Attenuation of fear conditioning by antisense inhibition of brain corticotropin releasing factor-2 receptor.

Corticotropin releasing factor (CRF) is an important regulator of the endocrine, behavioral, autonomic and immune responses to stress. Two high affinity CRF receptors have been identified, which are distributed in distinct anatomical regions. CRF(1) receptors have been relatively well characterized and antagonists to this receptor effectively block stress-induced behaviors in rodents. The function of CRF(2) receptors, which are highly expressed in limbic brain regions, is less well understood. Therefore, an antisense oligonucleotide approach was used to study the role of CRF(2) receptors in the lateral septum in rats. An antisense oligonucleotide directed against the CRF(2) receptor mRNA reduced expression of CRF(2) receptors by 60--80%. In shock-induced freezing tests, animals administered the antisense oligonucleotide exhibited a significant reduction in freezing duration. However, pain sensitivity and locomotor activity were unaltered. A four-base mismatch of the antisense sequence had no significant effects on CRF(2) receptor density and on freezing behavior. These data support the involvement of CRF(2) receptors in fear conditioning. CRF(1) receptor antagonists also reduce freezing in this test. Additional studies to determine the effects of simultaneous inhibition of both receptor subtypes show that rats receiving both CRF(2) receptor antisense oligonucleotide and CRF(1) receptor antagonist froze significantly less than animals treated with either agent alone. These results provide additional evidence for the role of CRF(2) receptors in mediating the stress-induced actions of endogenous CRF.

Regulation of the angiotensin type-1 receptor by antisense oligonucleotides occurs through an RNase H-type mechanism.

Multiple, diverse sites in the coding region of the angiotensin type-1 receptor mRNA were targeted with 2'-deoxyribonucleotide antisense oligonucleotides (ONs). The uptake of 1 microM concentration of these ONs into Chinese hamster ovary cells was facilitated by the use of cationic liposomes. The antisense sequences reduced binding of 125I-angiotensin II by 57-73%, while mismatch ONs and reverse sequence ONs produced little reduction in receptor binding. These reductions in AT1 receptor binding were accompanied by comparable decreases in AT1 receptor mRNA levels. Furthermore, mRNA cleavage fragments corresponding in size to 3'-cleavage fragments were observed with two of the antisense ONs, consistent with the involvement of an RNase H-type enzyme. When 2'-methoxyribonucleotide analogs of these same sequences were tested, AT1 receptor mRNA levels were unchanged even though small reductions in AngII binding were observed. Antisense effects seen with these 2'-methoxyribonucleotide sequences may have arisen through a translational arrest mechanism. Direct comparisons between 2'-deoxyribonucleotide analogs and their 2'-methoxyribonucleotide counterparts show that antisense effects are significantly larger when they are mediated through an RNase H-type mechanism. 2'-methoxyribonucleotide sequences were most effective when they were directed against the translation initiation codon.

Modification of phosphorothioate oligonucleotides yields potent analogs with minimal toxicity for antisense experiments in the CNS.

There is increasing evidence that phosphorothioate oligonucleotides infused into the brain can cause a host of undesired side effects which compromise the antisense experiment. In studies on the corticotropin releasing factor type-2 receptor, several phosphorothioate oligonucleotides administered intraventricularly produced significant weight loss in rats. Four different phosphodiester and phosphorothioate oligonucleotide analogs were examined to identify molecules which could eliminate these side effects while maintaining good potency for antisense inhibition. Of these, chimeric oligonucleotides consisting of a mixed phosphodiester-phosphorothioate backbone, and having 2'-methoxyribonucleotide modifications in 60% of the oligonucleotide were the most optimal. Rats treated with these chimeric oligonucleotides gained weight at rates identical to that of saline-treated controls. In addition, the antisense oligonucleotide but not the mismatch control sequence reduced corticotropin releasing factor type-2 receptor binding of 125iodo-sauvagine in the lateral septum by 40-60% after 5 daily injections. Increasing the dosing period to 9 days reduced receptor binding by 78%. Reductions in protein binding were accompanied by comparable reductions in the in situ hybridization signal of the corticotropin releasing factor type-2 receptor mRNA. However, when an oligonucleotide analog incapable of supporting ribonuclease H activity was used, neither protein nor RNA binding levels were changed compared to saline-treated controls. These results suggest that ribonuclease H or enzymes with similar activity are critical to the antisense inhibition observed in the lateral septum.

Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries.

Antisense experiments are often complicated by the lack of reliable methods for selecting effective antisense sequences. Chimeric oligodeoxynucleotide (ODN) libraries and ribonuclease H (RNase H) were used to identify regions on the 1253 nucleotide angiotensin type-1 receptor (AT1) mRNA that are accessible to hybridization with antisense ODNs. Phosphorothioate antisense ODNs targeted against accessible sites reduced AT1 receptor levels by at least 50% in cell culture. ODNs to 4 sites produced a 70% to 80% reduction. In contrast, most sequences targeted between accessible sites were ineffective. When injected into the brains of rats, ODNs targeted to accessible sites reduced AT1 (by 65%) but not AT2 receptor levels. Additionally, AT1 receptor function as measured by agonist-induced water intake, was significantly attenuated in these rats. ODNs directed between accessible sites were ineffective at suppressing water intake. RNA mapping can be applied to any RNA target to facilitate selection of multiple, active antisense sequences for cell culture and in vivo experiments.

Astrocytes increase barrier properties and ZO-1 expression in retinal vascular endothelial cells.

PURPOSE: Diabetic retinopathy and other diseases associated with retinal edema are characterized by increased microvascular leakage. Astrocytes have been proposed to maintain endothelial function in the brain, suggesting that glial impairment may underlie the development of retinal edema. The purpose of this study was to test the effects of astrocytes on barrier properties in retinal microvascular endothelial cells. METHODS: Bovine retinal microvascular endothelial cells were exposed to conditioned media from rat brain astrocytes. Transendothelial electrical resistance (TER) was determined on 24-mm Transwell (Cambridge, MA) polycarbonate filters with the End-Ohm device (World Precision Instruments, Sarasota, FL). ZO-1 protein content was quantified by microtiter enzyme-linked immunosorbent assay. RESULTS: Astrocyte-conditioned medium (ACM) significantly increased TER (P < 0.0001) and ZO-1 content (P < 0.01). Both serum-containing and serum-free N1B defined ACM increased ZO-1 expression, but heating abolished the effect. Serum-free ACM decreased cell proliferation by 16%. CONCLUSIONS: Astrocytes release soluble, heat-labile factors that increase barrier properties and tight junction protein content. These results suggest that astrocytes enhance blood-retinal barrier properties, at least in part by increasing tight junction protein expression. Our findings suggest that glial malfunction plays an important role in the pathogenesis of vasogenic retinal edema.

Histamine reduces ZO-1 tight-junction protein expression in cultured retinal microvascular endothelial cells.

We examined ZO-1 protein content in cultured retinal vascular endothelial cells to test the hypothesis that histamine alters tight-junction-protein expression. Histamine (10(-9) -10(-4) M) causes a reversible concentration-dependent reduction of ZO-1 protein content, mediated by both H1 and H2 receptors. Histamine reduces ZO-1 expression within the time associated with increased paracellular permeability. Tight-junction-protein alterations may be a novel explanation for the mechanism by which vasoactive agents increase microvascular permeability.