Control of sodium and potassium homeostasis by renal distal convoluted tubules.

Distal convoluted tubules (DCT), which contain the Na-Cl cotransporter (NCC) inhibited by thiazide diuretics, undergo complex modulation to preserve Na+ and K+ homeostasis. The lysine kinases 1 and 4 (WNK1 and WNK4), identified as hyperactive in the hereditary disease pseudohypoaldosteronism type 2, are responsible for activation of NCC and consequent hypokalemia and hypertension. WNK4, highly expressed in DCT, activates the SPAK/OSR1 kinases, which phosphorylate NCC and other regulatory proteins and transporters in the distal nephron. WNK4 works as a chloride sensor through a Cl- binding site, which acts as an on/off switch at this kinase in response to changes of basolateral membrane electrical potential, the driving force of cellular Cl- efflux. High intracellular Cl- in hyperkalemia decreases NCC phosphorylation and low intracellular Cl- in hypokalemia increases NCC phosphorylation and activity, which makes plasma K+ concentration a central modulator of NCC and of K+ secretion. The WNK4 phosphorylation by cSrc or SGK1, activated by angiotensin II or aldosterone, respectively, is another relevant mechanism of NCC, ENaC, and ROMK modulation in states such as volume reduction, hyperkalemia, and hypokalemia. Loss of NCC function induces upregulation of electroneutral NaCl reabsorption by type B intercalated cells through the combined activity of pendrin and NDCBE, as demonstrated in double knockout mice (KO) animal models, Ncc/pendrin or Ncc/NDCBE. The analysis of ks-Nedd-4-2 KO animal models introduced the modulation of NEDD4-2 by intracellular Mg2+ activity as an important regulator of NCC, explaining the thiazide-induced persistent hypokalemia.

Control of sodium and potassium homeostasis by renal distal convoluted tubules

Distal convoluted tubules (DCT), which contain the Na-Cl cotransporter (NCC) inhibited by thiazide diuretics, undergo complex modulation to preserve Na+ and K+ homeostasis. The lysine kinases 1 and 4 (WNK1 and WNK4), identified as hyperactive in the hereditary disease pseudohypoaldosteronism type 2, are responsible for activation of NCC and consequent hypokalemia and hypertension. WNK4, highly expressed in DCT, activates the SPAK/OSR1 kinases, which phosphorylate NCC and other regulatory proteins and transporters in the distal nephron. WNK4 works as a chloride sensor through a Cl- binding site, which acts as an on/off switch at this kinase in response to changes of basolateral membrane electrical potential, the driving force of cellular Cl- efflux. High intracellular Cl- in hyperkalemia decreases NCC phosphorylation and low intracellular Cl- in hypokalemia increases NCC phosphorylation and activity, which makes plasma K+ concentration a central modulator of NCC and of K+ secretion. The WNK4 phosphorylation by cSrc or SGK1, activated by angiotensin II or aldosterone, respectively, is another relevant mechanism of NCC, ENaC, and ROMK modulation in states such as volume reduction, hyperkalemia, and hypokalemia. Loss of NCC function induces upregulation of electroneutral NaCl reabsorption by type B intercalated cells through the combined activity of pendrin and NDCBE, as demonstrated in double knockout mice (KO) animal models, Ncc/pendrin or Ncc/NDCBE. The analysis of ks-Nedd-4-2 KO animal models introduced the modulation of NEDD4-2 by intracellular Mg2+ activity as an important regulator of NCC, explaining the thiazide-induced persistent hypokalemia.

Phylogenetically conserved sequences in the promoter of the rabbit sodium-hydrogen exchanger isoform 1 gene (NHE1/SLC9A1).

NHE1 (gene symbol SLC9A1) encodes an isoform of the amiloride-sensitive Na+-H+ exchanger that is present in many cells and the basolateral membrane of renal epithelia. Expression of NHE1 is modulated in response to chronic metabolic acidosis, growth factors, and phorbol esters. To begin examining the molecular basis for this regulation, a rabbit genomic clone that contained 6 kb of 5' flanking region, the first exon, and a portion of the first intervening sequence of NHE1 was obtained. The principal transcription start site in native rabbit tissues was located 798 bp 5' to the initiation codon. The sequence of the proximal 5' flanking region of rabbit NHE1 was similar to the human sequence and contained a TATA-box, (G + C)-boxes, homopyrimidine direct repeats, and a putative AP-1 site. When ligated to luciferase and transfected into porcine renal epithelial cells (LLC-PK1), 708 bp of proximal 5' flanking region exhibited orientation-dependent promoter activity.

Cloning, sequence, and tissue distribution of a rabbit renal Na+/H+ exchanger transcript.

Rabbit kidney expresses a transcript that is similar to the human growth-factor-activatable Na+/H+ exchanger. PCR and library screening were used to clone overlapping 2.5 kb, 1.4 kb, and 1.8 kb cDNAs that together contain the entire coding region (2448 bp) and 5' untranslated region (726 bp) and part of the 3' untranslated region (128 bp) of a rabbit renal Na+/H+ exchanger transcript. The nucleotide and inferred amino acid sequences are highly conserved between rabbit and human (88% nucleotide identity, 95% amino acid identity). In rabbit, the transcript is expressed in both epithelial and non-epithelial tissues, with highest expression in stomach, brain, kidney, lung and ileum, and minimal expression in liver and skeletal muscle.

Identification of clonally rearranged T-cell receptor beta chain genes in HTLV-I carriers as a potential instrument for early detection of neoplasia.

We analyzed the genetic recombination pattern of the T-cell receptor beta-chain gene (TCR-beta) in order to identify clonal expansion of T-lymphocytes in 17 human T-lymphotropic virus type I (HTLV-I)-positive healthy carriers, 7 of them with abnormal features in the peripheral blood lymphocytes. Monoclonal or oligoclonal expansion of T-cells was detected in 5 of 7 HTLV-I-positive patients with abnormal lymphocytes and unconfirmed diagnosis by using PCR amplification of segments of TCR-beta gene, in a set of reactions that target 102 different variable (V) segments, covering all members of the 24 V families available in the gene bank, including the more recently identified segments of the Vbeta-5 and Vbeta-8 family and the two diversity beta segments. Southern blots, the gold standard method to detect T-lymphocyte clonality, were negative for all of these 7 patients, what highlights the low sensitivity of this method that requires a large amount of very high quality DNA. To evaluate the performance of PCR in the detection of clonality we also analyzed 18 leukemia patients, all of whom tested positive. Clonal expansion was not detected in any of the negative controls or healthy carriers without abnormal lymphocytes. In conclusion, PCR amplification of segments of rearranged TCR-beta is reliable and highly suitable for the detection of small populations of clonal T-cells in asymptomatic HTLV-I carriers who present abnormal peripheral blood lymphocytes providing an additional instrument for following up these patients with potentially higher risk of leukemia.

Retinal lesions induce differential changes in the expression of flip and flop isoforms of the glutamate receptor subunit GluR1 in the chick optic tectum.

A sensitive RNase protection assay was employed to determine the levels of mRNA encoding the GluR1 subunit flip and flop isoforms in the chick optic tectum and forebrain. We found that the flip GluR1 mRNA predominates in the forebrain, whereas the flop variant is more strongly expressed in the optic tectum. A temporal analysis of GluR1 variants in the embryonic and adult chick brain revealed that the flip isoform is more highly expressed at E12 than at P15-21, whereas mRNA levels of the flop isoform are higher at P15-21 than at E12. To study the effect of deafferentation on GluR1 expression, unilateral retinal lesions were performed. Two days later the mRNA levels of GluR1 flip and flop variants were decreased in the deafferented tectum, especially for the flop isoform. However, 7 days after the lesion, the mRNA levels of both GluR1 isoforms were increased, especially for the flip isoform. These results reveal an important control of the retinal input upon the expression of the different GluR1 isoforms. Furthermore, they indicate a differential spatial and temporal regulation of the flip and flop splice variants, suggesting the existence of a mechanism regulating differential splicing or possibly differential RNA stability.

Expression of endo-oligopeptidase A in the rat central nervous system: a non-radioactive in situ hybridization study.

Endo-oligopeptidase A (EOPA, formerly EC 3.4.22.19), a thiol-activated oligopeptidase, is able to degrade both bradykinin and neurotensin, and also to convert enkephalin-containing peptides into enkephalins. The expression of this enzyme was studied in the rat brain by in situ hybridization using non-radiotopic probes. The distribution of EOPA transcripts included many regions of the rat central nervous system, with higher expression in some regions, such as the hippocampus, cerebellum, and basal nucleus of Meynert. The marked EOPA expression in these areas could be anticipated, since they are rich in neuropeptides that are known to be EOPA substrates in vitro. The data characterize a widespread occurrence of EOPA in the rat brain and reinforce the suggestion of a critical role for EOPA in peptide processing.

Molecular biology of renal Na(+)-H+ exchangers.

Na(+)-H+ exchangers are plasma membrane proteins that are responsible for Cl- and HCO3- reabsorption and regulation of intracellular pH in several nephron segments. Recently, a cDNA encoding a human Na(+)-H+ exchanger of unknown tissue origin was cloned, and Northern blot analysis revealed that structurally similar transcripts were expressed in rabbit kidney and porcine renal epithelial cells (LLC-PK1). To clone these renal transcripts we employed cDNA library screening and the polymerase chain reaction (PCR). We obtained 2.5 kb and 1.4 kb cDNAs corresponding to the 5' untranslated region and the membrane-associated domain of a rabbit renal Na(+)-H+ exchanger. From LLC-PK1 cells we obtained 1.5 kb and 1.3 kb cDNAs encoding the membrane-associated and cytoplasmic domains. The sequences of cDNAs from these three species were very similar and suggested a high degree of evolutionary conservation. Immunolocalization of synthetic oligopeptides derived from the deduced amino acid sequences indicated that the cloned cDNAs encoded the amiloride-sensitive form of the Na(+)-H+ exchanger present in basolateral membranes of epithelia. cDNAs were also used to study regulation of Na(+)-H+ exchanger gene expression in the kidney, and we found that metabolic acidosis stimulated both the transport rate and steady-state transcript levels of the basolateral Na(+)-H+ exchanger in LLC-PK1 cells.

H+ secretion in distal tubules: the effect of carbonic anhydrase in luminal perfusates.

1. The ability of distal tubules in control rats to acidify luminal fluids is questioned in view of the absence of bicarbonate reabsorption and of a disequilibrium pH as shown by the equilibrium electrode method. However, the ability to establish a transepithelial pH gradient has been shown previously. These discrepancies are investigated in the present work. 2. Luminal pH was measured in cortical distal segments of rat nephron during stationary perfusion with carbonic anhydrase (1 mg/ml)-containing solutions. During perfusion with bicarbonate Ringer, the stationary pH was significantly higher in the presence of the enzyme, suggesting the existence of an acid disequilibrium pH in these segments. 3. Acidification half-times were also longer in the presence of the enzyme, yielding bicarbonate reabsorption rates of 0.24 +/- 0.07 (N = 29)nmol cm-2s-1 in early and 0.32 +/- 0.09 (N = 28) in late distal tubules. 4. During luminal perfusion with phosphate Ringer, significant transepithelial pH gradients were also observed but were unaffected by the enzyme. Thus, a disequilibrium pH was detected in distal tubules when perfused with bicarbonate-containing solutions. This acid disequilibrium pH can inhibit distal bicarbonate reabsorption by creating an adverse pH gradient. 5. In conclusion, the ability of distal tubules to establish a significant pH gradient will contribute to the titration of non bicarbonate buffers, i.e., to titratable acid formation.

Gender differences in vascular expression of endothelin and ET(A)/ET(B) receptors, but not in calcium handling mechanisms, in deoxycorticosterone acetate-salt hypertension.

We determined if the increased vascular responsiveness to endothelin-1 (ET-1) observed in male, but not in female, DOCA-salt rats is associated with differential vascular mRNA expression of ET-1 and/or ET A/ET B receptors or with functional differences in Ca2+ handling mechanisms by vascular myocytes. Uninephrectomized male and female Wistar rats received DOCA and drinking water containing NaCl/KCl. Control rats received vehicle and tap water. Blood pressure and contractile responses of endothelium-denuded aortic rings to agents which induce Ca2+ influx and/or its release from internal stores were measured using standard procedures. Expression of mRNA for ET-1 and ET A/ET B receptors was evaluated by RT-PCR after isolation of total cell RNA from both aorta and mesenteric arteries. Systolic blood pressure was higher in male than in female DOCA rats. Contractions induced by Bay K8644 (which activates Ca2+ influx through voltage-operated L-type channels), and by caffeine, serotonin or ET-1 in Ca2+-free buffer (which reflect Ca2+ release from internal stores) were significantly increased in aortas from male and female DOCA-salt compared to control aortas. DOCA-salt treatment of male, but not female, rats statistically increased vascular mRNA expression of ET-1 and ET B receptors, but decreased the expression of ET A receptors. Molecular up-regulation of vascular ET B receptors, rather than differential changes in smooth muscle Ca2+ handling mechanisms, seems to account for the increased vascular reactivity to ET-1/ET B receptor agonists and higher blood pressure levels observed in male DOCA-salt rats.

Identification of nuclear factor-κB sites in the Slc2a4 gene promoter.

Glucose transporter GLUT4 protein, codified by Slc2a4 gene plays a key role in glycemic homeostasis. Insulin resistance, as in obesity, has been associated to inflammatory state, in which decreased GLUT4 is a feature. Inflammatory NF-κB transcriptional factor has been proposed as a repressor of Slc2a4; although, the binding site(s) in Slc2a4 promoter and the direct repressor effect have never been reported yet. A motif-based sequence analysis of mouse Slc2a4 promoter revealed two putative κB sites located inside -83/-62 and -134/-113 bp. Eletrophoretic mobility assay showed that p50 and p65 NF-κB subunits bind to both putative κB sites. Chromatin immunoprecipitation assay using genomic DNA from adipocytes confirmed p50- and p65-binding to Slc2a4 promoter. Moreover, transfection experiments revealed that NF-κB binds to the -134/-113bp region of the mouse Slc2a4 gene promoter, inhibiting the Slc2a4 gene transcription. The current findings demonstrate the existence of two κB sites in Slc2a4 gene promote, and that NF-κB has a direct repressor effect upon the Slc2a4 gene, providing an important link between insulin resistance and inflammation.

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells.

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5'-flanking sequence (5'FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5'FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5'FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5'FS are used as promoter, efficient transcription only occurs with 44 bp of 5'FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5'FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Regulation of rat proximal tubule Na/H exchange by protein kinase C.

The effect of protein kinase C (PKC) activation on fluid and bicarbonate transport in renal tubules has been discussed controversially. Stimulation and inhibition have been shown to depend on factors such as experimental model and exposure time to the mediator of enzyme activation. We studied the role of PKC activation by phorbol-12-myristate-13-acetate (PMA) and by 1,2-dioctanoyl glycerol (DOG) in proximal bicarbonate reabsorption (JHCO3-) by 'in vivo' stationary microperfusion and ion-exchange resin microelectrode determination of luminal pH. Both PMA (10(-8) mol/l) and DOG (10(-3) mol/l) added to lumen or to peritubular capillaries reduced the net JHCO3- significantly. When added to lumen, the inhibition was 44 and 32%, respectively. This reduction did not involve changes in lumen stationary pH, but was mediated by a marked increase in the halftime of luminal bicarbonate disappearance; from 4.22 +/- 0.23 to 6.27 +/- 0.51 s with PMA and from 3.90 +/- 0.25 to 6.33 +/- 0.48 s with DOG. This effect was intensified by 10(-6) mol/l okadaic acid, a phosphatase inhibitor (inhibition of JHCO3- increased to 61%), and reduced by 30% by 10(-6) mol/l H7, an inhibitor of PKC. H89, a protein kinase A inhibitor, did not affect the inhibitory action of PMA. Our data suggest that PKC activation reduces the rate of H ion secretion (bicarbonate reabsorption) in convoluted segments of rat renal proximal tubules and that phosphorylation of the Na+/H+ exchanger by this kinase is the cause of the reduction in net secretion of H ions.

Dinitrophenol effect on proximal tubular acidification in the rat.

The effect of 10(-3) M-dinitrophenol (DNP) on renal tubular acidification was studied in proximal tubules of rat kidneys perfused with mammalian Ringer solution. Alkaline (pH 7.8) or acid (pH 5.8) phosphate-buffered solutions were injected into the lumen, and pH changes recorded with antimony micro-electrodes. Luminal perfusion with DNP caused complex acidification or alkalinization curves, an initial rapid shift toward a higher than control pH being followed by a slower acidification. Acidification half-times of the initial phase (t1/2 = 1.6 s) were markedly shorter than controls (6.2 s). This response was probably due to transient action of DNP, since keeping constant peritubular DNP levels by capillary perfusion caused simple exponential pH curves. In such experiments luminal pH increased from pH 6.6-6.8 to 7.1-7.2, while acidification and alkalinization t1/2 decreased from about 7 s to 3-5 s. Secretory H-ion fluxes increased transiently and then fell below controls after a few minutes of perfusion, while H-ion efflux from the lumen increased progressively. These data suggest that, besides its known effect on cell metabolism, DNP acts directly on proximal tubular cell membranes, increasing the rate of passive H-ion equilibration, both mechanisms impairing the tubular capacity to maintain normal proximal pH gradients and fluxes.

Chronic effect of parathyroid hormone on NHE3 expression in rat renal proximal tubules.

BACKGROUND: The most abundant Na+/H+ exchanger in the apical membrane of proximal tubules is the type 3 isoform (NHE3), and its activity is acutely inhibited by parathyroid hormone (PTH). In the present study, we investigate whether changes in protein abundance as well as in mRNA levels play a significant role in the long-term modulation of NHE3 by PTH. METHODS: Three groups of animals were compared: (1) HP: animals submitted to hyperparathyroidism by subcutaneous implantation of PTH pellets, providing threefold basal levels of this hormone (2.1 U. h-1); (2) control: sham-operated rats in which placebo pellets were implanted; (3) PTX: animals submitted to hypoparathyroidism by thyroparathyroidectomy followed by subcutaneous implantation of thyroxin pellets, which provided basal levels of thyroid hormone. After eight days, we measured bicarbonate reabsorption in renal proximal tubules by in vivo microperfusion. NHE3 activity was also measured in brush border membrane (BBM) vesicles by proton dependent uptake of 22Na. NHE3 expression was evaluated by Northern blot, Western blot and immunohistochemistry. RESULTS: Bicarbonate reabsorption in renal proximal tubules was significantly decreased in HP rats. Na+/H+ exchange activity in isolated BBM vesicles was 6400 +/- 840, 9225 +/- 505, and 12205 +/- 690 cpm. mg-1. 15 s-1 in HP, sham, and PTX groups, respectively. BBM NHE3 protein abundance decreased 39.3 +/- 8.2% in HP rats and increased 54.6 +/- 7.8% in PTX rats. Immunohistochemistry showed that expression of NHE3 protein in apical BBM was decreased in HP rats and was increased in PTX rats. Northern blot analysis of total kidney RNA showed that the abundance of NHE3 mRNA was 20.3 +/- 1.3% decreased in HP rats and 27. 7 +/- 2.1% increased in PTX. CONCLUSIONS: Our results indicate that the chronic inhibitory effect of PTH on the renal proximal tubule NHE3 is associated with changes in the expression of NHE3 mRNA levels and protein abundance.

Proximal tubular HCO3-, H+ and fluid transport during maleate-induced acidification defect.

The mechanism of tubular acidification was studied in proximal tubular acidification defect induced in rats by acute parenteral infusion of maleate (200 mg/kg), which causes diuresis and bicarbonaturia. Proximal tubular bicarbonate reabsorption and H+ ion secretion were determined by stopped-flow microperfusion and measurement of luminal pH by Sb microelectrodes. Stationary pH increased in proximal tubule from 6.78 to 7.25 and bicarbonate reabsorption decreased from 1.32 to 0.51 nmol/cm2 X s. In these segments, mean cell PD fell from -66.6 to -20.2 mV, while Jv as estimated by the Gertz technique fell to 15% of controls. A similar impairment of acidification was observed during luminal and capillary perfusion with phosphate Ringer's. Since H+-ion efflux from the lumen was not significantly increased and both acidification and alkalinization half-times (t/2) were increased, no evidence for an increase in passive permeability for H+/HCO3- was obtained. The increased t/2 found during luminal perfusion with acid phosphate indicates, according to an electrical analog model, a reduction in pump series conductance. These results show that maleate affects both proximal Na+ and H+ transport; this effect may be ascribed to impairment of sodium-dependent transport systems in the brush-border membrane.

Ovarian hormones modulate endothelin-1 vascular reactivity and mRNA expression in DOCA-salt hypertensive rats.

We previously demonstrated a differential activation of the endothelin-1 (ET-1) pathway in male and female deoxycorticosterone (DOCA)-salt hypertensive rats, with the male rats exhibiting marked alterations in vascular and pressor responses to ET-1 and Suc-[Glu,(9)Ala(11,15)]-ET-1(8-21) (IRL-1620), an ET(B) agonist. Mechanisms underlying these gender differences are unclear, and we hypothesized that the ovarian hormones attenuate vascular ET(B) responses in female DOCA-salt rats. Female Wistar rats were randomized in 3 groups: sham-operated, ovariectomized (OVX), and OVX plus hormone replacement with estradiol (E) or estradiol/progesterone (EP). Two weeks later, rats were uninephrectomized and further randomized in DOCA-salt (subcutaneous injections of desoxycorticosterone and drinking water containing NaCl/KCl) and control normotensive (subcutaneous injections of vehicle and tap water). Blood pressure was evaluated both by direct and standard tail-cuff methods. Responses to IRL-1620 were evaluated in vivo/in situ in the mesenteric microcirculation. mRNA expression of ET-1 and ET(A/B) receptors was evaluated in mesenteric arteries by reverse transcription-polymerase chain reaction and expressed relative to GAPDH. OVX-DOCA rats developed a more severe form of hypertension than did DOCA rats. Treatment with E or EP restored blood pressure to levels observed in DOCA rats. In the mesentery, IRL-1620 induced vasodilatation in control rats, a mild vasoconstriction in DOCA rats, and marked vasoconstriction in OVX-DOCA rats. Both E and EP decreased IRL-1620-induced vasoconstriction in the DOCA group. In the normotensive group, OVX did not change blood pressure or IRL-1620-induced vasodilation. Removal of the ovaries increased ET-1 mRNA in arteries from DOCA and control rats, although treatment with E or EP reversed these changes. Vascular ET(B) receptor mRNA levels were greatly enhanced in OVX-DOCA but not OVX-control rats. Hormone replacement with E or EP restored ET(B) receptor expression in the DOCA group. A greater blood pressure-lowering effect of bosentan (ET(A)/ET(B) blocker) was observed in OVX-DOCA rats. The observation that OVX worsens hypertension as well as the altered ET(B) receptor-mediated responses and the effects of bosentan in female DOCA rats supports our suggestion that the ovarian hormones modulate ET-1/ET(B) receptor vascular responses/expression in DOCA-salt hypertension.

Species specificity of thimet oligopeptidase (EC 3.4.24.15).

The recombinant rat testes metallo-endooligopeptidase (EC 3.4.24.15) and the rabbit brain endooligopeptidase A (formerly EC 3.4.22.19) were compared, side-by-side, in view of their striking similarities in both the physicochemical features and the specificities for oligopeptides. Concerning the tissue distribution in rat and rabbit, no relation between the levels of enzyme activity in cytosol and the levels of metallo-endooligopeptidase 24.15 mRNA could be established. The results suggest that the predominant neuropeptide-metabolizing activity attributed to the metallo-endooligopeptidase 24.15 is performed by, at least, two distinct cytosolic enzymes, one predominant in rat testes and the other in rabbit brain and testes, and possibly also in rat brain. Both enzymes are activated by dithiothreitol and irreversibly inhibited by a SH-affinity labeling dynorphin-related compound, but they are not inhibited by EDTA in a concentration dependent manner. Both enzymes exhibit the same specificity toward several bioactive peptides, except for LH-RH and substance P, which are only hydrolysed by the rat testes enzyme. Taken together, these results lead us to conclude that it is unlikely that the recombinant rat testes metallo-endooligopeptidase 24.15 and the rabbit brain endooligopeptidase A are the same molecule although they might belong to the same family of oligopeptidases.