Collectivising the Kulaks? General practitioners and primary care groups in England.

It has previously been suggested that the English Department of Health seems, wittingly or not, to have drawn on the experiences of Stalinist Russia in devising policies for the National Health Service. The development of general practitioner fundholding during the 1990s could be compared to the Soviet support for the Kulaks (rich peasants with capital and entrepreneurship) in 1923. Both initiatives aimed to propel innovation and enhance productivity by giving increased market freedom to an elite group of entrepeneurial workers. Writing in 1991, Hughes and Dingwall speculated about the likelihood of general practitioners sharing the same fate as the Kulaks in 1928, namely forcible collectivisation. The current creation of Primary Care Groups (collections of about 50 general practitioners) raises the question of whether they are likely to be vulnerable to the same pathologies as collective agriculture, or has the metaphor become exhausted?

Hepatic angiotensin II nuclear receptors and transcription of growth-related factors.

OBJECTIVES: To investigate the influence of angiotensin II (All) receptors in isolated hepatic nuclei on other genes regulated by All and to determine whether the function of these intracellular receptors is influenced by alterations in the endocrine renin system. METHODS: Nuclei were isolated from hepatic tissue of normal and bilaterally nephrectomized or adrenalectomized Wistar rats. Following nuclear run-off, in the presence of varying All concentrations, specific messenger RNAs (mRNA) were determined by slot blot hybridization. Tissue levels of renin system components were measured by radioimmunoassay and nuclear receptors characterized by displacement of radiolabeled All with specific All receptor antagonists. RESULTS: All binding in the presence of DUP 753 and PD 123177 confirmed that nuclear All receptors can be classified as AT1 receptors and that as much as 10% of the specific binding is attributable to nuclear chromatin. All stimulated not only the production of mRNA for renin system components such as renin and angiotensinogen, but also that of mRNA for growth-related factors such as platelet-derived growth factor and the oncogene c-myc. Maximal stimulation occurred at 10(-9) mol/l All; higher concentrations reduced this response. After stimulation or suppression of the plasma renin system by adrenalectomy or bilateral nephrectomy, nuclei isolated from rat hepatic tissue contained elevated endogenous levels of growth-related and renin system mRNA including AT1 and AT2 All receptors. However, despite the level of receptor mRNA having been elevated, the total All receptor density of isolated nuclei decreased. In addition, after both maneuvers, isolated nuclei were refractory to All-induced gene transcription. CONCLUSION: The existence of mechanisms producing intracellular All and regulating its level, which in turn exert local regulatory responses via nuclear All receptors, lends significance to the presence of a functional intracrine renin system that could act in concert with or independently of the endocrine renin system.

Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y.

Neuropeptide Y (NPY), a 36-amino-acid transmitter distributed throughout the nervous system, is thought to function as a central stimulator of feeding behaviour. NPY has also been implicated in the modulation of mood, cerebrocortical excitability, hypothalamic-pituitary signalling, cardiovascular physiology and sympathetic function. However, the biological significance of NPY has been difficult to establish owing to a lack of pharmacological antagonists. We report here that mice deficient for NPY have normal food intake and body weight, and become hyperphagic following food deprivation. Mutant mice decrease their food intake and lose weight, initially to a greater extent than controls, when treated with recombinant leptin. Occasional, mild seizures occur in NPY-deficient mice and mutants are more susceptible to seizures induced by a GABA (gamma-aminobutyric acid) antagonist. These results indicate that NPY is not essential for certain feeding responses or leptin actions but is an important modulator of excitability in the central nervous system.

Nuclear angiotensin receptors induce transcription of renin and angiotensinogen mRNA.

The observation that nuclei from hepatic tissue exhibit specific angiotensin II (Ang II) binding led us to explore whether Ang II modulates mRNA in general, mRNA specific for renin system components, or both. Nuclei from hepatic tissue exhibited a single high-affinity (Kd = 0.4 nmol/L) Ang II-specific binding site, which was associated with increased RNA transcription. Whereas total RNA extracted from nuclei increased 1.5-fold in response to Ang II (10(-9) mol/L), specific mRNA for renin and angiotensinogen increased 7.8- and 2.5-fold, respectively. Ang II binding and induced transcription showed parallel Ang II dose responses that were both inhibited by 10(-5) mol/L DuP 753 or saralasin. Maximum Ang II binding and RNA transcription occurred at the same Ang II concentration (10(-9) mol/L). Higher doses of Ang II resulted in a progressive decrease in RNA transcription. Together, these results demonstrate that hepatic nuclei have functional Ang II-specific receptors. It is concluded that Ang II may elicit responses at nuclear receptors, which heretofore were associated only with Ang II receptors located on plasma membranes. However, the individual contribution of plasma and nuclear membrane Ang II receptors to the overall cellular Ang II transcriptional response and their possible interactions remain to be determined.

Improved techniques for successful neonatal rat surgery.

Problems encountered in neonatal rat surgery include mortality due to anesthesia and postoperative mortality due to cannibalism or neglect by the dam. We required a method of anesthesia which would enable us to perform complicated, lengthy, recovery eye surgery on day-old rat pups. Because ethical concerns have been raised regarding hypothermia, the currently recommended procedure for anesthesia of newborn rats, we adapted two effective techniques for anesthetizing adult rats for use in neonates. In the first of these methods, halothane was administered via a gas anesthetic machine which allowed for precise regulation of anesthetic levels. The second method employed diluted Innovar-Vet, a neuroleptanalgesic drug combination that is easily administered by injection, with oxygen supplementation. Because each surgical procedure required 30 to 45 minutes and was technically demanding, it was important to minimize the loss of experimental animals due to cannibalism. To accomplish this, we developed an easy, noninvasive method to encourage acceptance of surgically manipulated pups by the dam, which included hand gentling and olfactory conditioning of pregnant females. All pups (63/63) survived eye surgery under halothane anesthesia and of those examined 7 days later, 55/57 (97%) were alive and appeared normal. Of the pups treated with Innovar-Vet, 16/16 (100%) survived anesthesia and all were normal in appearance when examined 7 days later. Our results suggest that using these anesthetic methods coupled with appropriate conditioning of the dam and handling of the pups contribute to successful neonatal rat surgery.

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.

Influence of recombinant human erythropoietin on blood pressure and tissue renin-angiotensin systems.

In humans, blockade of the renin-angiotensin system with angiotensin converting-enzyme inhibitors (ANG CEI) prevents the rise in blood pressure associated with the administration of recombinant human erythropoietin (rhEPO). This study was conducted to determine whether rhEPO elevates blood pressure in normal Wistar rats and whether the renin-ANG system is affected. Groups of 10 rats each were given rhEPO, ANG CEI (enalapril), rhEPO + ANG CEI, or vehicle. Renin and/or renin substrate mRNA was measured in aortas, kidney, and heart; renin activity (PRA), inactive renin, and renin substrate were measured in plasma. rhEPO raised blood pressure in the normal rat without changing the plasma renin system. ANG CEI prevented this blood pressure rise. Renin-specific mRNA was increased by rhEPO in renal tissue, and renin substrate mRNA was significantly elevated in the kidney and aorta. mRNA for renin and renin substrate were not altered in the heart. In both aorta and kidney, a significant correlation was observed between renin substrate mRNA and blood pressure. The data indicate that rhEPO modulates specific tissue renin-ANG systems, which may contribute to blood pressure elevation.

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.

Comparison of the heat shock response in cultured cortical neurons and astrocytes.

Cultured cortical neurons and astrocytes were compared for synthesis of the major inducible 68 kDa heat shock protein. By one- and two-dimensional electrophoresis the inducible 68 kDa protein appeared similar, but astrocytes produced greater amounts of the protein by 3 h than did neurons. Antibodies raised against HeLa cell inducible 72 and constitutive 73 kDa heat shock proteins were used to characterizes the inducible heat shock proteins in neurons and astrocytes. Unlike the gels, major differences were noted of the major inducible heat shock protein in astrocytes compared with neurons when analyzed by Western immunoblots. Heat shock protein 68 kDa mRNA induction in neurons was less than astrocytes suggesting an attenuated inducible 68 kDa heat shock protein response in neurons. The neuronal protein may be a different isoform of the 70 kDa family of heat shock proteins.

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.

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.

Production of angiotensinogen by cultured rat aortic smooth muscle cells.

This study was conducted to further investigate angiotensinogen synthesis in rat aortic smooth muscle cells (SMC) grown in culture. tissue cultures maintained in defined medium neither grew nor synthesized angiotensinogen. However, in the presence of 5% homologous serum both cell proliferation and angiotensinogen synthesis became apparent. Substitution of normal control serum with that of bilaterally nephrectomized rats or animals given dexamethasone (10mg/kg, ip) led to a further significant increase in angiotensinogen production. In contrast, serum from adrenalectomized rats suppressed angiotensinogen synthesis below the rate observed with normal serum. A positive linear correlation (r = 0.96, p less than 0.01) was evident between the serum angiotensinogen level and the rate of de novo synthesis of this protein. No correlations were found between cell proliferation and either angiotensinogen synthesis or serum angiotensinogen levels. Dexamethasone added to serum did not stimulate the rate of angiotensinogen synthesis and appeared to inhibit cell proliferation. Stimulation or suppression of angiotensinogen synthesis was not accompanied by a statistically significant change in angiotensinogen specific mRNA. The data indicate a complex regulation of angiotensinogen in vascular smooth muscle cells in culture.

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.

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.

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.

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.