Functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors precedes the development of cholinergic phenotype in embryonic rat septal cells in culture.

We examined the development of cholinergic neuronal functions and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) responses in cultured embryonic rat septal cells. Choline acetyltransferase activity was increased from 4 to 6 days in culture and reached a plateau at day 8. Acetylcholine release was increased from 6 to 8 days in culture. AMPA-induced increase in intracellular Ca(2+) level was observed at 3 days in culture and most of the AMPA-responsive cells coincided with high-K(+) responsive cells. These results suggest that cholinergic neurons develop their neuronal functions about 8 days under cultured conditions, and functional expression of AMPA receptors precedes the cholinergic functional development.

Contractile responses of cultured bovine retinal pericytes to angiotensin II.

OBJECTIVE: To document that angiotensin (ANG) II contracts cultured bovine retinal pericytes via saralasin-sensitive receptors if the cells are prerelaxed. METHODS: Changes in the contractile tone were quantified as the changes in the summed length of wrinkles induced by pericytes cultured on the silicone surface. RESULTS: Angiotensin II (10(-5) mol/L) did not increase the contractile tone of cultured pericytes that were not prerelaxed. However, when the pericytes had been prerelaxed 41% with 10(-6)-mol/L sodium nitroprusside, ANG II at the range of 10(-7) to 10(-5) mol/L caused prompt, dose-related, significant (P<.01) contraction. It induced a maximum contraction (29.9%+/-5.2% [mean+/-SE]) at 10(-6) mol/L. This effect lasted at least 10 minutes. Angiotensin II receptor antagonist saralasin (10(-6) mol/L) abolished the contractile effect of ANG II (10(-6) mol/L), although by itself it did not affect the contractile tone. CONCLUSIONS: Angiotensin II contracts cultured pericytes through saralasin-sensitive ANG II receptors. If ANG II affects the contractile tone of pericytes in vivo, it may affect capillary caliber, resistance, and blood flow.

Suppression of CO2-induced relaxation of bovine retinal pericytes by angiotensin II.

PURPOSE: To determine whether angiotensin II (Ang II), a vasoconstrictive peptide, changes the relaxation effect of elevated partial pressure of carbon dioxide (PCO2) on pericytes. METHODS: The contractile tone of cultured bovine retinal pericytes was measured when the ambient PCO2 was elevated in the absence or the presence of Ang II. All experiments were performed in the bicarbonate-buffered solution at 37 degrees C. RESULTS: Ang II (10(-6) M) by itself did not increase the baseline tone of the pericytes. Raising the PCO2 from 5% to 20% acidified the solution (pH dropped 0.51 +/- 0.02 U) and caused a sustained and statistically significant 22.9% +/- 4.6% relaxation of pericytes within 5 minutes (n = 8). In the presence of Ang II (10(-6) M), the maximum relaxation induced by 20% PCO2 was only 12.6% +/- 4.5% (n = 6) at 3 minutes, and the relaxation was not sustained. The effect of Ang II was statistically significant. Pretreatment with the competitive Ang II receptor antagonist saralasin (10(-6) M) for 10 minutes completely abolished the effect of Ang II (10(-6) M) on the response of pericytes to 20% PCO2. Saralasin by itself had no effect. CONCLUSIONS: Ang II attenuated the relaxing response of pericytes to elevated PCO2 through saralasin-sensitive Ang II receptors. Results suggest that some vasoactive agents, such as Ang II, could affect the pericyte responses to metabolic needs as signaled by local PCO2. This experimental design may permit further investigation of the altered physiology of local blood flow regulation.

Adenosine-induced relaxation of cultured bovine retinal pericytes.

PURPOSE: To investigate the effect of adenosine on the contractile tone of cultured bovine retinal pericytes. METHODS: Changes in the contractile tone were quantified as the changes in the summed length of wrinkles induced by pericytes on the silicone surface on which the cells were grown. RESULTS: Adenosine at 10(-9) M had no effect. In the range of 10(-8) to 10(-4) M, adenosine caused relaxation of pericytes in a concentration-dependent manner. Complete relaxation was induced by 10(-5) M to 10(-4) M adenosine. The concentration of adenosine that produced 50% relaxation was 3 x 10(-7) M. At all concentrations, relaxation began within 1 minute, reached the maximum within 5 to 10 minutes, and persisted for at least 30 minutes. After a washout of 3 x 10(-7) M adenosine, the reduced contractile tone recovered to the original level in 10 minutes. The adenosine-induced relaxation (3 x 10(-7) M) was completely abolished in the presence of 8-phenyl theophylline (10(-5) M), a nonselective adenosine receptor antagonist. The selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) at 10(-6) M did not reduce the effect of adenosine (3 x 10(-7) M). Conversely, the selective A2 receptor antagonist CP-66,713 at 10(-8) M partially inhibited (and at 10(-7) M, completely inhibited) the relaxation induced by adenosine (3 x 10(-7) M). The adenosine receptor antagonists-8-phenyl theophylline (10(-5) M), DPCPX (10(-6) M), and CP-66,713 (10(-7) M) by themselves had no effect on the contractile tone of pericytes. CONCLUSIONS: Adenosine causes relaxation of pericytes through the activation of the adenosine A2 receptor. Adenosine, which accumulates under ischemic conditions, may help to regulate local capillary blood flow.

The Evi-1 zinc finger myeloid transforming protein binds to genomic fragments containing (GATA)n sequences.

The EVI1 gene is activated by chromosomal translocations and inversions in approximately 5% of human acute myeloid leukemia (AML) and by retroviral insertion in approximately 20% of murine myeloid leukemias. EVI1 encodes a nuclear DNA-binding protein having 10 zinc finger motifs in two noncontiguous domains consisting of an amino-terminal domain of seven fingers and a carboxyl domain containing three fingers. To evaluate the sequence specificity of Evi-1 binding and potentially identify genomic targets, whole-genome PCR was utilized to isolate multiple Sau3A fragments which specifically bind to the amino-terminal zinc finger domain. The majority of these clones represented single copy sequences and virtually all contained variable numbers of repeats of the GATA motif, the target sequence for the erythroid-specific transcription factor GATA-1. GST/Evi-1 fusion proteins containing the amino-terminal domain of zinc fingers bound the GATA motif in these clones as well as to those present in the human gamma-globin promoter, similar to the binding of purified GATA-1 protein. By obtaining corresponding large genomic clones for eight of these fragments, transcription units were found associated with two. One corresponded to the glyceraldehyde-3-phosphate dehydrogenase gene and its expression was not affected by Evi-1. The second is a novel gene whose expression is repressed in murine myeloid cell lines that express Evi-1.

Selective prostaglandin D2 receptor stimulation elicits ocular hypotensive effects in rabbits and cats.

The effects of the selective prostaglandin D2 (DP) receptor agonists, 572C85 ((+/-)-5-(3-carboxypropylthio)-1-(2-cyclohexyl-2-hydroxyethyl- amino)hexahydrocyclopenta(d)imidazol-2(1H)-one) and 192C86 ((+/-)-5-(3-carboxypropylthio)-1-(2-cyclohexyl-2-hydroxyethylidene - amino)-3-ethylhexahydrocyclopenta(d)imidazol-2(1H)-one), were determined on intraocular pressure regulation in rabbits and cats. 572C85 (50 micrograms) in rabbits maximally lowered intraocular pressure by 4.3 mm Hg, and significantly for 4 h compared to control. In cats 572C85 had a similar effect. 192C86 (50 micrograms) reduced intraocular pressure by 2.8 mm Hg for 2 h in rabbits. Following exposure to the specific DP receptor antagonist, BW A868C ((+/-)-3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethylamin o)- hydantoin; 50 micrograms), which had no effect on intraocular pressure by itself, 572C85 (50 micrograms) did not reduce intraocular pressure in rabbits and cats. The intraocular pressure lowering effect of the mixed DP and EP receptor agonist, BW245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)-hydantoin; 50 micrograms), in cats was suppressed by only 64% by BW A868C (50 micrograms). These results clearly show that the DP receptors in rabbit and cat eyes are involved in intraocular pressure regulation. However, under baseline conditions DP receptor activity does not contribute to this regulation.

Selective suppression by bunazosin of alpha-adrenergic agonist evoked elevation of intraocular pressure in sympathectomized rabbit eyes.

PURPOSE: To determine whether there is alpha 1-adrenergic receptor heterogeneity associated with the regulation of intraocular pressure (IOP) and mydriasis in rabbits, the authors tested the hypothesis by characterizing the ability of the selective alpha 1-adrenergic antagonist, bunazosin, to block the ocular hypertensive and mydriatic responses to alpha 1-adrenoceptor stimulation by either norepinephrine (NE) or phenylephrine (PE). METHODS: The effects of topical application of bunazosin on IOP and pupillary diameter were measured in unilateral superior cervical ganglionectomized (SCGX) rabbits after exposure to either NE or PE. RESULTS: Bunazosin (0.1%) alone only lowered the IOP in the normal eye and did not elicit a pupillary response on either side. NE (0.01-1.0%) by itself caused a concentration-dependent rise in IOP on both sides, but mydriasis did not occur on the normal side. In SCGX eyes, the sensitivity of the IOP response to NE increased tenfold over that measured on the normal side. Unlike on the normal side, concentration-dependent mydriatic responses occurred with 0.1 and 1% NE. After pretreatment with bunazosin (0.1%), neither NE (0.1%) nor PE (0.1%) evoked a rise in IOP. However, the mydriatic response to either one of these agonists in the SCGX eyes was less affected. By contrast, pretreatment with the alpha 2-adrenergic antagonist, 0.5% yohimbine, did not change the IOP increase elicited by 0.1% NE. CONCLUSIONS: These results suggest that the alpha 1-adrenergic receptors that regulate IOP and pupillary diameter are different from one another in the rabbit.

Expression of the Evi-1 zinc finger gene in 32Dc13 myeloid cells blocks granulocytic differentiation in response to granulocyte colony-stimulating factor.

Expression of the Evi-1 gene is frequently activated in murine myeloid leukemias by retroviral insertions immediately 5' or 90 kb 5' of the gene. The Evi-1 gene product is a nuclear, DNA-binding zinc finger protein of 145 kDa. On the basis of the properties of the myeloid cell lines in which the Evi-1 gene is activated, it has been hypothesized that its expression blocks normal differentiation. To explore this proposed role, we have constructed a retrovirus vector containing the gene and examined its effects on an interleukin-3-dependent myeloid cell line that differentiates in response to granulocyte colony-stimulating factor (G-CSF). Expression of the Evi-1 gene in these cells did not alter the normal growth factor requirements of the cells. However, expression of the Evi-1 gene blocked the ability of the cells to express myeloperoxidase and to terminally differentiate to granulocytes in response to G-CSF. This effect was not due to altered expression of the G-CSF receptor or to changes in the initial responses of the cells to G-CSF. These results support the hypothesis that the inappropriate expression of the Evi-1 gene in myeloid cells interferes with the ability of the cells to terminally differentiate.

The Evi-1 zinc finger myeloid transforming gene is normally expressed in the kidney and in developing oocytes.

Activation of the Evi-1 zinc finger gene is commonly associated with the transformation of murine leukemias and is involved in some cases of human AML involving rearrangements at chromosome 3q25. To determine the normal function of the gene, we have looked for expression in a variety of cell lines and tissues. The predominant sites of expression of the gene are in the kidney and ovary. In the kidney, expression is localized to the renal tubules in the corticomedullary junction. In the ovary, high levels of the Evi-1 protein are found in the cytoplasm of developing oocytes. The latter result suggests a potential role for the Evi-1 gene product in early oocyte development.

Evidence for the involvement of cyclic GMP in adenosine-induced, age-dependent vasodilatation.

1. Adenosine-induced dilatation of rat aorta was present in aorta taken from 4 week-old rats, attenuated with increase in age of rats to 8 weeks, and was virtually absent in the aorta from 12 week-old rats. 2. Removal of the endothelium by mechanical rubbing attenuated adenosine-induced dilatation. 3. Haemoglobin and methylene blue partly reversed the adenosine-induced endothelium-dependent dilatation. 4. The order of potency of adenosine derivatives was 5'-(N-ethylcarboxamido)adenosine (NECA) greater than 2-phenylaminoadenosine (CV-1808) greater than 2-chloroadenosine greater than N6-([R]-[-]-phenylisopropyl)adenosine (R-PIA) greater than adenosine greater than N6-cyclohexyladenosine (CHA) greater than N6-([S]-[+]-phenylisopropyl)adenosine (S-PIA), indicating that adenosine receptors mediating the dilatation are of the A2 subtype. 5. [3H]-NECA bound to preparations of membranes from rats of 4 weeks old; it was displaced more effectively by NECA and the A2 ligand CV-1808 than by the A1 ligands CHA and S-PIA. ligands CHA and S-PIA. 6. The number but not affinity of specific binding sites for NECA decreased considerably with increase in age of rats to 8 weeks, and binding sites for [3H]-NECA were hardly detected in membrane preparations from rats of 20 weeks old. 7. Adenosine caused a marked increase in cyclic GMP production, but did not induce an increase in the cyclic AMP level. 8. This increase in cyclic GMP production induced by adenosine was abolished by methylene blue or 8-phenyltheophylline, or by removal of the endothelium. 9. The age-associated decrease in adenosine-induced dilatation was found to be associated with a reduction in the formation of cyclic GMP, but not of cyclic AMP. 10. These results suggest that adenosine causes dilatation via A2 receptors by inducing production of an endothelium-derived relaxing factor (EDRF), which in turn stimulates soluble guanylate cyclase, and so increases production of cyclic GMP. It is also suggested that the main reason for the age-associated decrease in adenosine-induced dilatation is a decrease in the number of A2-receptors or the ability of the endothelium to produce EDRF, leading to decreased production of cyclic GMP.

Identification, nuclear localization, and DNA-binding activity of the zinc finger protein encoded by the Evi-1 myeloid transforming gene.

Activation of the Evi-1 zinc finger gene is a common event associated with transformation of murine myeloid leukemias. To characterize the gene product, we developed antisera against various protein domains. These antisera primarily detected a 145-kilodalton nuclear protein that bound double-stranded DNA. Binding was inhibited by chelating agents and partially restored by zinc ions.

Insertional mutagenesis and transformation of hematopoietic stem cells.

The mechanisms that are involved in the control of the normal differentiation of hematopoietic progenitor cells are largely unknown. Moreover, little is known concerning the types of genes that can alter the ability of hematopoietic progenitors to differentiate and cause transformation. One approach to the latter has been to use retroviral induced IL-3-dependent myeloid leukemia cell lines to identify transforming genes by their activation through insertional mutagenesis. This approach has implicated alterations in c-myb in transformation and has identified a novel transcriptional factor of the zinc finger family that is frequently activated in murine myeloid leukemias and in some cases of human AML. Using this approach it should be possible to identify additional myeloid transforming genes. The identification and characterization of the genes will provide important information and approaches to the study of the regulation of differentiation in normal hematopoiesis.

Phenotypes and mechanisms in the transformation of hematopoietic cells.

Interleukin 3 (IL-3) is a growth factor that supports the proliferation of early hematopoietic stem cells, as well as cells that are committed to a variety of the myeloid lineages. The mechanisms by which IL-3 functions have been studied through the use of a series of IL-3-dependent cell lines isolated from myeloid leukemias or long-term bone marrow cultures. A variety of studies have implicated tyrosine phosphorylation in IL-3 signal transduction. One of the substrates of phosphorylation is a 140 kDa, IL-3-binding protein that is speculated to be the biologically relevant IL-3 receptor. IL-3, through tyrosine phosphorylation, supports viability and growth through the regulation of transcription of a series of genes including c-myc and c-pim-1. The c-myc gene contributes to viability, in part, by regulating the transcription of the ornithine decarboxylase gene. The role of growth factors in differentiation is less clear. By studying IL-3-dependent myeloid leukemia cell lines, two genes have been identified whose altered expression is associated with blocking the ability of the cells to differentiate. The c-myb gene is a nuclear DNA binding protein that has been implicated in myeloid transformation in a number of systems. The Evi-1 gene is a novel gene of the zinc finger family of transcriptional activators. Possible mechanisms by which these genes interfere with normal differentiation are discussed.

Presence of a novel spleen focus-forming virus-specific glycoprotein (gp51) in a Friend leukemia cell line and its decrease during erythrodifferentiation.

All Friend leukemia cell lines induced by polycythemic strains of Friend leukemia virus complex express an appreciable amount of spleen focus-forming virus (SFFV)-coding envelope gene (env)-related glycoprotein with a molecular weight of 55 kilodaltons (gp55). A clonal, highly differentiation-inducible Friend leukemia cell line, T3-C1-2-O(2-O), possesses not only gp55 but also gp51 as SFFV-specific gene products. The peptide map of gp51 is quite similar to that of gp55 and the difference in their molecular weights is primarily dependent on their oligosaccharide content. In the differentiation-induced state, gp51 becomes far less detectable than gp55 in 2-O cells. The biological significance of this novel SFFV-coding glycoprotein is discussed.