Macrophage inhibitory cytokine-1/growth differentiation factor-15 as a predictor of colonic neoplasia.

BACKGROUND: Serum macrophage inhibitory cytokine-1 (MIC-1/GDF15) concentration has been associated with colonic adenomas and carcinoma. AIMS: To determine whether circulating MIC-1/GDF15 serum concentrations are higher in the presence of adenomas and whether the level decreases after excision. METHODS: Patients were recruited prospectively from a single centre and stratified into five groups: no polyps (NP); hyperplastic polyps (HP); sessile serrated ademona (SSA); adenomas (AP); and colorectal carcinoma (CRC). Blood samples were collected immediately before and 4 weeks after colonoscopy. MIC-1/GDF15 serum levels were quantified using ELISA. RESULTS: Participants (n=301) were stratified as: NP; n=116 (52%), HP; n=37 (12%), SSA; n=19 (7%), AP; n=68 (23%); and CRC; n=3 (1%). Patients were excluded from the study due to nondiagnostic pathology (n=9, 3%) and exclusion criteria (n=20, 6%). In the 272 remaining subjects (M=149; F=123), age (P=.005), history of colonic polyps (P=.003) and family history of colonic polyps (P=.002) were associated with presence of adenomas. Baseline median MIC-1/GDF15 serum levels increased significantly from NP 609 (460-797) pg/mL, HP 582 (466-852) pg/mL, SSA 561 (446-837) pg/mL to AP 723 (602-1122) pg/mL and CRC 1107 (897-1107) pg/mL; (P<.001). In the pre- and postpolypectomy paired adenoma samples median MIC-1/GDF15 reduced significantly from 722 (603-1164) pg/mL to 685 (561-944) pg/mL (P=.002). A ROC analysis for serum MIC-1/GDF15 to identify adenomatous polyps indicated an area under the curve of 0.71. CONCLUSIONS: Our data suggest that serum MIC-1/GDF15 has the diagnostic characteristics to increase the detection of colonic neoplasia and improve screening.

Identification of patterns of transmission of Salmonella within swine production systems using pulsed field gel electrophoresis (PFGE) and repetitive sequence polymerase chain reaction (REP-PCR): a quantitative analysis.

Pulsed field gel electrophoresis (PFGE) using 3 enzymes (Spe I, Xba I, Avr II) and repetitive sequence polymerase chain reaction (REP-PCR) with 3 primers (BOX, ERIC, REP) were compared with respect to their validity as a method for identifying transmission of Salmonella on swine farms. Sixty-eight isolates of Salmonella were obtained from feces of swine, cats, mice, and birds, insect body parts, water and floor samples, and boot scrapings collected on 9 swine farms in Illinois USA. Genetic distances between isolates were calculated using the Dice matching coefficient. Cluster analysis of distance matrices was conducted using the UPG-MA algorithm. There was no significant difference between PFGE and REP-PCR in the genetic diversity detected; however, REP-PCR differentiated between 14 pairs of isolates which PFGE identified as identical. There were no significant differences between PFGE and REP-PCR in identifying all or most close genetic links as isolates from the same farm, the same building, and from the same sampling visit, suggesting ecological validity for both methods. Thus, REP-PCR should be considered as an acceptable and perhaps preferable alternative to PFGE as a genotyping method for studies of Salmonella transmission.

Increased coronary effects of stimulation of endothelin-B receptor in experimental hypercholesterolemia.

BACKGROUND: Vasoconstriction in response to endothelin-1 has been shown to be primarily related to its effects on the endothelin-A receptor. Experimental hypercholesterolemia is associated with an increase in coronary vasoconstrictor response to endothelin-1 in vivo, although the relative contributions of subtypes of endothelin receptor in this model remain unknown. OBJECTIVE: To test the hypothesis that there is an increase in coronary vasoconstriction in response to stimulation of endothelin-B receptor in hypercholesterolemia, which might be related to attenuation of activity of endothelin-derived relaxing factor. METHODS: We infused 5 ng/kg/min sarafotoxin, a specific endothelin-B receptor agonist, or 50 micrograms/kg/min NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthase, into the left anterior descending coronary arteries of pigs before and after feeding them a cholesterol-rich diet for 10 weeks. RESULTS: There was a significant increase in serum level of cholesterol. After 10 weeks, infusion of sarafotoxin resulted in an accentuated decrease in coronary blood flow (CBF) compared with baseline (decreases by 60 +/- 7 versus 34 +/- 6%, P < 0.05). There was no significant difference between the effects on diameter of coronary arteries for the two time periods. The effect of L-NMMA on CBF was attenuated after 10 weeks (by 5 +/- 10.1 versus 45.6 +/- 4.7%, P < 0.05). Endothelin-receptor status of epicardial coronary arteries remained unchanged. Sarafotoxin and L-NMMA were co-infused at the above-mentioned doses into normolipidemic animals; the decrease in CBF in response to this co-infusion was comparable to the decrease observed with sarafotoxin alone in hypercholesterolemic animals (decreases of 67 +/- 5 versus 60 +/- 7, NS). CONCLUSIONS: The present results demonstrate that selective stimulation of the endothelin-B receptor increases coronary vasoconstriction in experimental hypercholesterolemia, primarily at the level of the microvasculature. These findings may be related to the attenuation of activity of endothelin-derived relaxing factor in this model, and support the hypothesis that endothelin-B receptor plays a role in the regulation of coronary vascular tone in pathophysiologic states.

Distribution and function of recombinant endothelial nitric oxide synthase in transplanted hearts.

Introducing recombinant genes into donor hearts may offer a therapeutic intervention that could potentially attenuate the complications of heart transplantation, including rejection, infection and accelerated atherosclerosis. In the cardiovascular system, reduced bioactivity of endothelial nitric oxide is a feature of atherosclerosis and vascular injury. Nitric oxide is an arterial vasodilator that also inhibits proliferation of vascular smooth muscle cells and platelet aggregation. Experiments were designed to determine the distribution of adenoviral-mediated transfer of recombinant endothelial nitric oxide synthase gene (eNOS) and the effect of recombinant gene expression on the function of transplanted hearts. Adenoviral vectors for (a) bovine eNOS (AdeNOS) or (b) beta-galactosidase (AdLacZ; control) were infused into two groups (n = 12, per group) of explanted rat hearts. The transduced hearts were then implanted heterotopically into the abdomen of syngeneic recipient rats. After four days, the hearts were excised and examined for distribution and function of the recombinant genes. Polymerase chain reaction (PCR) verified the presence of the recombinant eNOS gene in eNOS-transduced but not in beta-galactosidase-transduced hearts; reverse transcriptase-PCR identified mRNA for eNOS in AdeNOS-transduced hearts. NOS activity (conversion of tritiated L-arginine to citrulline) was greater in homogenates of AdeNOS-compared to AdLacZ-transduced hearts. Positive immunoreactivity for eNOS was present in cardiomyocytes predominantly in eNOS-transduced hearts. Myocardial contractility and coronary blood flow, as determined using a Langendorff preparation, were not different between hearts transduced with AdeNOS or AdLacZ. These results suggest that, up to four days post transplantation, adenoviral-mediated transfer of eNOS into transplanted hearts is possible. However, expression of the recombinant protein did not result in measurable changes in myocardial contractility or coronary perfusion.

Gender differences and endothelium- and platelet-derived factors in the coronary circulation.

1. Experiments were designed to determine whether or not interactions of platelets with coronary arteries are affected by gender or oestrogen-status. 2. Platelets and right coronary arteries were isolated from sexually mature male, female and ovariectomized pigs. Arteries were suspended in organ chambers for the measurement of isometric force. Responses of rings, with and without endothelium, were evaluated to aggregating platelets and the platelet products 5-hydroxytryptamine (5-HT) and adenosine diphosphate (ADP). 3. Release of 5-HT, thromboxane A2 (TXA2) and prostacyclin were measured from platelets. 4. Platelets caused relaxations of rings with endothelium from all pigs. However, in rings without endothelium, consistent contractions were observed only in rings from male pigs. 5. The release of 5-HT and prostacyclin was greatest from platelets of ovariectomized pigs compared with male and female pigs. Release of TXA2 was greatest from platelets of male pigs. 6. Endothelium-dependent relaxations to ADP and contractions to 5-HT were similar among the three groups. 7. These results suggest that there may be gender-specific differences in vasomotor responses to autogenous platelets but not to the platelet-derived products 5-HT and ADP. Furthermore, there are gender differences in platelets in the release of cyclo-oxygenase metabolites of arachidonic acid and 5-HT. These products could contribute to gender differences in response to injury in the coronary circulation.

Anesthetic management of Takayasu's arteritis for extra-anatomic thoraco-abdominal aorto-aortic bypass: a case report.

Takayasu's arteritis, also known as Takayasu's syndrome, is a chronic inflammatory disease, which primarily affects large vessels including the aorta and its main branches. The anesthetic management for the patient presenting with Takayasu's arteritis is complicated by the multiple organ systems affected by the disease. A 59-year-old Chinese female with the diagnosis of Takayasu's arteritis presented with severe progressive occlusion of the thoracic and abdominal aorta and bilateral renal artery stenosis, necessitating an extra-anatomic aorto-aortic bypass and bilateral aorto-renal bypass. The patient tolerated the operative procedure without any untoward events. This case report discusses the pathophysiology of Takayasu's arteritis and the anesthetic management of aortic surgery specific to this particular disease process.

Gender and relaxation to C-type natriuretic peptide in porcine coronary arteries.

Experiments were designed to determine whether or not relaxations of coronary arterial smooth muscle to C-type natriuretic peptide (CNP) vary according to gender, and if so, to determine mechanisms for the differences. Rings of coronary arteries without endothelium from sexually mature male and female Yorkshire pigs were suspended in organ chambers for measurement of isometric force. Cumulative concentration-responses to CNP (10(-9)-10(-7) M) were obtained in the absence and presence of either K+ channel blockers (charybdotoxin, apamine, or glibenclamide, 10(-7) M) or the clearance-receptor antagonist C-ANP (10(-6) M) during contractions to prostaglandin F2alpha (2 microM). Relaxations to CNP were significantly less in arteries from male compared with female pigs and were significantly attenuated by charybdotoxin and glibenclamide in both sexes. However, apamine reduced relaxations to CNP only in arteries from female pigs. C-ANP significantly potentiated relaxations to CNP only in arteries from male pigs. In separate experiments, cyclic guanosine monophosphate (cGMP) was measured by radioimmunoassay at specified times after the addition of CNP (10(-7) M). Peak increases in cGMP were greater and occurred earlier in arteries from female than from male pigs; these differences were eliminated by the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (10(-4) M). These results demonstrate three mechanisms that contribute to gender differences in CNP-mediated relaxation of coronary arterial smooth muscle: activation of low conductance Ca2+-activated K+ channels, natriuretic peptide clearance receptors, and activity/regulation of phosphodiesterases.

Adrenomedullin-mediated relaxations in veins are endothelium-dependent and distinct from arteries.

In arteries, adrenomedullin (ADM) causes relaxations of rings with and without endothelium by stimulating accumulation of cyclic nucleotides resulting from activation of the ADM and calcitonin gene-related peptide (CGRP) receptors. Experiments were designed to determine the mechanism(s) of relaxation to ADM in veins. Rings of canine femoral vein with and without endothelium were suspended in organ chambers for measurement of isometric force. Rings were contracted with prostaglandin F2alpha (2 x 10(-6) M), and cumulative dose-responses to ADM (10(-11) to 10(-7) M) were obtained in the absence or presence of indomethacin (10(-5) M), indomethacin + N(G)-monomethyl-L-arginine (10(-4) M), methylene blue (10(-5) M), particulate guanylate cyclase inhibitor HS-142-1 (10(-5) M), tetraethylammonium (TEA, 10(-2) M), CGRP-receptor antagonist (CGRP 8-37, 10(-6) M), ADM-receptor antagonist (ADM 26-52, 10(-6) M), diphenhydramine (10(-6) M), 8-phenyltheophylline (3 x 10(-6) M), or superoxide dismutase (150 U/ml) plus catalase (1,200 U/ml). ADM produced concentration-dependent relaxations only in veins with endothelium. Relaxations to ADM in rings with endothelium were significantly inhibited only by methylene blue and HS-142-1. In separate experiments, incubation of rings with ADM (10(-8) M) and 3-isobutyl-1-methyl-xanthine (10(-4) M) for 3 min did not significantly affect the accumulation of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP). These data suggest that ADM-mediated relaxation in veins is endothelium dependent and is not associated with activation of CGRP receptors or currently defined ADM receptors. Further, relaxations are not mediated by nitric oxide, indomethacin-sensitive prostanoids, TEA-sensitive hyperpolarizing factors, oxygen free radicals, or accumulation of cyclic nucleotides.

Gender differences in endothelium-dependent relaxations do not involve NO in porcine coronary arteries.

Experiments were designed to determine whether normal fluctuations in endogenous sex steroid hormones and/or gender affect endothelium-dependent relaxations in coronary arteries, and, if so, to identify endothelium-derived factors contributing to these differences. Coronary arteries from sexually mature, gonadally intact male and female pigs or ovariectomized pigs were prepared either for study of isometric force in organ chambers or for measurement of prostanoids and activity of nitric oxide (NO) synthase. In organ chamber studies, neither the magnitude nor the sensitivity of endothelium-dependent relaxations correlated with endogenous estrogen or progesterone in female pigs. However, relaxations to bradykinin and UK-14304 were significantly greater and/or shifted leftward in arterial rings from female compared with male pigs. Indomethacin (10(-5) mol/l) increased endothelium-dependent relaxations only in arteries from male pigs. N(G)-monomethyl-L-arginine reduced endothelium-dependent relaxations to a similar extent in coronary arteries from either sex. Neither production nor response to thromboxane A2 or prostacyclin differed in coronary arteries from male compared with female pigs. Activity for calcium-dependent or -independent NO synthase was similar in both sexes. These results suggest that normal fluctuations in endogenous sex steroid hormones do not affect endothelium-dependent relaxations in coronary arteries from female pigs. There are, however, gender differences in endothelium-dependent relaxations that are indomethacin sensitive and may be due to cyclooxygenase products other than thromboxane A2 or prostacyclin.

Gender and transcriptional regulation of NO synthase and ET-1 in porcine aortic endothelial cells.

Experiments were designed to determine whether normal fluctuations in sex steroid hormones alter gene transcription for endothelial nitric oxide synthase (NOS) and preproendothelin-1 (prepro-ET-1). Aortic endothelial cells were removed from adult, gonadally intact male and female or ovariectomized Yorkshire pigs. Endothelial cells were prepared for Northern blot analysis, Western blot analysis or enzyme activity. Nitric oxide products (NOx) and endothelin-1 (ET-1) in plasma were measured by chemiluminescence and radioimmunoassay, respectively. Northern blot analysis identified single bands corresponding to endothelial NOS and prepro-ET-1. Quantification of the blots showed an increase in expression of mRNA for both endothelial NOS and prepro-ET-1 in ovariectomized pigs compared with gonadally intact male and female pigs. There were no differences in amount of endothelial NOS protein identified by Western blot analysis among groups. On the contrary, plasma concentrations of NOx were significantly decreased in ovariectomized pigs, and there were no differences either in the concentrations of ET-1 in the plasma or extracts from the coronary arteries. These results suggest that expression of endothelial NOS and prepro-ET-1 may be regulated at transcriptional level by ovarian hormones. In addition, the ovarian hormones may regulate production of these endothelium-derived factors at the posttranscriptional level.

Adventitial gene transfer of recombinant endothelial nitric oxide synthase to rabbit carotid arteries alters vascular reactivity.

BACKGROUND: Adventitial gene transfer may serve as a tool to study vascular biology and may have therapeutic potential. We investigated the hypothesis that adenovirus-mediated transfer of the gene for endothelial nitric oxide synthase (eNOS) to the adventitia would alter vascular reactivity. METHODS AND RESULTS: Rabbit carotid arteries were surgically isolated and adenoviral vectors encoding eNOS (AdeNOS) or beta-galactosidase instilled into the periarterial sheath at a concentration of 1 x 10(10) pfu/mL. Arteries were harvested 4 days later for immunostaining, NOS enzymatic assay, measurement of cGMP, and vasomotor studies. Transgene expression in the adventitia was confirmed by histochemistry for beta-galactosidase and immunostaining for eNOS with a monoclonal antibody. Calcium-dependent NOS enzymatic activity and cGMP levels were significantly greater in the AdeNOS-transduced arteries. Maximal contractions to phenylephrine (10(-5) mol/L) were diminished in the AdeNOS-transduced arteries (4.6+/-0.2 versus 5.6+/-0.2 g; P<.05), but in the presence of the eNOS inhibitor N(G)-monomethyl-L-argininc (3x10(-4) mol/L) there was no difference between the two groups (7.1+/-0.2 versus 7.5+/-0.3 g; P=NS). Relaxations to calcium ionophore obtained during submaximal contractions to phenylephrine were significantly enhanced in the AdeNOS-transduced arteries (-log EC50, 7.77+/-0.08 versus 7.45+/-0.07; P<.02). CONCLUSIONS: We conclude that eNOS gene transfer to the adventitia alters vascular reactivity, as demonstrated by diminished contractile responses to phenylephrine and enhanced relaxations to calcium ionophore. This may represent a therapeutic strategy for vascular diseases characterized by decreased bioavailability of NO.

Enhanced endothelin-mediated coronary vasoconstriction and attenuated basal nitric oxide activity in experimental hypercholesterolemia.

BACKGROUND: Experimental hypercholesterolemia is associated with coronary vasomotor dysfunction. This study was designed to test the hypothesis that experimental hypercholesterolemia is characterized by altered coronary vasomotor responses to endothelin and inhibition of the endogenous NO pathway. METHODS AND RESULTS: Endothelin-1 (ET-1) at 5 ng x kg(-1) x min(-1) or N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthase (NOS), at 50 microg x kg(-1) x min(-1) was infused into the left anterior descending coronary artery in pigs before and after 10 weeks of cholesterol diet. There was a significant increase in serum cholesterol. At 10 weeks, ET-1 resulted in an accentuated decrease in coronary blood flow (CBF) and coronary artery diameter (CAD) compared with baseline (-88+/-6% versus -45+/-9%, P<.05, and -77+/-14% versus -18+/-8%, P<.05, respectively) and an increase in coronary vascular resistance (CVR) (242+/-18% versus 110+/-17%, P<.05); ET receptor density and binding affinity in epicardial coronary arteries were unchanged. The effect of L-NMMA on CBF, CAD, and CVR was attenuated at 10 weeks (-7+/-8% versus -48+/-4%, -2+/-3% versus -17+/-5%, and 16+/-10% versus 125+/-32%; each P<.05). Immunohistochemistry staining for constitutive NOS revealed a decrease in immunoreactivity in the coronary arteries of hypercholesterolemic pigs. CONCLUSIONS: The present study demonstrates an enhanced coronary vasoconstrictive response to pathophysiological doses of endothelin and an attenuated response to the inhibition of endogenous NO activity, suggesting an alteration in coronary vascular reactivity in experimental hypercholesterolemia.

Characterization of endothelin receptors in human varicose veins.

PURPOSE: Experiments were designed to characterize endothelin receptors in human varicose veins. Three groups of veins were studied: (1) varicose vein (VV) tributaries of the greater saphenous vein from patients who were undergoing vein stripping for primary varicosity; (2) greater saphenous veins (SVs) from the same patients; and (3) greater saphenous veins from patients without varicosity who were undergoing arterial reconstruction (control). METHODS: Veins were either cut into rings and suspended in organ chambers for measurement of isometric force, prepared for receptor binding of membrane proteins, or were prepared for measurement of preproendothelin mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Endothelin-1 (10(-11) to 10(-7) mol/L) produced similar concentration-dependent contractions in rings with or without endothelium. Maximal tensions were significantly greater in control veins compared with either SVs or VVs. Sarafotoxin S6c (10(-11) to 3 x 10(-7) mol/L), which is selective for the endothelin-B receptor, also produced concentration-dependent increases in tension in all veins. Sarafotoxin S6c responses in VVs were shifted significantly rightward compared with either SVs or control. Maximal tensions to sarafotoxin S6c also were significantly greater in control veins compared with either SVs or VVs. In receptor binding studies, the number of binding sites as defined by competitive inhibition of 125I-endothelin-1 by endothelin-1 was less in VVs than control veins. Competitive inhibition of 125I-endothelin-1 with endothelin-3 (both A and B receptors) or sarafotoxin S6c (B receptors only) suggests that the difference in receptor number between varicose and nonvaricose veins is attributable to differences in the endothelin-B receptor subtype. Binding affinities were not significantly different for either of the receptor subtypes in all veins studied. Preproendothelin mRNA as quantitated by RT-PCR tended to be higher in VVs compared with either SVs or control veins. CONCLUSIONS: Decreased contractions to endothelin-1 in both varicose and saphenous veins of patients with primary varicosity may be associated with a decrease in the number of receptors. These receptors may be downregulated in response to increased production of endothelin-1, which is regulated at the transcriptional level.

Characteristics of endothelin receptors in acutely rejecting transplanted lungs.

Experiments were designed to characterize endothelin receptors in bronchi and parenchyma of transplanted lungs during acute rejection. Third-order bronchi from autografted or allografted lungs were either cut into rings and suspended in organ chambers for the measurement of isometric force or frozen for isolation of membrane proteins. Lung parenchyma was prepared for histology or isolation of membrane protein. The grade of rejection was 2.74+/-0.17 (n= 19) in allotransplanted lungs; evidence of infection was present in 58% of the transplanted lungs. In organ chamber experiments, endothelin 1 (which stimulates endothelin A receptors) caused comparable contraction of bronchi from autotransplanted and allotransplanted rejecting lungs. Endothelin 3 (which stimulates endothelin A and B receptors) caused contractions of bronchi from autotransplanted lungs which were not different from those caused by endothelin 1. In contrast, contractions caused by endothelin 3 were reduced in bronchi from rejecting allotransplanted lungs. The magnitude of contractions caused by endothelin 3 was reduced further when infection was present with rejection. Competitive inhibition of 125I-endothelin 1 by endothelin 3 was significant for a two-site binding model in membranes prepared from all bronchi and lung parenchyma. The total number of binding sites (Bmax) was reduced significantly in bronchi and parenchyma from rejecting lungs with or without infection. The relative proportions of high-affinity and low-affinity binding sites did not change. Affinities of both high- and low-affinity receptors were not altered with rejection. These results indicate that at least two subtypes of endothelin receptors are present on canine bronchial smooth muscle and parenchyma. The number of endothelin receptors associated with bronchial contractions is reduced with rejection of lung allografts.

Endothelin receptors are modulated in association with endogenous fluctuations in estrogen.

Experiments were designed to determine whether endogenous physiological fluctuations in sex steroid hormones affect expression or functional responses of endothelin receptors. Coronary arteries from sexually mature male, female, and ovariectomized pigs were prepared either for receptor binding or measurement of isometric force in organ chambers. Competitive binding of 125I-labeled endothelin-1 was significant for a one-site model with unlabeled endothelin-1 and a two-site model with unlabeled endothelin-3 and sarafotoxin S6c in all pigs. The total number of binding sites for all endothelin ligands was not different between male and female pigs. Binding affinities for the high-affinity binding site for both endothelin-3 and sarafotoxin S6c were significantly greater (lower inhibition constant) in membranes prepared from female pigs with high endogenous estrogen. In organ chamber experiments, contractions to endothelin-1 but not endothelin-3 or sarafotoxin S6c were significantly greater in coronary arterial rings from female compared with male pigs and were not affected significantly by removal of the endothelium or by treatment of the rings with either indomethacin (10(-5) mol/l) or the combination of indomethacin and NG-monomethyl-L-arginine (10(-4) mol/l). These results suggest endogenous fluctuations in estrogen are associated with an increase in affinity of a high-affinity endothelin receptor in coronary arterial smooth muscle of female pigs. In addition, independent of endogenous estrogen status, coronary arteries from female pigs generate significantly greater contractions to endothelin-1 compared with male pigs. This phenomenon occurs at the level of smooth muscle and is not dependent on the endothelium or synthesis of nitric oxide or prostaglandins.

Modulation of endothelium-derived nitric oxide in canine femoral veins.

Experiments were designed to determine whether nitric oxide was the mediator of increased endothelium-dependent relaxations in veins proximal to an arteriovenous fistula. A fistula was prepared between femoral arteries and veins in dogs. After 6 wk, veins proximal to the fistula were removed, cut into rings, and suspended for the measurement of isometric force in organ chambers. In some rings the endothelium was removed deliberately. NG-monomethyl-L-arginine (L-NMMA) caused contraction in three of six fistula-operated veins with and without endothelium. In rings contracted submaximally with prostaglandin F2 alpha, acetylcholine and the alpha 2-adrenergic agonist UK-14,304 cause e tylcholine and the alpha 2-adrenergic agonist UK-14,304 caused endothelium-dependent, concentration-dependent relaxations that were greater in fistula compared with sham-operated veins. These relaxations were reduced by L-NMMA. Calcium ionophore A23187 caused comparable endothelium-dependent relaxations in fistula- and sham-operated veins that were unaffected by L-NMMA. There were no differences in either calcium-dependent or -independent activity of nitric oxide synthase isolated from fistula- and sham-operated veins. Positive staining for nitric oxide synthase was present in both the endothelium and media of fistula-operated veins. These results indicate that nitric oxide mediates increased endothelium-dependent relaxations to acetylcholine and alpha 2-adrenergic agonists in fistula-operated veins. Therefore, chronic increases in blood flow and oxygen tension modify selectively receptor-coupled production of nitric oxide in endothelium and smooth muscle of veins.

Gender differences in response to endothelin-1 in coronary arteries: transcription, receptors and calcium regulation.

1. Experiments were designed to determine how sex hormonal status may influence production and response to endothelins. 2. Circulating concentrations of endothelin-1 were not different between sexually mature male and female pigs and did not differ between females of low- and high-oestrogen status. 3. Expression of messenger RNA for preproendothelin was not different in aortic endothelial cells derived from sexually mature male and female pigs. 4. Endothelin-1, endothelin-3 and sarafotoxin S6c caused concentration-dependent contractions of rings of coronary arteries suspended for the measurement of isometric force. Only in response to endothelin-1 were contractions greater in arteries from female than in male pigs. This difference was not related to oestrogen levels in females. 5. The intrinsic force-calcium relationship defined in smooth muscle cells permeabilized with either triton-X or beta-escine was not different in coronary arteries from male and female pigs. The calcium sensitivity was increased to the same extent by endothelin-1 in smooth muscle from male and female pigs. 6. Competitive inhibition of radiolabelled endothelin-1 by unlabelled endothelin-1 was not different in membranes prepared from coronary arteries from male and female pigs. However, competitive inhibition of radiolabelled endothelin-1 by unlabelled endothelin-3 showed two binding sites. The affinity of the high affinity binding site was increased only in females with high oestrogen levels. 7. These results suggest that there are gender differences in contractions of porcine coronary arteries to endothelin-1. These differences may not relate to transcriptional regulation for the production of the peptide in endothelial cells, to the regulation of the number or affinity of endothelin receptors or to intrinsic calcium-sensitivity of the contractile proteins. Rather they are probably due to differences in the regulation of intracellular calcium.

Chronic increases in blood flow upregulate endothelin-B receptors in arterial smooth muscle.

Experiments were designed to characterize endothelin receptors in arteries after chronic increases in blood flow. A fistula was created between the femoral artery and vein in one hindlimb of dogs; contralateral blood vessels were sham operated. Sham- and fistula-operated arteries were removed 6 wk postoperatively. Some arteries were prepared for measurement of isometric force or for isolation of membrane proteins. Other arteries were used for histological staining with an endothelin-B (ETB) receptor antibody. In arteries suspended for the measurement of isometric force, endothelin-1 produced concentration-dependent increases in tension that were significantly greater in fistula- than in sham-operated arteries without endothelium. The ETB-receptor-selective peptide sarafotoxin S6c produced concentration-dependent increases in tension only in fistula-operated arteries. In receptor-binding studies of membrane proteins, Scatchard analysis of saturation binding with 125I-labeled endothelin-1 (125I-endothelin-1) indicated that the total number of receptors was greater in fistula-operated arteries; affinity was threefold less in fistula- than in sham-operated arteries. Competitive displacement of 125I-endothelin-1 by endothelin-3 was significant for a two-site model in membranes prepared from sham-and fistula-operated arteries. Competitive inhibition of 125I-endothelin-1 binding by sarafotoxin S6c was significant for a one-site binding model in all arteries. Sarafotoxin S6c binding sites were elevated significantly in fistula-operated arteries. Immunohistochemical staining for the ETB receptor was significantly greater in both the endothelium and smooth muscle of fistula- than in sham-operated arteries. These results suggest that chronic increases in blood flow upregulate endothelin receptors, including ETB receptors in arterial smooth muscle.