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Plasma leptin levels are elevated in obesity suggesting a pathophysiologic role of this hormone in obesity and related disorders, such as hypertension. Furthermore, despite excess leptin levels, leptin satiety action is blunted in obesity suggesting the occurrence of central leptin resistance. As leptin acts on the kidney to induce natriuresis, renal leptin receptor alterations could lead to a defect in sodium excretion and hence to hypertension. Therefore, the present study investigated renal leptin receptor (Ob-Ra and Ob-Rb) mRNA and leptin binding capacities in diet-induced hypertension. Feeding male, female, and testosterone-treated female rats a cafeteria diet for 10 weeks increased body fat mass, plasma insulin, and leptin levels.Furthermore, although male and testosterone-treated female cafeteria-fed rats were hypertensive, the female rats fed the same diet failed to develop elevated blood pressure. In renal medulla, Ob-Ra and Ob-Rb mRNA levels were unchanged after cafeteria diet feeding in all groups; however, binding analysis revealed Ob-R protein down-regulation exclusively in hypertensive rats. Moreover, renal Ob-R densities were inversely correlated to plasma leptin concentrations in male rats and testosterone-treated female rats but not in female rats. These findings demonstrate the existence of differences in renal Ob-R binding capacities, which are correlated to hypertension.

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A method was devised for directing RNA polymerase on a single promoter site on T7 DNA. Initiation coniplexes were formed on each of the three main promoter sites using one dinucleotide plus one nucleoside triphosphate. The ternary initiation complexes are resistant to rifampicin action, to inhibition by (rI), at 0 "C and are stable at high salt concentrations. A minimum of a trinucleotide is required to form a stable ternary complex. To determine which promoter site was selected by RNA polymerase during initiation, the (rI),-resistant RNA was digested by RNAse 111 to generate three characteristic initiator RNA fragments, resolved by gel electrophoresis. The three major promoter sites could be selected individually by using different primer and substrate combinations :ApC + ATP selected promoter A3, CpG + CTP selected A2 and CpC + ATP specified preferentially A,. A number of primer-substrate combinations specified each site at low salt concentration but the substrate requirement became very stringent at high salt concentration, suggesting that the postulated local opening of the promoter site could be more or less extensive, depending on the ionic strength. The minimum opening observed at high salt concentration corresponded to the insertion of a leader trinucleotide sequence. The promoter region melted by RNA polymerase at low salt concentration was (G + C)-rich and corresponded to about 9 to 11 base pairs. Sequences of the melting recognition regions were tentatively inferred from the results.Transcription of T7 genome, a linear duplex DNA molecule containing approximately 40 000 base pairs has been extensively investigated as a simplified model of genetic expression. The finding that one large RNA chain is transcribed in v i m from the r-strand of T7 DNA by the host RNA polymerase first suggested the existence of a unique early promoter site [l] located at the left end of the DNA molecule [2,3]. More refined studies recently indicated that RNA synthesis was initiated at three closely spaced sites in the early promoter region [4,5]. Three strong binding sites of Escherichiu coli RNA polymerase were located by electron microscopy at 1.15, 1.4 and 1.65 "/, of T7 unit length from the left end of the molecule [6].Selective initiation with dinucleotides [ S ] and binding studies [7] as well as the analysis of transcripts in vivo [8,9] also revealed the existence of additional secondary sites for binding of RNA polymerase and Abbreviation. (rI),,, poly(inosinic acid). Enzymes. RNA polymerase (EC 2.7.7.6); RNAase 111 (EC 3.4.3.1.4.24).RNA chain initiation within the early region. The forthcoming step should now provide information firstly on the nature of the DNA sequences recognized by the enzyme and secondly on the relative efficiency of the different sites to direct RNA chain initiation. As a prerequisite for the isolation of RNA polymerase binding sites on T7 DNA we looked for conditions which would selectively stabilize RNA polymerase on a single promoter. Since the formation of an "initiation complex" is known...

Although obesity is associated with a state of leptin resistance, it has been suggested that leptin may contribute to the pathogenesis of obesity-related hypertension. In previous studies, we reported that cafeteria diet feeding induces hyperleptinaemia and hyperinsulinemia in both male and female rats, with hypertension occurring only in male rats. However, when female rats were neonatally treated with testosterone (T), these animals develop hypertension when fed the cafeteria diet. These observations led us to investigate leptin signaling and some neuropeptides that are leptin targets in the hypothalamus of male, intact female, and T-treated female cafeteria diet-fed rats. A decrease in the hypothalamic leptin receptors (Ob-Ra and Ob-Rb) and pro-opiomelanocortin (POMC) mRNA was observed only in male hypertensive cafeteria diet-fed rats. Although no alterations in Ob-R occurred in both groups of female cafeteria diet-fed rats, the hyperleptinaemic state of these animals had no influence on POMC mRNA levels. In intact female rats, expression of the suppressors of cytokines signaling SOCS-1, SOCS-2, SOCS-3, and cytokine inhibitor signaling were unaltered, whereas in T-treated females SOCS-3 was overexpressed. Finally SOCS-1 mRNA level was increased only in male rats. Because hyperinsulinemia was reported to counteract the leptin-induced stimulation of the sympathetic tone and because SOCS-1 and -3 are potential inhibitors of insulin signaling, our results suggest that the hypothalamic overexpression of SOCS-1 or SOCS-3 found in male or Ttreated female rats after cafeteria diet feeding could block the negative influence of the hyperinsulinemia on the central pressor action of leptin, thereby contributing to their hypertensive state.Obesity is associated with profound alterations of cardiovascular functions, including an increase in blood pressure. In the central nervous system, the hypothalamus is an important structure that regulates food intake and blood pressure. Thus, hypothalamic lesions induce not only obesity, but also sex-related differences in hypertension (Baylis et al., 1996). Leptin, an adipocyte-derived hormone, regulates food intake and neuroendocrine functions and stimulates sympathetic nerve activity (Dunbar et al

Factors influencing promoter site selection by Escherichia coli RNA polymerase were investigated using T7 DNA as template. The utilization of the three major early promoters A1, A2 and A3, was followed by processing the RNA transcripts with RNAase 111, which generates the three corresponding initiator RNA fragments. The three promoters proved to be functionally distinct. A strong differential effect of temperature and ionic strength on promoter selection was observed. The transition temperature of promoters A1, A2 and A3 was measured directly by preincubating, at different temperatures, RNA polymerase and T7 DNA, with primer-substrate combinations which selected each site independently. The transition temperature of the three sites was markedly different. Promoter A3 was used predominantly at low temperature, whereas A1 or Az promoters were gradually activated by increasing the temperature. The temperature response curves were strongly dependent upon the salt concentration. On the other hand, challenge experiments with rifampicin or poly (inosinic acid) showed that, once preinitiation complexes are formed by incubating RNA polymerase with DNA at 37 'C, the three sites A1, A2 and A3 promote chain initiation with equal efficiency and at the same rate.Bacterial RNA polymerase is responsible for transcription of most of the RNA species formed in the cell [l]. This implies that the enzyme, with or without specifier proteins, is able to recognize many promotor sites in bacterial and phage DNAs. In addition, it is becoming clear that multiple forms of promoter sites exist, which are recognized with different efficiencies. The primary sequence of a number of RNA polymerase binding sites or promoter regions have been determined [2 -61. On the whole, besides some sequence homology found in the non-transcribed region, these promoter sequences are strikingly different.The T7 system lends itself to a comparison of different promoters since three closely spaced promoter sites govern the reading of a unique transcription unit [7,8]. An interesting question concerning these three sites is whether they are functionally identical in terms of affinity for RNA polymerase and efficiency of chain initiation.In the present paper, the method of visualization of the small RNA initiator fragments, following RNAase I11 processing of the RNA transcript, was Abbreviation. (rI)n, poly(inosinic acid). Enzymes. RNA polymerase (EC 2.7.7.6); RNAase I11 (EC 3.1.4.24).used to study the factors influencing promoter site selection [7,9,10]. It is shown that the utilization of T7 DNA early promoters is differently affected by alterations in ionic strength and temperature. In contrast, once the enzyme . DNA preinitiation complexes are formed, the rate of chain initiation appears to be the same at the three sites. MATERIALS AND METHODS MaterialsE. coli RNA polymerase, RNAase 111, T7 DNA, 3',5'-dinucleotides and (rI),, were prepared or obtained as previously described [9]. Unlabelled ATP and CTP used for formation of initiation complexes were purified b...

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This study investigated the incidence of cafeteria-diet induced hypertension on hypothalamic tyrosine hydroxylase (TH) and ␣ 2 -adrenoceptor subtype gene expression in male, female, and neonatally testosterone-imprinted female rats. After 10 weeks of cafeteria diet, all these rats were hyperleptinemic. In contrast, males and testosterone-treated females developed hypertension, whereas intact females remained normotensive. In these rats, cafeteria diet up-regulated TH gene expression only in males and testosterone-treated females. On the other hand, cafeteria diet differentially affected hypothalamic gene expression of ␣ 2 -adrenoceptor subtypes. In fact, this diet increased ␣ 2A -adrenoceptor mRNA levels only in intact normotensive females. In contrast, gene expression of the ␣ 2B -adrenoceptor was up-regulated only in male and testosterone-treated female cafeteria-fed rats. Furthermore, an ␣ 2C -adrenoceptor gene over-expression was also induced, but only in male cafeteriafed rats. If one assumes that the up-regulations in TH and ␣ 2B -adrenoceptor gene expression are indicative of increased sympathetic nervous activity, then, these altered gene expressions could be responsible for the maintenance of high blood pressure in male and testosterone-treated female cafeteria-fed rats. Conversely, in intact females, the absence of these overexpressions and the up-regulation of the ␣ 2A -adrenoceptor gene expression could reflect an adaptive response to the diet and, consequently, could be protective against cafeteria dietinduced hypertension. Moreover, neonatal testosterone imprinting in females could have induced an irreversible android susceptibility to the cafeteria diet, leading to the onset of hypertension.