Direct measurements of nitric oxide release in relation to expression of endothelial nitric oxide synthase in isolated porcine mitral valves

The aim of this study was to measure the direct release of nitric oxide (NO) from the porcine mitral valve using a NO microelectrode. Furthermore, the expression and localization of endothelial nitric oxide synthase (eNOS) in the mitral valve was studied using immunohistochemistry, Western blotting and RT-PCR. Results show that bradykinin increases NO release from mitral valves (DeltaBradykinin: 33.71 +/- 10.41 nm NO, P < 0.001, n = 10), whereas N-nitro-l-arginine methyl esther (l-NAME) decreases NO release when compared with basal level (Deltal-NAME: 82.69 +/- 15.66 nm NO, P < 0.005, n = 4). Both protein and mRNA expression of eNOS in mitral valves and in isolated valvular endothelial cells suggest that the NO release is mainly associated with the mitral valve endothelium. It is concluded that direct NO release from porcine mitral valves coincides with eNOS expression. This study documents useful techniques for investigations into the role of local NO release in mitral valve diseases.     

Gene expression of endothelin-1 and its receptors in the heart of broiler chickens with T(3)-induced pulmonary hypertension

To investigate the effect of T₃-induced pulmonary hypertension on endothelin (ET) production and genes expression of ET-1, ET_A and ET_B receptors (ET_AR and ET_BR) during rearing, semiquantitative RT-PCR and enzyme immunometric assay were performed in the heart ventricles and serum, respectively. The ET-1 and its receptor genes were expressed in the right and left ventricles of control and T₃-treated broilers at 12, 28 and 49 days of age. There were significant (P < 0.05) reductions of the relative amounts of ET-1 (in both ventricles) and ET_AR (in the right ventricle) mRNAs at 28 and 49 days of age, in T₃-treated broilers compared to controls. The relative amounts of ET_BR mRNA in the right and left ventricles did not significantly differ between control and T₃-treated broilers at any age. The serum level of ET was significantly (P < 0.05) increased in T₃-treated chickens at 28 and 49 days of age when compared with that of the control. It is concluded that ET-1, ET_AR and ET_BR genes are normally expressed in the heart ventricles of broilers. It is likely that increased serum level of ET and decreased ET-1/ET_AR genes expression in the ventricles are involved in the heart dysfunction of broiler chickens with developmental pulmonary hypertension.     

Fasting influences steroidogenesis, vascular endothelial growth factor (VEGF) levels and mRNAs expression for VEGF, VEGF receptor type 2 (VEGFR-2), endothelin-1 (ET-1), endothelin receptor type A (ET-A) and endothelin converting enzyme-1 (ECE-1) in newly formed pig corpora lutea

This study was designed to verify whether fasting influences vascular endothelial growth factor (VEGF) production and VEGF, VEGF receptor-2 (VEGFR-2) as well as endothelin (ET) system members (endothelin converting enzyme-1, ECE-1; ET-1; endothelin receptor type A, ET-A) mRNA expression in pig corpora lutea; furthermore, we wanted to assess whether fasting affects steroidogenesis in luteal cells. Eight prepubertal gilts were induced to ovulate and were randomly assigned to two groups: (A) n = 4, normally fed; and (B) n = 4, fasted for 72 h starting 3 days after ovulation. At the end of fasting, ovaries were removed from all the animals and corpora lutea (CLs) were collected. VEGF and steroid levels in luteal tissue were determined by ELISA and RIA, respectively; VEGF, VEGFR-2, ET-1, ET-A and ECE-1 mRNAs expression was measured by real-time PCR. VEGF protein levels were similar in the two groups, while all steroid (progesterone, testosterone, estradiol 17beta) concentrations were significantly (P < 0.001) higher in CLs collected from fasted animals compared with those from normally fed gilts. VEGF, VEGFR-2, ET-1 and ECE-1 (but not ET-A) mRNA expression was significantly lower (P < 0.05) in fasted versus normally fed animals. The overall conclusion is that all the parameters studied are affected by feed restriction, but the mechanisms activated at luteal level are possibly not fully adequate to compensate for nutrient shortage.     

Oxidative Stress and Interaction of Endothelin Receptors in Airways of Clinically Healthy Horses

Endothelin-1 (ET-1), a physiological as well as an inflammatory mediator, causes contraction of airway smooth muscles by binding to endothelin-A (ETA) and endothelin-B (ETB) receptors. We investigated the interaction between endothelin receptors on contractions and oxidative stress of bronchial rings of clinically healthy horses. Rings for response studies were set in tissue chambers containing oxygenated Tyrode’s solution. Concentration-response relationships were determined for ET-1 on control rings, rings incubated with ETA antagonist (BQ123), rings incubated with ETB antagonists (IRL 1038 and BQ788), and rings incubated with all antagonists combined. For oxidative stress studies, rings were incubated for 30 minutes separately with ET-1, ETA antagonist + ET-1, ETB antagonists + ET-1, and all three antagonists + ET-1. The control rings were not treated with any agents. Electron paramagnetic resonance (EPR) method was used to measure total reactive oxygen species (ROS), superoxide, and peroxynitrite. Results showed that ET-1 caused dose-dependent contractions which were increased by ETA blockade and decreased by ETB blockade. Combined blockade significantly inhibited the response to ET-1. Regarding the oxidative stress, ET-1 and its antagonists significantly reduced the total ROS. Superoxide production was significantly decreased by ETA antagonist and completely abolished when ETA and ETB antagonists were combined. Peroxynitrite production was not changed significantly. The study suggested that an interaction between ET receptors exists in the expression of contractile responses and oxidative stress. ET-1 attenuated oxidative stress by decreasing total ROS. ETA receptors may be primarily responsible for superoxide production. It appears that ET-1 does not affect peroxynitrite production in normal equine tissues.     

The yin and yang of corpus luteum-derived endothelial cells: balancing life and death

A dense network of capillaries irrigates the corpus luteum (CL) allowing an intricate cross talk between luteal steroiodgenic and endothelial cell (EC) types. Indeed, luteal endothelial cells (LEC) play pivotal roles throughout the entire CL life-span. Microvascular endothelial cells are locally specialized to accommodate the needs of individual tissues, therefore unraveling the characteristics of LEC is imperative in CL physiology. Numerous studies demonstrated that endothelium-derived endothelin-1 (ET-1) is upregulated by the luteolytic hormone-prostaglandin F2alpha (PGF2alpha) and functions as an important element of the luteolytic cascade. To have a better insight on its synthesis and action, members of ET system (ET-1, ET converting enzyme -ECE-1 and ET(A) and ET(B) receptors) were quantified in LEC. The characteristic phenotype of these cells, identified by high ET-1 receptor expression (both ET(A), ET(B)) and low ET-1 and ECE-1 levels, was gradually lost during culture suggesting that luteal microenvironment sustains the selective phenotype of its resident endothelial cells. Proper vascularization and endothelial cell activity per se are essential for normal CL function. Therefore, factors affecting vascular growth are expected to play major role in the regulation of luteal function. Concomitantly with the angiogenic process, luteal PGF2alpha and its receptors (PGFR) are induced and maintained during most of the CL life-span, suggesting a possible role of PGF2alpha in LEC proliferation and function. Dispersed LEC expressed PGFR and incubation with the prostaglandin stimulated mitogen-activated protein kinase (MAPK) signaling cascade. PGF2alpha activated p42/44 MAPK phosphorylation also in long-term cultured LEC. In this cell type, PGF2alpha increased cell number, 3H-Thymidine incorporation and cell survival. Additionally, PGF2alpha rapidly and transiently stimulated the expression of immediate-early response genes, i.e. c-fos and c-jun mRNA, further suggesting a mitogenic effect for this prostaglandin in LEC. These data imply that PGF2alpha may assume different and perhaps opposing roles depending on luteal microenvironment.     

Elevated endothelin-1 expression in dogs with heartworm disease

We explored the involvement of endothelin-1 (ET-1) in the pathophysiology of dog dirofilariasis (heartworm disease caused by Dirofilaria immitis) by analyzing mRNA levels of preproendothelin-1 (PPET-1), the precursor form of ET-1, in cardiopulmonary organs as well as ET-1 peptide levels in plasma. To determine the cDNA sequence and primary protein structure of dog PPET-1, we performed molecular cloning of the full-length cDNA. Based on the determined sequence information, comparative expression analysis of PPET-1 mRNA was carried out by real-time polymerase chain reaction on cardiopulmonary organs from healthy (n=5) and filarial (n=5) dogs. Filarial dogs showed a significantly (p<0.05) higher mRNA expression level in the heart (about one hundred times) and lung (about ten times) than healthy dogs. Analysis of plasma ET-1 levels in healthy (n=10) and filarial (n=10) dogs showed that filarial dogs (6.9+/-2.7 pg/ml) have significantly (p<0.01) increased plasma ET-1 levels compared with healthy dogs (1.4+/-0.3 pg/ml). To assess the pathophysiological significance of ET-1 in dirofilariasis relative to other cardiopulmonary disorders, plasma ET-1 levels determined in dogs diagnosed with mitral regurgitation (n=10), tricuspid regurgitation (n=5), ventricular septal defect (n=5), and patent ductus arteriosus (n=5) were compared to plasma ET-1 levels in filarial dogs. Filarial dogs, which commonly develop serious pulmonary hypertension, exhibited by far the highest ET-1 levels of the disease states examined. Based on the fact that ET-1 is a potent bioactive mediator that induces vasoconstriction and promotes vascular remodeling, these findings suggest that ET-1 plays an important role in the pathophysiology of dog dirofilariasis as an aggravating factor by inducing pulmonary hypertension.     

Up-regulation of Endothelin-1 and Endothelin type A receptor genes expression in the heart of broiler chickens versus layer chickens

To compare Endothelin (ET) production and genes expression of ET-1 and ET_A receptor (ET_AR) between broiler and layer chickens during rearing, semi-quantitative RT-PCR and enzyme immunometric assay were performed in the heart ventricles and serum. There were gradual elevations of ET-1 and ET_AR mRNAs in the left ventricle of broiler and layer chicken groups that were mainly significant (P < 0.05) at 28, 35 and 42 days of age with compared to previous days whereas were not significant between two groups.These gradual elevations of ET-1 and ET_AR mRNAs were also observed in the right ventricle that were significant (P < 0.05) at 28, 35 and 42 days of age in broilers and 42 days of age in layers with compared to previous days. Increasing of these mRNAs in the right ventricle of broiler chickens were significantly (P < 0.05) more than layer chickens at 28, 35 and 42 days. Serum ET in broilers was significantly (P < 0.05) higher than layer chickens at 28 and 42 days of age. It is concluded that circulating ET and cardiac ET-1, ET_AR genes expression is higher in broiler chickens than in layer chickens particularly after 21 days of age. It is probably that these breed differences make broiler chickens to be more susceptible to Endothelin related-cardiomyopathies such as congestive heart failure and ascites.     

A prospective study of canine infective endocarditis in northern California (1999-2001): emergence of Bartonella as a prevalent etiologic agent

A prospective study was performed (June 1999 to May 2001) to determine the incidence of infective endocarditis (IE) due to Bartonella in dogs in northern California and to compare these patients with other dogs with IE. IE was diagnosed antemortem based on clinical signs and echocardiography in 18 dogs. The etiologic agent was Bartonella sp. in 5 dogs (28%) and was diagnosed by high seroreactivity to Bartonella (titer > 1:512; range, 1:1,024-1:4,096); and confirmed postmortem by positive polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) from the infected valve and partial DNA sequencing of the citrate synthase gene (glt A). Conventional bacteria were causative agents in 7 dogs (39%). An etiologic agent was not identified in 6 dogs (33%). Bartonella vinsonii berkhoffii (n = 3), B clarridgeiae (n = 1), and a B clarridgeiae-like organism (n = 1) were identified. Blood culture was positive only for the IE case due to B clarridgeiae. All dogs with IE due to Bartonella were also seroreactive to Anaplasma phagocytophilum. All dogs with IE due to Bartonella had lesions only on the aortic valve. Of the cases of IE not due to Bartonella, 31% involved the aortic valve, 61% the mitral valve, and 8% both valves. Dogs with mitral valve IE lived longer than all dogs with aortic valve IE (P = .004) and dogs with IE of the aortic valve due to Bartonella (P = .002). In conclusion, Bartonella is a common cause of IE in dogs of northern California. A high Bartonella serologic titer (> 1:512) is useful antemortem to diagnose aortic valve IE due to Bartonella.     

Mitral valve stenosis in three cats

Two-dimensional and M-mode echocardiography were used to diagnose mitral stenosis in two cats with heart failure. This appeared to be related to mitral valve complex malformation. Ultrasound findings included thickened mitral valve leaflets with inhibited mobility, reduced mitral valve orifice size, abnormal upward (cranial) motion of the caudal mitral leaflet during diastole and severe left atrial enlargement. Colour-flow and spectral Doppler imaging helped characterise this condition. Colour-flow Doppler mapping showed turbulence and increased mitral filling velocity (aliasing) at the site of stenosis and related jets of mitral regurgitation. Spectral Doppler recordings showed increased diastolic mitral filling velocities with spectral broadening and prolonged pressure half-time. Mitral stenosis should be included in the differential diagnosis of cats with severe left atrial enlargement when congenital or acquired mitral valve disease is detected; it may represent an advanced form of mitral valve complex malformation in some cases.     

Prostaglandin F2alpha (PGF2alpha) independent and dependent regulation of the bovine luteal endothelin system

We have examined the genes of the endothelin system that are targets for regulation by prostaglandin F2alpha (PGF2alpha). The effects of a luteolytic dose of PGF2alpha ) on the mRNA encoding endothelin converting enzyme-1 (ECE-1), pre-pro endothelin-1 (pp ET-1) and the ET receptors ETA, ETB, in bovine corpus luteum (CL) during the early (days 1 and 4), mid (day 10) or late (day 17) luteal phases were examined. The effect of the PGF(2alpha) treatment on ECE-1 protein, Big ET-1 and the biologically active mature ET-1 peptide were also examined. Most importantly, the direct ECE-1 activity was determined. Before day 10 of the cycle, in a PGF2alpha-independent manner, the amounts of mRNA encoding ET-1, ECE-1, ETA, and ETB were increased steadily from day 1. After day 10 of the cycle, expression of mRNA encoding pp ET-1 and ETA acquired responsiveness to exogenous PGF2alpha and both genes were up-regulated by the PGF2alpha treatment. This effect of PGF2alpha was also detected for the proteins corresponding to the mature ET-1. The enzymatic activity of ECE-1 remained unchanged throughout the lifespan of the CL in spite of the detected changes in mRNA and protein. The results suggest that the luteal endothelin system is regulated in a PGF2alpha-independent and -dependent manner. Importantly, an alteration in luteal ET-1 availability is most likely achieved by modulating the expression of mRNA encoding pp ET-1 and not by the amount or activity of ECE-1. This interpretation is supported by the observation that the activity of ECE-1 remained unchanged throughout the ovarian cycle. The combined effects of greater ET-1 availability and gene expression encoding the ETA receptor in the late luteal phase could render the CL, at this developmental stage, more sensitive or responsive to ET-1. If the luteal tissue is responsive to the available ET-1 during the early phase of the ovarian cycle, an additional role for ET-1 should be considered beyond mediating the luteolytic actions of PGF2alpha. Agents blocking the actions of ET-1 might be the best approach to interfere with the luteal ET system and test its physiological role(s) in vivo.     

Digital blood flow and plasma endothelin concentration in clinically endotoxemic horses

OBJECTIVE: To measure plasma endothelin-1 (ET-1) concentrations and digital blood flow in clinically endotoxemic horses. ANIMALS: 36 adult horses that underwent emergency celiotomy for primary gastrointestinal tract disease. PROCEDURE: On days 2 and 5 following surgery, Doppler ultrasonographic digital arterial blood flow measurements were obtained. Hematologic and biochemical analyses were performed, and plasma concentrations of ET-1 and endotoxin (lipopolysaccharide) were determined. A scoring system based on 9 clinical variables was used to assign horses to group B (quartile with greatest cumulative score) or group A (remaining 3 quartiles). Follow-up at 2.5 years was obtained by telephone questionnaire. RESULTS: For all horses on day 2, median (interquartile values) plasma ET-1 concentrations were 1.4 (0.8, 1.7) pg/mL, whereas on day 5, plasma ET-1 concentrations were 1.0 (0.5, 1.6) pg/mL. On day 2, digital blood flow was 0.057 (0.02, 0.07) mL/min in group A horses and 0.035 (0.02, 0.03) mL/min in group B horses. On day 5, plasma ET-1 concentration was significantly (73%) higher in group B horses, compared with group A horses. Thirty of 36 horses were alive at 2.5 years; group A horses were more likely to have survived (odds ratio, 25; 95% confidence interval, 2.4 to 262). Significant associations were found between an increase in digital pulses, hoof wall temperatures, or both and increased digital blood flow (0.14 vs 0.04 mL/min) on day 2 and increased digital arterial diameter (0.32 vs 0.23 cm) on day 5. CONCLUSIONS AND CLINICAL RELEVANCE: Horses with more severe endotoxemia had decreased digital blood flow, increased plasma ET-1 concentrations, and decreased long-term survival.     

Clinical, echocardiographic, and Doppler imaging characteristics of mitral valve stenosis in two dogs.

Mitral stenosis was diagnosed noninvasively by echocardiography and Doppler imaging in 2 Bull Terriers. Two-dimensional echocardiography revealed severe atrial and moderate left ventricular dilatation; severely reduced mitral valve opening excursion; doming of the cranial mitral valve leaflet into the left ventricle during diastole; thickened, nodular cranial mitral valve leaflets; and reduced mitral valve orifice. M-mode echocardiographic findings additionally indicated greatly diminished mitral valve E to F slope and abnormal caudal mitral valve leaflet motion. Color flow Doppler imaging revealed bright bursts of color with aliasing originating from the stenotic mitral valve orifice, extending into the left atrium during systole, and into the left atrium during diastole. Spectral Doppler recordings revealed transvalvular mitral valve gradients and prolonged pressure half-times. Necropsy performed on 1 dog revealed extremely thickened, nodular, and stiff mitral valves with short, thickened, and fused chordae tendineae. The diagnosis of mitral valve stenosis was easily facilitated with diagnostic ultrasonography.     

A cooperative action of endothelin-1 with prostaglandin F(2alpha) on luteal function in the cow

Prostaglandin F(2alpha) (PGF(2alpha)) is the primary luteolysin in the cow, and luteal endothelin-1 (ET-1) interacts with PGF(2alpha) during the process of luteolysis. In contrast, a developing corpus luteum (CL) is refractory to exogenous administration of PGF(2alpha). Thus, the present study was aimed to investigate the functional relationship between ET-1 and PGF(2alpha) in the mid-CL (PGF(2alpha)-sensitive) and early-CL (PGF(2alpha)-refractory). In the mid-CL model, cows (n = 6/treatment) were assigned to receive one of five types of treatments on day 10 of the estrous cycle: (1) an injection of saline; control, (2) a 500 microg of PGF(2alpha) analogue (sufficient dose to induce luteolytis); full-PG, (3) an intraluteal injection of 0.25 mg ET-1; ET-1, (4) a 125 micro g of PGF(2alpha) (insufficient dose to induce luteolytis); 1/4PG or (5) an intraluteal injection of 0.25 mg ET-1 after administration of a insufficient dose of PGF(2alpha) analogue; 1/4PG/ET. In the early-CL model, cows were assigned to receive one of two types of treatments on day 5 of the estrous cycle: (1) a sufficient dose of PGF(2alpha) analogue; PG (n = 5) or (2) an intraluteal injection ET-1 after a sufficient dose of PGF(2alpha); PG/ET (n = 7). In the mid-CL model, 1/4PG/ET resulted in a rapid reduction of progesterone (P) concentrations similar to that in full-PG from the next day. However, the levels of P in 1/4PG/ET (1.5-2.5 ng/ml) kept significantly higher than that in full-PG (< 0.5 ng/ml). ET-1 or 1/4PG did not decrease plasma P concentrations (4-6 ng/ml). The plasma ET-1 levels increased with the full-PG administration. In the early-CL model, both treatments had no effect on plasma P increase and ET-1 levels. The overall results indicate that the intraluteal ET-1 injection after administration of insufficient dose of PGF(2alpha) induces the depression of P secretion in vivo during the mid luteal phase in the cow, supporting the concept that ET-1 is one of a local mediator of functional luteolysis in the cow. The result further indicates that the early-CL is not only PG-refractory but also ET-1-refractory.     

内皮素对哈萨克公羊生理生化指标的影响

为探究内皮素(ET)对羊生理生化指标的影响,选取5只新疆哈萨克公羊,采用不完全拉丁方试验设计,每只羊一次性静脉注射0.7nmol/kg ET-1或ET-3,对照组注射1g/L BSA生理盐水。结果显示,注射ET-1或ET-3后同对照组比,心率降低21%~36%(P0.01),红细胞总数增加10%~14%(P0.01),血红蛋白含量增加14%~17%(P0.05),红细胞比容增加14%~16%(P0.05);血糖含量增高27%~53%(P0.05),而且从这些指标改变的幅度和持续时间看,ET-1比ET-3作用强。在整个试验过程中未观察到羊呼吸频率、白细胞总数及分类计数和血浆蛋白浓度的影响(P0.05)。研究结果提示,ET可调控反刍动物心血管活动、红细胞比容,并参与血糖稳态调节,而且这些作用可能主要是通过内皮素A受体(ET-A)介导的。