of CD3 and CD68 infiltrating cells, a robust infiltration of CD20 B-cells in allogeneically transplanted rats, even at early time points after transplantation was detected. Additionally an MHC class I expression could reliable be seen in allogeneically transplanted rats. The infiltration of B-cells and the reliable antigen presentation might act as a silent subclinical trigger for subsequent chronic rejection and premature graft loss.

Multilevel regulation of HIF-1 signaling by TTP.

Hypoxia-inducible factor-1 (HIF-1) is a well-studied transcription factor mediating cellular adaptation to hypoxia. It also plays a crucial role under normoxic conditions, such as in inflammation, where its regulation is less well understood. The 3'-untranslated region (UTR) of HIF-1α mRNA is among the most conserved UTRs in the genome, hinting toward posttranscriptional regulation. To identify potential trans factors, we analyzed a large compilation of expression data. In contrast to its known function of being a negative regulator, we found that tristetraprolin (TTP) positively correlates with HIF-1 target genes. Mathematical modeling predicts that an additional level of posttranslational regulation of TTP can explain the observed positive correlation between TTP and HIF-1 signaling. Mechanistic studies revealed that TTP indeed changes its mode of regulation from destabilizing to stabilizing HIF-1α mRNA upon phosphorylation by p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2. Using a model of monocyte-to-macrophage differentiation, we show that TTP-driven HIF-1α mRNA stabilization is crucial for cell migration. This demonstrates the physiological importance of a hitherto-unknown mechanism for multilevel regulation of HIF-1α in normoxia.

Functional role of α1-adrenoceptor subtypes in murine ophthalmic arteries.

PURPOSE: To identify the α(1)-adrenoceptor (α(1)-AR) subtypes mediating vascular adrenergic responses in murine ophthalmic arteries. METHODS: Expression of mRNA was quantified for individual α(1)-AR subtypes in murine ophthalmic arteries using real-time PCR. To assess the functional relevance of α(1)-ARs for mediating vascular responses, ophthalmic arteries from mice deficient in one of the three α(1)-AR subtypes (α(1A)-AR(-/-), α(1B)-AR(-/-), and α(1D)-AR(-/-), respectively) and wild-type controls were isolated, cannulated with micropipettes, and pressurized. Changes in luminal artery diameter in response to the α(1)-AR agonist phenylephrine, the sympathetic transmitter noradrenaline, and to the nonadrenergic vasoconstrictor arginine vasopressin (AVP) were measured by video microscopy. RESULTS: Using real-time PCR, mRNA for all three α(1)-AR subtypes was detected in ophthalmic arteries from wild-type mice. In functional studies, phenylephrine and noradrenaline produced dose-dependent constriction of ophthalmic arteries that was similar in wild-type, α(1B)-AR(-/-), and α(1D)-AR(-/-) mice. Strikingly, responses to phenylephrine and noradrenaline were almost completely abolished in α(1A)-AR(-/-) mice. In contrast, the nonadrenergic agonist AVP produced dose-dependent vasoconstrictor responses that did not differ between any of the mouse genotypes tested. CONCLUSIONS: These findings provide evidence that the α(1A)-AR subtype mediates adrenergic vasoconstriction in murine ophthalmic arteries.

Globular and full-length adiponectin induce NO-dependent vasodilation in resistance arteries of Zucker lean but not Zucker diabetic fatty rats.

BACKGROUND: Adiponectin increases nitric oxide (NO) production in endothelial cell cultures and is reduced in the circulation of obese and diabetic patients, but its functional effect on resistance arteries is not yet studied in detail. METHODS: We assessed the direct vasodilatory response of isolated mesenteric resistance arteries of Zucker diabetic fatty (ZDF) rats and Zucker lean (ZL) rats to globular adiponectin (gAd) and full-length adiponectin (fAd) and tested the effect of additional reactive oxygen species (ROS) inhibitors in vitro. Serum adiponectin and insulin levels were measured by ELISA. The mRNA expressions of the adiponectin receptors and the downstream signaling molecules adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1), adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2), and endothelial NO synthase (eNOS) in mesenteric resistance arteries were quantified by real-time reverse transcriptase PCR. RESULTS: Both gAd and fAd induced a relevant dose-dependent vasodilation in ZL, but not in hypoadiponectinemic ZDF rats. This effect was totally blunted by L-nitroarginine-methyl-ester indicating NO dependency. The addition of ROS inhibitors could not improve the vasodilatory effect of adiponectin. Vasodilatory response to acetylcholine was reduced in ZDF rats, which could not be enhanced by low-dose adiponectin. Adiponectin receptor 1 (AdipoR1) was higher expressed than adiponectin receptor 2 (AdipoR2) with no significant differences between both animal groups, but APPL1 was significantly decreased in ZDF rats. The eNOS expression was not significantly different between ZL and ZDF rats. CONCLUSIONS: Adiponectin exerts a NO-dependent vasodilation in resistance arteries of normoglycemic ZL rats, but not diabetic ZDF rats. This may contribute to endothelial dysfunction in ZDF rats. Alterations in the expression of APPL1 may be involved in the observed insensitivity to adiponectin in ZDF rats.

Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles.

PURPOSE: To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles. METHODS: Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor-deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy. RESULTS: Only mRNA for the M(3) receptor was detected in retinal arterioles. Thus, M(3) receptor-deficient mice (M3R(-/-)) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R(-/-) mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R(-/-), mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non-subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. CONCLUSIONS: These findings provide evidence that endothelial M(3) receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase.