Diversity of ARSACS mutations in French-Canadians.

BACKGROUND: The growing number of spastic ataxia of Charlevoix-Saguenay (SACS) gene mutations reported worldwide has broadened the clinical phenotype of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). The identification of Quebec ARSACS cases without two known SACS mutation led to the development of a multi-modal genomic strategy to uncover mutations in this large gene and explore phenotype variability. METHODS: Search for SACS mutations by combining various methods on 20 cases with a classical French-Canadian ARSACS phenotype without two mutations and a group of 104 sporadic or recessive spastic ataxia cases of unknown cause. Western blot on lymphoblast protein from cases with different genotypes was probed to establish if they still expressed sacsin. RESULTS: A total of 12 mutations, including 7 novels, were uncovered in Quebec ARSACS cases. The screening of 104 spastic ataxia cases of unknown cause for 98 SACS mutations did not uncover carriers of two mutations. Compounds heterozygotes for one missense SACS mutation were found to minimally express sacsin. CONCLUSIONS: The large number of SACS mutations present even in Quebec suggests that the size of the gene alone may explain the great genotypic diversity. This study does not support an expanding ARSACS phenotype in the French-Canadian population. Most mutations lead to loss of function, though phenotypic variability in other populations may reflect partial loss of function with preservation of some sacsin expression. Our results also highlight the challenge of SACS mutation screening and the necessity to develop new generation sequencing methods to ensure low cost complete gene sequencing.

Oxytocin gene expression in rat uterus.

The neurohypophyseal hormone oxytocin (OT) is the most potent uterotonic agent known and is used to induce labor. Yet, endogenous circulating OT appears not to participate in the induction of labor. As shown here, the finding of OT messenger RNA and peptide in the uterus suggests a solution for this paradox. During gestation, rat uterus OT messenger RNA increased more than 150-fold and, at term, exceeded hypothalamic OT messenger RNA by 70-fold. Thus, during parturition, OT may act primarily as a local mediator and not as a circulating hormone.

Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions.

Recent studies suggest that increased activity of cyclin-dependent kinase 5 (Cdk5) may contribute to neuronal death and cytoskeletal abnormalities in Alzheimer's disease. We report here such deregulation of Cdk5 activity associated with the hyperphosphorylation of tau and neurofilament (NF) proteins in mice expressing a mutant superoxide dismutase (SOD1(G37R)) linked to amyotrophic lateral sclerosis (ALS). A Cdk5 involvement in motor neuron degeneration is supported by our analysis of three SOD1(G37R) mouse lines exhibiting perikaryal inclusions of NF proteins. Our results suggest that perikaryal accumulations of NF proteins in motor neurons may alleviate ALS pathogenesis by acting as a phosphorylation sink for Cdk5 activity, thereby reducing the detrimental hyperphosphorylation of tau and other neuronal substrates.

Effect of a nonselective endothelin antagonist on vascular remodeling in deoxycorticosterone acetate-salt hypertensive rats. Evidence for a role of endothelin in vascular hypertrophy.

We have previously shown that the endothelin content in arteries of deoxycorticosterone acetate (DOCA)-salt hypertensive rats is increased. We designed this study to examine, using the new orally active nonselective endothelin receptor antagonist bosentan, whether this increase in vascular endothelin may contribute to elevated blood pressure and vascular hypertrophy in DOCA-salt hypertensive rats. Rats received bosentan (100 mg/kg body wt per day) for 3 weeks mixed with their food. Systolic blood pressure of DOCA-salt hypertensive rats rose to 197 +/- 5 mm Hg, and that of bosentan-treated DOCA-salt hypertensive rats was 177 +/- 4 mm Hg (P < .01). Mesenteric resistance arteries were studied on a wire myograph. The media width, ratio of media width to lumen diameter, and cross-sectional area of the media of resistance arteries of bosentan-treated DOCA-salt hypertensive rats were significantly smaller than those of untreated DOCA-salt hypertensive rats. The lumen diameter and cross-sectional area of the media of vessels of bosentan-treated rats were not different from those of uninephrectomized control rats. Vasoconstrictor responses, which were altered in DOCA-salt hypertensive rats, approached control in the bosentan-treated rats. We conclude that these results with a nonselective endothelin receptor antagonist may suggest a role for endothelin in the elevation of blood pressure and vascular hypertrophy and remodeling in DOCA-salt hypertensive rats.

Increased expression of endothelin-1 gene in blood vessels of deoxycorticosterone acetate-salt hypertensive rats.

We have recently shown that the content of immunoreactive endothelin-1 is increased in acid extracts from blood vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats compared with uninephrectomized control rats. We have also found by immunohistochemistry a significant increase in immunoreactive endothelin-1 in endothelial cells of aorta and mesenteric arteries of DOCA-salt hypertensive rats. In the present study, we investigated preproendothelin-1 gene expression in blood vessels of DOCA-salt hypertensive rats and uninephrectomized control rats. Northern blot analysis using a specific 32P-labeled complementary RNA probe for rat preproendothelin-1 of 319 base pairs revealed a fourfold to fivefold increase in abundance of preproendothelin-1 messenger RNA transcripts in both aorta and mesenteric arteries from DOCA-salt hypertensive rats. Thus, increased immunoreactive endothelin-1 content in blood vessels of DOCA-salt hypertensive rats is secondary to increased preproendothelin-1 gene expression. Exaggerated expression of the preproendothelin-1 gene in mineralocorticoid hypertension may contribute to the maintenance of elevated blood pressure.

Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats.

Endothelin-1 (ET-1) is a powerful vasoconstrictor peptide produced in the endothelium of blood vessels that may play an important role in the control of local blood flow and could be involved in the pathogenesis of hypertension. We investigated immunoreactive ET-1 (ir-ET-1) levels in acid extracts from blood vessels of deoxycorticosterone acetate (DOCA)-salt and spontaneously hypertensive rats. We found that segments of thoracic aorta and the mesenteric vascular bed contain significantly more ir-ET-1 (11.84 +/- 0.84 and 17.30 +/- 1.89 fmol, respectively) than uninephrectomized control rats (1.78 +/- 0.20 and 9.19 +/- 0.63 fmol, respectively; p < 0.001). High performance liquid chromatography showed that ir-ET-1 of blood vessels of DOCA-salt hypertensive rats eluted in the same position as synthetic ET-1. Significantly increased ir-ET-1 was localized by immunohistochemistry in endothelial cells of aorta and large and small mesenteric arteries of DOCA-salt hypertensive rats. In contrast to the latter, in spontaneously hypertensive rats, vascular content of ir-ET-1 was similar to that of blood vessels of Wistar-Kyoto control rats, at both 6 and 16 weeks of age. High levels of vascular ET-1 may explain the downregulation of vascular endothelin receptors previously described in DOCA-salt hypertensive rats. Furthermore, this suggests that ET-1 may be involved in the maintenance of high blood pressure in mineralocorticoid hypertension.

Deoxycorticosterone acetate plus salt induces overexpression of vascular endothelin-1 and severe vascular hypertrophy in spontaneously hypertensive rats.

Endothelin-1 gene expression is enhanced in the aorta and mesenteric arteries, and possibly other vessels, of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In contrast, endothelin-1 gene expression is normal or reduced in spontaneously hypertensive rats (SHR). Severe vascular hypertrophy is present in DOCA-salt hypertensive rats but not in SHR. In this study we investigated whether treatment of SHR with DOCA and salt would result in enhanced endothelin-1 expression and at the same time in severe vascular hypertrophy. Increased abundance of endothelin-1 mRNA was found in the aorta and the mesenteric arterial bed of SHR treated simultaneously with DOCA and salt but not when rats were treated with either separately. The wet weight of the aorta and of the mesenteric arterial bed, media thickness, media cross-sectional area, and media-to-lumen ratio of mesenteric small arteries of DOCA-salt-treated SHR were exaggerated beyond what could be explained by the elevation of blood pressure, relative to SHR treated with salt or with DOCA, which did not overexpress vascular endothelin-1. In conclusion, SHR may exhibit enhanced expression of the endothelin-1 gene in blood vessels when treated with DOCA and salt, and associated with this there is severe vascular hypertrophy. These data support the hypothesis of a role of endothelin-1 in vascular hypertrophy.

Cytosolic calcium changes induced by angiotensin II in neonatal rat atrial and ventricular cardiomyocytes are mediated via angiotensin II subtype 1 receptors.

We determined the effects of angiotensin II (Ang II) on cytosolic free calcium concentrations ([Ca2+]i) in the absence and presence of the selective angiotensin subtype 1 (AT1) receptor antagonist losartan or the selective AT2 antagonist PD 123319 in cultured neonatal rat atrial and ventricular cardiomyocytes. We also Ang II receptor density, affinity, and mRNA expression. [Ca2+]i was measured in single cells microphotometrically and by fluorescent digital imaging with fura 2 methodology. Receptor parameters were assessed by competitive binding studies with 125I-[Sar1,Ile8]Ang II in the presence of increasing concentrations of [Sar1,Ile8]Ang II, losartan, and PD 123319. AT1 receptor (types AT1A and AT1B) mRNA abundance was measured by reverse transcription-polymerase chain reaction. Ang II produced concentration-dependent increases in [Ca2+]i values in atrial and ventricular cells were similar but Ang II (10-9 mol/L)-induced [Ca2+]i changes were significantly greater in atrial compared with ventricular cells Ang II responses were blocked by losartan (10-7 mol/L) but not PD 123319 (10-7 mol/L). Binding studies demonstrated a single class of high-affinity. Ang II binding sites on cardiomyocyte membranes (Kd = 0.71 +/- 0.11 mumol/L). 125I-[Sar1,Ile8]Ang II was displaced by losartan but not by PD 123319. AT1 receptor mRNA was detected by reverse transcription-polymerase chain reaction in cells from atria and ventricles. In atrial cardiomyocytes, both AT1A and AT1B receptor genes were expressed, whereas in ventricular cardiomyocytes, only the AT1A receptor gene was expressed. These data demonstrate that neonatal cardiomyocytes possess Ang II receptors of the AT1 receptor subtype that are linked to [Ca2+]i signaling pathways. The different Ang II-induced [Ca2+]i responses between atrial and ventricular cells may be related to differences in the distribution of AT1 receptor subtype subvariants.

Abnormal adrenal and vascular responses to vasopressin mediated by a V1-vasopressin receptor in a patient with adrenocorticotropin-independent macronodular adrenal hyperplasia, Cushing's syndrome, and orthostatic hypotension.

The elucidation of gastric inhibitory polypeptide-dependent Cushing's syndrome suggested that ectopic expression or increased responsiveness of other adrenal hormone receptors may underlie ACTH-independent macronodular adrenal hyperplasia (AIMAH) or adrenocortical tumors. We studied a 36-yr-old woman with Cushing's syndrome, AIMAH, and orthostatic hypotension. During upright posture, cortisol and aldosterone were stimulated despite suppression of ACTH and renin. Arginine vasopressin (AVP, 10 U im), under dexamethasone suppression, increased plasma cortisol (3.4-fold), aldosterone (67-fold), and androgens in this patient but not in controls. ACTH 1-24, but not desmopressin acetate, angiotensin II, isoproterenol, or other hormones stimulated steroidogenesis in vivo. Plasma AVP was undetectable initially and increased suboptimally during posture tests after bilateral adrenalectomy. AVP stimulated cortisol production more in dispersed cells from the AIMAH than from a normal adult adrenal (424 vs. 135% at 10 nmol/L). Adrenal V1-AVP receptor presence and mediation of response were shown by RT-PCR and by binding and [Ca+2]i studies. Post adrenalectomy, orthostatic hypotension persisted; a prolonged vasoconstrictive response to AVP was found in vitro in the patient's sc small arteries. We propose that altered adrenal and vascular responses of the V1-AVP receptor-effector pathway underlie this new syndrome.

Vascular binding sites and biological activity of vasopressin in DOCA-salt hypertensive rats.

In order to understand the regulation of vascular vasopressin receptors in hypertension, vasopressin (AVP) binding sites and the pressor response to AVP in the perfused mesenteric vasculature of DOCA-salt hypertensive rats, sodium-loaded and DOCA-treated rats were investigated. The binding capacity for AVP (Bmax) was significantly reduced (P less than 0.05) in uninephrectomized, DOCA-treated rats (70 +/- 17 fmol/mg protein) and in DOCA-salt hypertensive rats (90 +/- 9 fmol/mg protein) with respect to uninephrectomized rats (130 +/- 32 fmol/mg protein) or uninephrectomized salt-loaded rats (155 +/- 47 fmol/mg protein), with no change in affinity. In these rats with lower receptor density, however, the maximal pressor response to AVP in the perfused mesenteric vascular bed was increased (P less than 0.05). In DOCA-salt hypertensive rats plasma AVP was higher than in the other groups. In similarly treated rats with intact kidneys, which therefore did not become hypertensive, receptor density was significantly decreased after combined DOCA-salt treatment, together with an exaggerated pressor response to AVP and increased plasma AVP concentrations. These results suggest that AVP receptors are down-regulated when there is an increment in the plasma concentration of AVP, although other factors may also play a role. Biological responses to AVP are, however, increased in spite of decreased receptor density and this phenomenon is independent of the elevation in blood pressure and results from an exaggerated response mediated by post-receptor mechanisms.

Blood pressure-independent effect of angiotensin AT1 receptor blockade on renal endothelin-1 production in hypertensive uremic rats.

OBJECTIVE: We recently reported that treatment of uremic rats with reduced renal mass with the angiotensin II (Ang II) subtype 1 receptor (AT1) antagonist losartan reduces endothelin-1 (ET-1) levels in blood vessels and in glomeruli. Although this suggests an important role for Ang II in the modulation of ET-1 production, the concomitant decrease in blood pressure may also be involved. The present study was designed to investigate whether the modulation of ET-1 production in uremic rats is related to tissue-specific effects of AT1 receptor blockade or to the antihypertensive effect of losartan. DESIGN: One week after renal mass reduction, uremic rats were treated with the conventional triple therapy (TRx) [reserpine (5 mg/l), hydralazine (80 mg/l) and hydrochlorothiazide (25 mg/l)] or losartan (20 mg/kg per day) for 6 weeks. Immunoreactive-ET-1 (ir-ET-1) levels in plasma and urine, as well as in vascular and renal tissues were measured by a specific radioimmunoassay after sample extraction and purification. RESULTS: Before treatment, systolic blood pressure was significantly higher in uremic animals compared to sham-operated controls (165+/-4 versus 123+/-2 mmHg, respectively; P < 0.01). Treatment with the TRx or with losartan normalized systolic blood pressure in uremic rats, whereas it was further increased in untreated uremic animals. At week 6, serum creatinine, proteinuria and urinary ET-1 and transforming growth factor-beta1 (TGF-beta1) excretion, as well as vascular and glomerular ET-1 content were increased in uremic rats compared to the controls (P < 0.01). Treatment of uremic rats with the TRx or with losartan reduced ET-1 content in the thoracic aorta and the mesenteric arterial bed (P < 0.01). However, losartan, but not the TRx, significantly attenuated the rise of serum creatinine, proteinuria and urinary ET-1 and TGF-beta1 excretion, as well as ET-1 content in glomeruli of uremic rats. Compared with the controls, renal preproET-1 mRNA expression was also significantly higher in uremic rats. Treatment of uremic rats with losartan prevented renal preproET-1 mRNA overexpression, indicating that changes in glomerular ET-1 content and urinary ET-1 excretion were related to modulation of renal ET-1 production. CONCLUSIONS: These findings indicate that the effect of losartan on ET-1 production in peripheral blood vessels may be mediated, in part, by the reduction of blood pressure. In contrast, the reduction of renal ET-1 production is mediated by tissue-specific effects of AT1 receptor blockade, and may contribute to the renal protective effects of losartan.

Transient involvement of endothelin in hypertrophic remodeling of small arteries.

OBJECTIVE: This study was designed to evaluate the capacity of norepinephrine (NE) to induce hypertrophic remodeling of small arteries in rats, and to determine the involvement of endothelin (ET) to initiate and maintain it. DESIGN AND RESULTS: Treatment with NE (2.5 microg/kg per min) for 14 or 28 days produced a similar inward hypertrophic remodeling, characterized by a smaller lumen, but increased media thickness and cross-sectional area. Arterial stiffness was reduced. Histological evaluation confirmed the hypertrophic nature of remodeling. Concomitant administration of LU135252 (ET-receptor antagonist) for the first 14 days of NE administration prevented the development of hypertrophy, without altering arterial mechanics. Treatment with the same antagonist from day 14 to day 28 of NE or angiotensin II (Ang II) treatment failed to regress established vascular hypertrophy. In contrast, normalization of arterial structure was observed with prazosin, an alpha-adrenergic blocker. Endothelin content in small mesenteric arteries showed a transient elevation following chronic NE administration. CONCLUSIONS: Increased circulating NE levels are associated with hypertrophic remodeling of small arteries, in which ET plays an initiating role. However, the maintenance of vascular hypertrophy is ET-independent, either in the presence of augmented circulating levels of NE or Ang II. Thus, early rather than late treatment with ET-receptor antagonists may be a preferable approach to limit small artery-mediated end-organ damage in cardiovascular diseases.

In situ hybridization shows increased endothelin-1 mRNA levels in endothelial cells of blood vessels of deoxycorticosterone acetate-salt hypertensive rats.

Endothelin-1 is a potent vasoconstrictor peptide produced in blood vessels and other tissues that may play an important role in the control of local blood flow and could be involved in the pathogenesis of hypertension. Our previous studies have documented increases in endothelin-1 peptide content and gene expression in mesenteric arteries and thoracic aorta of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Although changes in endothelin-1 were observed in the blood vessels of hypertensive rats, the exact cellular sites of these changes were not identified clearly. In the present study we investigated endothelin-1 gene expression in DOCA-salt hypertensive rats by in situ hybridization histochemistry using a high specific activity 35S-labeled complementary RNA probe. Robust increases in endothelin-1 mRNA levels were observed in both mesenteric blood vessels and aorta of DOCA-salt hypertensive rats as compared with the vessels from the uninephrectomized control rats. In both cases it was shown clearly that these increased endothelin-1 mRNA levels only originated in the endothelial cell layer, not in the underlying smooth muscle cells. Higher expression levels of endothelin-1 mRNA by the endothelial cells of DOCA-salt hypertensive rats may play an important role in vascular hypertrophy and in the maintenance of elevated blood pressure in this and perhaps other models of experimental hypertension.

Inducible nitric oxide synthase in vascular smooth muscle cells from prehypertensive spontaneously hypertensive rats.

The inducible nitric oxide synthase (iNOS) present in vascular smooth muscle cells (VSMC) may play a role in the generation of nitric oxide (NO) in the vascular wall, regulating blood vessel tone in normotension and hypertension. In this study the effect of interleukin (IL)-1 beta, a cytokine that induces iNOS, on NO generation (measured as nitrite), cyclic guanosine monophosphate (cGMP) generation, and steady-state abundance of iNOS mRNA were examined in VSMC from 3 week old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, during the period preceding the elevation of blood pressure. With cell density dependent variations in nitrite production eliminated, VSMC from SHR and WKY did not differ in NO generation except after prolonged incubation (30 h), when SHR cells produced less NO. However, cGMP concentrations associated with IL-1 beta stimulation were significantly smaller in SHR VSMC than in cells from WKY. IL-1 beta stimulation resulted in increased abundance of iNOS mRNA to the same extent in both WKY and SHR VSMC. Inhibitors of NOS, NG-monomethyl-L-arginine (L-NMMA) and N omega-nitro-L-arginine methyl ester (L-NAME), did not block the induction of iNOS mRNA, although nitrite production and cGMP generation were inhibited. The protein synthesis inhibitor cycloheximide and the RNA synthesis inhibitor actinomycin-D almost completely blocked the production of nitrite in cells from both strains of rats. Actinomycin-D completely blocked the induction of iNOS mRNA by IL-1 beta in cells from both strains of rats, whereas cycloheximide partially blocked its synthesis in WKY, but had no significant effect on IL-1 beta induced expression of iNOS mRNA in SHR VSMC. Thus, IL-1 beta controls iNOS gene expression at the transcriptional level, and an intermediate labile protein, whose synthesis is inhibited by cycloheximide, is required for IL-1 beta stimulated induction of iNOS mRNA transcription in WKY cells but not in SHR. We conclude that although iNOS is expressed to similar extent in VSMC of prehypertensive SHR and WKY and similar amounts of NO are initially generated, there are differences between the VSMC of SHR and WKY in the regulation of the transcription of iNOS mRNA, there is a lower sustained production of NO, and there is a reduced generation of cGMP in response to IL-1 beta stimulated NO production. These differences between VSMC from prehypertensive SHR and WKY may indicate a pathophysiological role of iNOS in early blood pressure elevation in SHR.

Effects of losartan and captopril on endothelin-1 production in blood vessels and glomeruli of rats with reduced renal mass.

Recently, we have reported that endothelin-1 (ET-1) production is increased in blood vessels and glomeruli of rats with chronic renal failure. This study was design to investigate the role of angiotensin II (Ang II) in endogenous ET-1 production in rats with reduced renal mass. One week after subtotal (5/6) nephrectomy, uremic rats were divided into three groups, and received either no treatment, the Ang II subtype 1 receptor (AT1) antagonist losartan (10 mg/kg/day), or the angiotensin-converting enzyme inhibitor (ACE-I) captopril (30 mg/kg/day) for 6 weeks. Sham-operated rats were used as controls and received no treatment. The levels of immunoreactive ET-1 (ir-ET-1) in plasma and urine, as well as in vascular and renal tissues, were determined by radioimmunoassay (RIA) after extraction. In uremic rats, losartan and captopril completely prevented the increase in systolic blood pressure. At week 6, plasma ir-ET-1 was similar in the different groups of uremic rats and in the controls. However, ir-ET-1 concentration in the mesenteric arterial bed, the thoracic aorta, preglomerular arteries, and glomeruli, as well as urinary ir-ET-1 excretion were significantly greater in uremic-untreated rats compared to controls (P < .01). Treatment of uremic rats with losartan or captopril reduced irET-1 concentration in the thoracic aorta and preglomerular arteries (P < .05), but ir-ET-1 concentration in the mesenteric arterial bed was unchanged. Although both drugs completely prevented the increase in proteinuria, losartan but not captopril significantly reduced ir-ET-1 concentration in glomeruli (P < .05) and normalized urinary ir-ET-1 excretion. This indicates that increased ET-1 production in blood vessels and glomeruli of uremic rats is modulated, at least in part, by Ang II through the AT1 receptor. The beneficial effects of the AT1 antagonist losartan could be attributable to the attenuation of Ang II-induced ET-1 production in this rat remnant kidney model of chronic renal failure, whereas those of the ACE-I captopril are not related to changes in ET-1 production in glomeruli.

Endothelin influences pHi of human platelets through protein kinase C mediated pathways.

Stimulation of human platelets with endothelin raises cytosolic pH (pHi). In order to determine whether this effect is mediated via protein kinase C and Na(+)-H+ linked pathways, the effects of staurosporine and calphostin C (protein kinase C inhibitors) and 5-(N,N-hexamethylene) amiloride (Na(+)-H+ exchange blocker) on endothelin-induced pHi responses in human platelets were assessed. In addition, platelet endothelin receptor subtypes were determined pharmacologically using the selective ETA receptor antagonist BQ-123 and the ETB receptor agonist sarafotoxin S6c. pHi was measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets obtained from 15 healthy subjects. Endothelin-1 at a fixed concentration of 10(-9) M significantly increased pHi from 7.11 +/- 0.01 ([H+]i = 77 +/- 0.9 nM) to 7.19 +/- 0.04 ([H+]i = 64 +/- 0.9 nM) (p < 0.01). The pHi-stimulating effect of endothelin-1 was inhibited by 10(-7) M staurosporine, calphostin C and 5-(N,N-hexamethylene) amiloride. BQ-123 (10(-7) M) abolished the pHi responses to endothelin-1, whereas sarafotoxin S6c had no effect on platelet pHi. These data suggest that in vitro effects of endothelin-1 on platelet pHi are receptor-mediated via a pathway involving protein kinase C. Platelet endothelin receptors appear to be of the ETA subtype.

Expression of endothelin-1 gene in blood vessels of adult spontaneously hypertensive rats.

Endothelins are potent vasoconstrictor peptides produced by the endothelium of blood vessels and other tissues, which could play a potentially important pathogenic role in hypertension. In some hypertensive models, endothelin production in blood vessels is enhanced. We have therefore investigated endothelin-1 gene expression in arteries of 17-week old spontaneously hypertensive rats (SHR) with established hypertension and Wistar-Kyoto control rats (WKY). The abundance of endothelin-1 mRNA in aorta and mesenteric arteries was evaluated by Northern blot analysis. Endothelin-1 mRNA expression was found to be reduced in the aorta of SHR in comparison to the age-matched WKY, whereas it was similar in mesenteric arteries in both strains. These results agree with the vascular content of immunoreactive endothelin-1 which we previously reported, and indicate that reduced or normal vascular endothelin-1 content in SHR is not the result of rapid turnover of the peptide in SHR blood vessels. Together with normal plasma immunoreactive endothelin-1 and normal or reduced responsiveness of blood vessels of SHR to endothelin-1, this indicates that vascular endothelin-1 does not appear to play an important role in the pathogenesis of the elevation of blood pressure in this model of genetic hypertension.

Effects of monovalent and divalent cations and of guanine nucleotides on binding of vasopressin to the rat mesenteric vasculature.

The rat mesenteric vasculature contains high affinity binding sites specific for [3H]Arg8-vasopressin which mediate its vasoconstrictor action. We have investigated the in vitro effect of monovalent and divalent cations and guanine nucleotides on the interactions between [3H]Arg8-vasopressin and its receptor in this preparation. Binding was increased by divalent cations from fourfold in the presence of Mg2+ at 5 mM to ninefold in the presence of Mn2+ at 5 mM. The potency order of divalent cations to increase binding was Mn2+ greater than Co2+ greater than Ni2+ greater than Mg2+ greater than Ca2+ approximately equal to control without cations. Addition of Na2+ or other monovalent cations (K+, Li+, and NH4+) in the presence or absence of divalent cations reduced binding significantly. Analysis of saturation binding curves showed a single high affinity site. In the presence of 5 mM Mn2+, binding capacity (Bmax) increased to 139 +/- 23 fmol/mg protein. Receptor affinity was enhanced (KD decreased to 0.33 +/- 0.07 nM). In presence of 5 mM Mg2+ or 150 mM Na+, Bmax and affinity were reduced. The addition of 100 microM GTP or its nonhydrolyzable analogue, Gpp(NH)p, reduced receptor affinity in the presence of Mn2+ + Na+, Mg2+, and Mg2+ + Na+, but not in the presence of Mn2+ alone. Computer modeling of competition binding curves demonstrated that in contrast with saturation studies, the data were best explained by a two-site model with high affinity, low capacity sites and low affinity, high capacity sites. Mn2+ or Mn2+ + Na+ with or without guanine nucleotides resulted in a predominance of high affinity sites. GTP or Gpp(NH)p in the presence of Mg2+ or Mg2+ + Na+ induced a reduction of affinity of the high affinity binding sites and the number of these sites. In the presence of Mg2+ + Na+ and guanine nucleotides, high affinity sites were maximally decreased. An association kinetic study indicated that the association rate constant (K+1) was increased by divalent cations and reduced by guanine nucleotides, without change in the dissociation rate constant (K-1). The equilibrium dissociation constant (KD) calculated with these rate constants (K-1/K+1) was similar to that obtained in saturation experiments at steady state. Dissociation kinetics were biphasic, indicating the presence of two receptor states, one of high and one of low affinity, associated with a slow and a rapid dissociation rate. Cations and guanine nucleotides interact with one or more sites closely associated with vasopressin receptors, including possibly with a GTP-sensitive regulatory protein, to modulate receptor affinity for vasopressin.

Rat amnion: a novel site of oxytocin production.

Fetal membranes, in addition to serving as passive barriers, possess secretory capacities and have a role in amniotic fluid production. In the present study, we demonstrate that rat amniotic/chorionic membranes have the capacity to synthesize oxytocin (OT). Analysis of RNA extracted from rat amnion/chorion membranes by RNA blotting and polymerase chain reaction (PCR) revealed the presence of a single species of OT mRNA. The transcript was smaller than the hypothalamic OT transcript and equaled the size of uterine and placental OT mRNA. The abundance of amniotic OT mRNA decreased by 37% from Day 14 to Day 21 of pregnancy. Over the same time, amniotic/chorionic OT immunoreactivity, as assessed by RIA, increased from 0.17 +/- 0.03 to 1.36 +/- 0.18 ng/g tissue weight. HPLC analysis of amniotic/chorionic membrane extracts revealed two peaks of immunoreactive OT (ir-OT). The first peak (36% of total ir-OT) co-eluted with synthetic OT, while the second peak (64%) corresponded to a noncovalent complex, most likely between OT and neurophysin-I. Using the selective OT receptor ligand, 125I-d(CH2)5 [Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT, we detected high numbers of OT binding sites in rat uterine tissues, but no OT binding sites in amniotic/chorionic or placental membranes. We conclude that the rat amniotic/chorionic membranes represent an extra-hypothalamic site of OT gene expression in the rat during pregnancy and thus represent a possible source of OT present in amniotic fluid.