Effects of volume change on circulating immunoreactive atrial natriuretic factor in rats.

The mammalian atrial hormone atrial natriuretic factor (ANF) has been shown to have potent natriuretic and diuretic actions as well as vasodilator effects when released into the circulation. To investigate how the levels of the circulating form of this peptide change with alteration of intravascular fluid volume, we measured immunoreactive ANF in the plasma of Wistar rats after acute saline load, acute furosemide treatment, and chronic water restriction. Circulating levels of immunoreactive ANF increased significantly (p less than 0.001) 1 minute after acute saline load and returned to normal levels within 5 minutes. Volume contraction induced by furosemide treatment of chronic water restriction significantly reduced the circulating immunoreactive ANF. These data indicate that acute volume expansion causes an immediate release of immunoreactive ANF into the general circulation and acute volume contraction results in a decline of circulating levels of immunoreactive ANF, which is maintained during chronic volume contraction. These results suggest that the atria detect alterations in intravascular fluid volume and respond by changing the levels of ANF acutely as well as chronically and thereby participate in the regulation of body fluids and, perhaps, of blood pressure.

Characterization of the major 68 kDa heat shock protein in a rat transformed astroglial cell line.

The heat shock response in a transformed astrocyte line was compared with nontransformed astrocytes. The synthesis of HSP 68, the major inducible heat shock protein (HSP 68) was induced by a non-lethal 45 degrees C, 10 min heat shock. Although the incorporation of [35S]methionine into HSP 68 suggested that similar amounts of protein were being synthesized after heat shock, Western immunoblotting demonstrated striking differences in the HSP immunostaining between the two cell types. By one- and 'two-dimensional gel electrophoresis the major 68 kDa heat shock protein (HSP 68) was similar in both cell types. However, HSP 68 from heat shocked, transformed astrocytes did not immunostain with the monoclonal antibody, C-92, which is specific for the major inducible heat shock protein of HeLa cells. In contrast HSP 68 from heat shocked, nontransformed astrocytes immunostained quite well. A polyclonal antibody raised against the inducible 72 kDa heat shock protein of HeLa cells immunostained the HSP 68 from both astrocytes and transformed astrocytes. Analysis of the mRNA from the two cell types after heat shock revealed two bands of approximately 2.5 and 2.8 kb in astrocytes but only a single 2.5 kb band in the heat shocked transformed astroglia. These results suggest that structural differences in the HSP 68 may be present in the transformed astrocytes compared to the normal astrocytes.

Inhibition of epidermal growth factor-mediated DNA synthesis by a specific tyrosine kinase inhibitor in vascular smooth muscle cells of the spontaneously hypertensive rat.

Aortic vascular smooth muscle cells isolated from spontaneously hypertensive rats (SHR) grow nearly twice as fast in vitro as cells isolated from several normotensive control strains of rats. We have previously shown that DNA synthesis in SHR cells from both young and adult animals in response to epidermal growth factor is selectively enhanced compared with normotensive controls, suggesting that epidermal growth factor may be at least partly responsible for the enhanced growth rate. To determine whether the enhanced DNA synthesis in response to epidermal growth factor in SHR cells is mediated via an enhanced epidermal growth factor receptor tyrosine kinase, we measured thymidine incorporation in epidermal growth factor-stimulated vascular smooth muscle cells in the presence of the highly specific tyrosine kinase inhibitor genistein. The 50% inhibitory dose (IC50) of genistein was higher for the SHR vascular smooth muscle cells than for the normotensive Wistar rat (NBR; National Institutes of Health Black rat). This suggests that the increased DNA synthesis in response to epidermal growth factor in SHR cells is a result of higher receptor tyrosine kinase activity initiating further intracellular signals.

Monoclonal antibodies to pig angiotensinogen recognize minor idiotypes.

We have produced monoclonal antibodies to a highly purified pig (P) angiotensinogen preparation and characterized their ability to bind [125]I-P- angiotensinogen. Lymphocytes of RBF/Dn mice immunized with P-angiotensinogen were fused with FOX-NY myeloma cells and clones were isolated by binding to [125]I-P-angiotensinogen and by an immunodot blot assay. Three of 16 clones which recognized P-angiotensinogen were characterized. Isolated monoclonal antibodies bound only 10-15% of the total [125]I-P-angiotensinogen; however, the bound counts could be displaced with unlabelled P-angiotensinogen. None of the monoclonals inhibited the cleavage of P-angiotensinogen by homologous renin, nor did they bind to the NH-terminal angiotensin I (ANG I) peptide. Little or no binding was detected to angiotensinogens in human, monkey, rat, rabbit, sheep or bovine serum. Mixtures of the clones and analysis of the immune complexes by PAGE indicated that different binding sites on different P-angiotensinogen were detected by some of the monoclonals, while the same or competing sites were recognized by others. No combination of clones tested significantly increased the amount of P-angiotensinogen bound. We interpret these findings to indicate that monoclonal antibodies to 'purified' pig P-angiotensinogen recognize species-specific minor epitope subsets of the protein, but not antigenic determinants common to all.

Chimeric rats from the fusion of genetically hypertensive and normotensive rat embryos.

Structural changes in the cardiovascular musculature of the SHR during the development of hypertension appears to involve both prehypertensive hyperplastic cellular growth and hypertension induced cellular hypertrophy. The genetic factors determining these changes are not fully known, but may involve altered growth control. The role of genetic determination on the development of hypertension in the SHR was investigated by producing chimeric rats composed of a mixture of genetically homozygous SHR and normotensive cells. Preimplantation 8-cell embryos isolated from SHR and the normotensive NBR (NIH Black Wistar) rat strains were aggregated in vitro and cultured to the blastocyst stage before implantation into surrogate mothers. Chimeric rats born to the surrogate females were raised to 36-40 wks of age and the development of hypertension monitored by tail cuff pressure (BP). BP in the chimeras varied from 115 to 205 mm Hg (146 +/- 25). Heart weights were positively correlated with BP, r = 0.76, p less than 0.05, while only a marginal and non-significant correlation of aortic weight was found (r = 0.53). The renin-angiotensin system was normal in the chimeras. This model may prove useful in determining the extent of genetically mediated cellular events in the development of hypertrophy and hypertension in the SHR.

Poly(A) length, cytoplasmic adenylation and synthesis of poly(A)+ RNA in early mouse embryos.

The poly(A) content of early mouse embryos fluctuates widely: after a transient increase in the one-cell embryo, there is a 70% drop in the two-cell and an approximately fivefold increase between the two-cell and early blastocyst stages (L. Pikó and K. B. Clegg, 1982, Dev. Biol. 89, 362-378). To shed light on the significance of these changes, we analyzed the size distribution of total poly(A) from embryos at different stages of development by gel electrophoresis and hybridization with [3H]poly(U). The number-average size of poly(A) tracts varies only slightly, from 61 to 77 nucleotides, indicating that the changes in poly(A) content are due primarily to changes in the number of poly(A) sequences, i.e., the number of poly(A)+ mRNA. From these data, the number of poly(A)+ mRNA can be estimated as follows: ovulated egg, 1.7 x 10(7); one-cell embryo, 2.4 x 10(7); late two-cell, 0.7 x 10(7); late eight-cell, 1.3 x 10(7); and early blastocyst, 3.4 x 10(7). These results suggest the elimination of the bulk of maternal poly(A)+ mRNA at the two-cell stage, to be replaced by newly synthesized mRNA derived from the embryonic genome. To study the synthesis of poly(A)+ mRNA, we cultured mouse embryos in vitro with [3H]adenosine and analyzed the labeled poly(A)+ RNA as to molecular size, length of the poly(A) tail, and relative distribution of label in poly(A) vs internal locations. We observed an active incorporation of label into large-molecular-weight (average size about 2 kb) poly(A)+ RNA at all stages from the one-cell to the blastocyst. However, in the one-cell embryo, about 70% of the label was localized in the poly(A) tail, suggesting cytoplasmic polyadenylation, and only about 30% was localized in the remainder of the molecule, suggesting the complete new synthesis of a small amount of poly(A)+ RNA. Differences in the size distribution of the labeled poly(A) as compared with the total poly(A) in the one-cell embryo indicate that the labeling is not due to a general turnover of poly(A) tails, but rather to the polyadenylation of previously nonpolyadenylated, stored RNA. Significant new synthesis of poly(A)+ RNA is evident from the two-cell stage onward and most likely accounts for the sharp rise in the number of poly(A)+ RNA molecules by the early blastocyst stage.

Quantitative aspects of RNA synthesis and polyadenylation in 1-cell and 2-cell mouse embryos.

Mouse embryos at the late 1-cell and late 2-cell stages were labelled with [3H]adenosine for periods of up to 320 min during which the specific activity of the ATP pool was constant. The time course of the molar accumulation of adenosine was calculated for tRNA, high-molecular-weight poly(A)- RNA and poly(A) tails versus internal regions of poly(A)+ RNA. Most of the adenosine incorporation into tRNA is due to turnover of the 3'-terminal AMP but some new synthesis of tRNA also appears to take place in both 1-cell and 2-cell embryos at a rate of about 0.2 pg/embryo/h. In the poly(A)- RNA fraction, an unstable component which is assumed to be heterogeneous nuclear RNA is synthesized at a high rate and accumulates at a steady-state level of about 1.5 pg/embryo in the 1-cell embryo and about 3.0 pg/embryo in the 2-cell embryo. Both 1-cell and 2-cell embryos synthesize relatively stable heterogeneous poly(A)- RNA, assumed to be mRNA, at a rate of about 0.3 pg/embryo/h; 2-cell embryos also synthesize mature ribosomal RNA at a rate of about 0.4 pg/embryo/h. Internally labelled poly(A)+ RNA is synthesized at a low rate in the 1-cell embryo, about 0.045 pg/embryo/h, but the rate increases to about 0.2 pg/embryo/h by the 2-cell stage. A striking feature of the 1-cell embryo is the high rate of synthesis of poly(A) tails, about 2.5 X 10(6) tails/embryo/h of an average length of (A)43, due almost entirely to cytoplasmic polyadenylation. This and other evidence suggests a turnover of the poly(A)+ RNA population in 1-cell embryos as a result of polyadenylation of new RNA sequences and degradation of some of the pre-existing poly(A)+ RNA. In the 2-cell embryo, the rate of synthesis of poly(A) tails (average length (A)93) is estimated at about 0.8 X 10(6) tails/embryo/h and a significant fraction of poly(A) synthesis appears to be nuclear.

Selectively enhanced stimulation of DNA synthesis by EGF in vascular smooth muscle cells from young and adult SHR.

Aortic vascular smooth muscle cells isolated from spontaneously hypertensive rats (SHR) replicate in vitro nearly twice as fast as cells isolated from several normotensive control strains of rats. Serum-derived peptide growth factors are known to stimulate cells to enter the DNA synthetic phase of the cell cycle and subsequent mitosis. We have examined the effect of several peptide growth factors to stimulate [3H]thymidine incorporation into DNA in smooth muscle cells isolated from adult (24 wk, hypertensive) SHR and age matched normotensive NIH Black Wistar (NBR) control rats. Our results indicate that the response of the SHR cells to epidermal growth factor (EGF) is selectively enhanced compared to the control NBR cells. PDGF also stimulated DNA synthesis but no significant difference between SHR and NBR was observed. Nerve growth factor and endothelial derived growth factor were not mitotic on either cell line. Additionally, we have found that SHR cells, isolated from young early hypertensive weanling animals before a significant elevation in pressure has occurred, divide at the same rate as adult SHR cells normotensive strains. These results are consistent with the view that genetic changes affecting the cellular response to EGF may influence the development of early hypertensive hyperplasia in the SHR which in concert with other factors aggravates the later development of hypertension.

Regulation of heat shock protein synthesis in rat astrocytes.

Rat forebrain astrocytes synthesize heat shock proteins with molecular weights 97, 89, 70, 68, and 30-34 kilodaltons. The stress inducible 68-kDa heat shock protein (HSP-68) was vigorously expressed by astrocytes in culture after a 45 degrees C, 20 min heat shock. HSP-68 synthesis was poorly inducible by a second heat shock given 16 hr after the initial heat shock. Decreased [35S]methionine incorporation into HSP-68 correlated with low levels of HSP-68 mRNA present after the second heat shock. The data suggest that control of HSP-68 mRNA levels by transcriptional/posttranscriptional mechanisms is a major site for regulation of HSP-68 synthesis.