Atriopeptin 2 is hydrolysed by cardiac but not pulmonary isozyme of angiotensin-converting enzyme

Hydrolysis of Bz-Gly-Ser-Phe-Arg, C-terminal fragment of atriopeptin 2, by human cardiac angiotensin-converting enzyme has been studied. The KM for the reaction was 10(-4) M. The effect of concentration of NaCl on activity of cardiac angiotensin-converting enzyme has been determined, which allowed to regard Bz-Gly-Ser-Phe-Arg as bradykinin-like substrates. It was demonstrated that cardiac, but not pulmonary isozyme of angiotensin-converting enzyme specifically hydrolyses atriopeptin 2.     

Inhaled NO inhibits platelet aggregation and elevates plasma but not intraplatelet cGMP in healthy human volunteers

Effects of inhaled nitric oxide (NO) on human platelet function are controversial. It is uncertain whether intraplatelet cGMP mediates the effect of inhaled NO on platelet function. We investigated the effect of 30 ppm inhaled NO on platelet aggregation and plasma and intraplatelet cGMP in 12 subjects. We performed platelet aggregation studies by using a photooptical aggregometer and five agonists (ADP, collagen, epinephrine, arachidonic acid, and ristocetin). During inhalation, the maximal extent of platelet aggregation decreased by 75% with epinephrine (P < 0.005), 56% with collagen (P < 0.005), and 20% with arachidonic acid (P < 0.05). Responses to ADP (8% P > 0.05) and ristocetin (5% P > 0.05) were unaffected. Platelet aggregation velocity decreased by 64% with collagen (P < 0.005), 60% with epinephrine (P < 0.05), 33% with arachidonic acid (P < 0.05), and 14% with ADP (P > 0.05). Plasma cGMP levels increased from 2.58 +/- 0.43 to 9.99 +/- 5.57 pmol/ml (P < 0.005), intraplatelet cGMP levels were unchanged (means +/- SD: 1.96 +/- 0.58 vs. 2.71 +/- 1.67 pmol/109 platelets; P > 0.05). Inhaled NO inhibits platelet aggregation via a cGMP independent mechanism.     

Atriopeptin II elevates cyclic GMP, activates cyclic GMP-dependent protein kinase and causes relaxation in rat thoracic aorta

Synthetic atriopeptin II, an atrial natriuretic factor with potent vasodilatory effects, was studied in isolated strips of rat thoracic aorta to determine its actions on contractility, cyclic nucleotide concentrations and endogenous activity of cyclic nucleotide-dependent protein kinases. Atriopeptin II was found to relax aortic strips precontracted with 0.3 microM norepinephrine whether or not the endothelial layer was present. Relaxation to atriopeptin II was closely correlated in a time- and concentration-dependent manner with increases in cyclic GMP concentrations and activation of cyclic GMP-dependent protein kinase (cyclic GMP-kinase). The threshold concentration for all three effects was 1 nM. Atriopeptin II (10 nM for 10 min) produced an 80% relaxation, an 8-fold increase in cyclic GMP concentrations and a 2-fold increase in cyclic GMP-kinase activity ratios. Atriopeptin II did not significantly alter cyclic AMP concentrations or cyclic AMP-dependent protein kinase activity. These data suggest that cyclic GMP and cyclic GMP-kinase may mediate vascular relaxation to a new class of vasoactive agents, the atrial natriuretic factors. Similar effects have been observed with the nitrovasodilator, sodium nitroprusside, and the endothelium-dependent vasodilator, acetylcholine. Therefore, a common biochemical mechanism of action that includes cyclic GMP accumulation and activation of cyclic GMP-kinase may be involved in vascular relaxation to nitrovasodilators, endothelium-dependent vasodilators and atrial natriuretic factors.     

Amplification of sumatriptan-induced contraction in rabbit saphenous vein but not in basilar artery

The modulation of serotonin (5-HT(1B/1D)) receptor-induced vascular contractility by histamine and U-46619 was compared in the rabbit basilar artery and saphenous vein. In the saphenous vein, histamine (5 x 10(-7) M) significantly increased the potency (from pEC(50) of 6.0 to 6.8) and efficacy (from 52.3% to 88.2%) of sumatriptan. Likewise, U-46619 (5 x 10(-9) M) also increased the potency (from pEC(50) of 5.9 to 6.6) and efficacy (from 51.8% to 92.1%) of sumatriptan in the saphenous vein. In contrast, equieffective concentrations of histamine (10(-7) M) and U-46619 (3 x 10(-9) M) failed to amplify contraction to sumatriptan in the basilar artery. Contraction to sumatriptan was inhibited by nitrendipine (10(-7) M) in the basilar artery but not in the saphenous vein, suggesting that different contractile signaling mechanisms are operating in these tissues. Furthermore, U-46619- and thrombin-induced contractility in the basilar artery were also not amplified by histamine, suggesting that lack of amplification of contraction in the basilar artery was not restricted to sumatriptan but was rather a characteristic of this cerebral vessel. These data suggest that in the in vivo plasma milieu sumatriptan will more markedly contract the peripheral saphenous vein than the basilar artery, a cerebral blood vessel.     

Domperidone elevates rat plasma beta-endorphin-immunoreactivity when administered peripherally but not intracerebroventricularly

Domperidone, a dopamine receptor antagonist which apparently does not penetrate the blood-brain barrier in rats was administered to adult males. Domperidone 500 micrograms and 100 micrograms, given through intracarotid cannula, significantly elevated plasma beta-endorphin-immunoreactivity (beta-EP-I) at +15 min. To show that only a peripheral site(s) of action is implicated, domperidone was given to rats by cannulae implanted into both lateral ventricles. Plasma beta-EP-I was unaffected by this route of administration. These results suggest that plasma beta-EP-I is tonically inhibited by dopamine acting at site(s) outside of the blood-brain barrier.     

A comparative study of glyceryl trinitrate biotransformation and glyceryl trinitrate induced relaxation in bovine pulmonary artery and vein

Recent evidence supports the hypothesis that the mechanism by which glyceryl trinitrate induces relaxation of vascular smooth muscle involves the biotransformation of glyceryl trinitrate. This study was conducted to determine if there was a direct correlation between the capacity of vascular smooth muscle preparations to biotransform glyceryl trinitrate and their sensitivity to the relaxant effect of this organic nitrate. Isolated bovine pulmonary arteries and veins were contracted submaximally and cumulative dose-response relationships to glyceryl trinitrate were obtained; the vein was approximately 10 times more sensitive than the artery to glyceryl trinitrate induced relaxation. In a separate series of experiments, these vascular tissues were contracted submaximally and incubated with 0.5 microM [14C]glyceryl trinitrate for 2 min, during which glyceryl trinitrate induced relaxation was monitored. At 2 min, tissue samples were taken for determination of glyceryl trinitrate and glyceryl-1,2- and 1,3-dinitrate content by thin-layer chromatography and liquid scintillation spectrometry. Biotransformation of glyceryl trinitrate to glyceryl dinitrate occurred concomitantly with relaxation of these blood vessels. The concentration of glyceryl dinitrate in the vein was significantly less than that in the artery (p less than or equal to 0.05), even though significantly greater relaxation of the vein than the artery was observed (p less than or equal to 0.05). From these data, a simple linear relationship between glyceryl trinitrate biotransformation and relaxation is not apparent.     

Manganese superoxide dismutase is not protective in bovine pulmonary artery endothelial cells at systemic oxygen levels

Bovine pulmonary artery endothelial cells (BPAEC) are extremely sensitive to oxygen, mediated by superoxide production. Ionizing radiation is known to generate superoxide in oxygenated aqueous media; however, at systemic oxygen levels (3%), no oxygen enhancement is observed after irradiation. A number of markers (cell growth, alamarBlue, mitochondrial membrane polarization) for metabolic activity indicate that BPAEC maintained under 20% oxygen grow and metabolize more slowly than cells maintained under 3% oxygen. BPAEC cultured in 20% oxygen grow better when they are transiently transfected with either manganese superoxide dismutase (MnSOD) or copper zinc superoxide dismutase (CuZnSOD) and exhibit improved survival after irradiation (0.5-10 Gy). Furthermore, X irradiation of BPAEC grown in 20% oxygen results in very diffuse colony formation, which is completely ameliorated by either growth in 3% oxygen or overexpression of MnSOD. However, MnSOD overexpression in BPAEC grown in 3% oxygen provides no further radioprotection, as judged by clonogenic survival curves. Radiation does not increase apoptosis in BPAEC but inhibits cell growth and up-regulates p53 and p21 at either 3% or 20% oxygen.     

Acute systemic hypoxia elevates venous but not interstitial potassium of dog skeletal muscle

Potassium release through ATP-sensitive potassium (K(ATP)) channels contributes to hypoxic vasodilation in the skeletal muscle vascular bed: It is uncertain whether K(ATP) channels on muscle cells contribute to the process. Potassium from muscle cells must cross the interstitial space to reach the vascular tissues, whereas that from vascular endothelium would have a higher concentration in venous blood than in interstitial fluid. We determined the effect of systemic hypoxia on arterial, venous, and interstitial potassium in the constant-flow-perfused gracilis muscles of anesthetized dogs. Hypoxia reduced arterial Po(2) from 138 to 25 and Pco(2) from 28 to 26 mmHg. Arterial pH and potassium were well correlated (r(2) = 0.9): Both increased in early hypoxia and decreased during the postcontrol. In denervated muscles, perfusion pressure decreased from 95 to 76 mmHg by the end of the hypoxic period; neither venous nor interstitial potassium was elevated. In innervated muscles, perfusion pressure increased from 110 to 172 mmHg by the 11th min of hypoxia and then decreased to 146 mmHg by the end of the hypoxic period; venous potassium increased from 5.0 to 5.3 mM, but interstitial potassium remained unchanged. Glibenclamide abolished both the increase in venous potassium and the hypoxic vasodilation in the innervated muscle. Thus skeletal muscle cells were unlikely to have contributed to the release of potassium, which was suggested to originate from vascular endothelium. The sympathetic nerve supply may play a direct or indirect role in the opening of K(ATP) channels under hypoxic conditions.     

Femoral arterial injection of adenosine in humans elevates MSNA via central but not peripheral mechanisms

MacLean, D. A., B. Saltin, G. Rådegran, and L. Sinoway. Femoral arterial injection of adenosine in humanselevates MSNA via central but not peripheral mechanisms.J. Appl. Physiol. 83(4):1045-1053, 1997.The purpose of the present study was to examinethe effects of femoral arterial injections of adenosine on musclesympathetic nerve activity (MSNA) under three different conditions.These conditions were adenosine injection alone, adenosine injectionafter phenylephrine infusion, and adenosine injection distal to a thighcuff inflated to arrest the circulation. The arterial injection ofadenosine alone resulted in a fourfold (255 ± 18 U/min) increaseabove baseline (73 ± 12 U/min; P < 0.05) in MSNA with an onset latency of 15.8 ± 0.8 s from thetime of injection. The systemic infusion of phenylephrine resulted in an increase (P < 0.05) in meanarterial pressure of ~10 mmHg and a decrease(P < 0.05) in heart rate of8-10 beats/min compared with baseline values before phenylephrineinfusion. After adenosine injection, the onset latency for the increasein MSNA was delayed to 19.2 ± 2.1 s and the magnitude of increasewas attenuated by ~50% (123 ± 20 U/min) compared with adenosineinjection alone (P < 0.05). When acuff was inflated to 220 mmHg to arrest the circulation and adenosinewas injected into the leg distal to the inflated cuff, there were nosignificant changes in MSNA or any of the other measured variables.However, on deflation of the cuff, there was a rapid increase(P < 0.05) in MSNA, with an onsetlatency of 9.1 ± 0.9 s, and the magnitude of increase (276 ± 28 U/min) was similar to that observed for adenosine alone. These datasuggest that ~50% of the effects of exogenously administered adenosine are a result of baroreceptor unloading due to a drop in bloodpressure. Furthermore, the finding that adenosine did not directlyresult in an increase in MSNA while it was trapped in the leg but thatit needed to be released into the circulation suggests that adenosinedoes not directly stimulate thin fiber muscle afferents in the leg ofhumans. In contrast, it would appear that adenosine exerts its effectsvia some other chemically sensitive pool of afferents.

     

Exercise training elevates RMR during moderate but not severe dietary restriction in obese male rats

This study examined the effects of 11 wk of exercise training (E) on resting O2 uptake (RMR) and the composition of diet-induced weight loss in obese male Sprague-Dawley rats (n = 48). The rats underwent one of three levels of dietary treatment: ad libitum (AL), moderate restriction (MR), and severe restriction (SR). Compared with AL-CC (cage confined; 647 +/- 13 g), the AL-E (84% of AL-CC), MR-CC (77%), MR-E (77%), SR-CC (63%), and SR-E (63%) groups were all reduced in body mass. At the MR level, E significantly reduced fat mass (FM; MR-CC 73 +/- 5, MR-E 45 +/- 5 g) and increased water mass (WM; MR-CC 307 +/- 5, MR-E 329 +/- 5 g) compared with CC. In contrast, no significant differences existed between the SR-CC and SR-E groups. Exercise training did not result in conservation of protein mass at any level of dietary intake. Exercise training significantly elevated RMR (on a kg0.75 basis) by approximately 7% for the AL-E and MR-E groups compared with their respective dietary counterparts. These findings may help reconcile the conflicting results present in the literature with respect to the effects of exercise training during diet-induced weight loss. That is, studies using relatively severe dietary restrictions are less likely to demonstrate exercise-induced changes in RMR, FM, and WM than those employing moderate dietary restrictions.     

The ANF-C receptor is not linked to adenylyl cyclase inhibition in bovine pulmonary artery endothelial cells.

The possible inhibition of adenylyl cyclase activity by atrial peptides selective for the ANF-C receptor was investigated in bovine pulmonary artery endothelial cells. In these cells isoprenaline, guanine nucleotide and forskolin dose-dependently increased activity over basal levels. In the presence of rANF(99-126), these dose-dependent increases were not reduced, nor were they affected by the ANF-C receptor selective analogue C-ANF(102-121). Furthermore, the selective analogues rANF(103-123) and des[Cys105,Cys121]rANF104-126 had no effect on basal or stimulated adenylyl cyclase activity. It can be concluded that ANF-C receptors are not linked to inhibition of adenylyl cyclase in these cells.     

Zinc-related metallothionein metabolism in bovine pulmonary artery endothelial cells

Bovine pulmonary artery endothelial cells (BPAEC) were cultured in vitro under a variety of conditions to investigate how metallothionein (MT) might participate in zinc homeostasis. Experimental conditions included 10% serum to ensure that the in vitro environment would be a better reflection of the in vivo situation than with protein-free medium. MT was increased by acutely high zinc concentrations (100-200 micromol/L) in the extracellular environment. MT was relatively insensitive to moderate changes in zinc concentration (2-50 micromol/L), even after prolonged exposure for 7 to 12 days. BPAEC had reduced MT content when grown in medium containing serum that had been dialyzed to remove components with a molecular mass of less than 1,000, including zinc. Because the principal source of the major minerals in the experimental medium was not the serum, their concentrations in the final medium were not significantly influenced by serum dialysis. Restoring the zinc concentration in the medium containing the dialyzed serum did not restore MT content in BPAEC, suggesting that some small molecular weight molecule other than zinc established their basal MT content. This study did not identify these putative factors in serum, but hormones are likely candidates. Forty-eight-hour incubations of BPAEC with interleukin (IL-6) or dexamethasone increased cellular MT; however, 17beta-estradiol decreased MT, and IL-1 and adenosine 3',5'-cyclic phosphate (cAMP) had no discernible effect. We conclude that extracellular zinc concentrations have relatively little impact on the cellular concentrations of MT and zinc of BPAEC in vitro. Zinc homeostasis by BPAEC is not maintained by changing the MT concentration in response to changes in the extracellular zinc environment. (J. Nutr. Biochem. 10:00-00, 1999).     

Angiotensin II but not potassium induces subcellular redistribution of protein kinase C in bovine adrenal glomerulosa cells

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) between cytosol and membrane fractions was examined in bovine adrenal glomerulosa cells treated with angiotensin II or potassium. Protein kinase C was isolated from cytosol and from detergent-solubilized particulate fractions by DEAE-cellulose chromatography. A major peak of activity for both the soluble and particulate forms of adrenal glomerulosa protein kinase C was eluted at 0.05-0.09 M NaCl. The soluble and particulate forms were found to constitute about 95 and 5%, respectively, of the total enzyme activity in unstimulated cells. A second peak of kinase activity was eluted with 0.15-0.19 M NaCl, which was not dependent on the presence of phospholipids. Exposure of isolated cells for 20 min to 10(-8) M angiotensin II resulted in a decrease in cytosolic activity to 30-40% of control values, and in a corresponding increase in protein kinase C activity associated with the particulate fraction. This hormone-induced redistribution was found to be dose-dependent with an ED50 of 2 nM for angiotensin II, and it occurred rapidly, reaching a plateau within 5-10 min. It was prevented by the specific antagonist [Sar1,Ala8]angiotensin II. By contrast, stimulation with 12 mM KCl did not change the subcellular distribution of protein kinase C activity. These results suggest that redistribution of protein kinase C represents an early step in the post-receptor activation cascade following angiotensin II, but not potassium stimulation of adrenal glomerulosa cells.     

Targeted blocking of gene expression for CGRP receptors elevates pulmonary artery pressure in hypoxic rats

We previously described the protection by calcitonin gene-related peptide (CGRP) against hypoxic pulmonary hypertension. Here, we examine the roles of its putative receptor RDC-1 and receptor activity-modifying protein (RAMP) 1 in mediating this protection by selectively inhibiting their synthesis. RAMP1 is an accessory protein for another putative CGRP receptor, calcitonin receptor-like receptor. Antisense oligodeoxyribonucleotides (ASODNs, 5 mg.kg-1.day-1 or 5 and 10 mg.kg-1.day-1 for RDC-1) targeting RAMP1 and RDC-1 mRNAs were chronically infused to the pulmonary circulation of male Sprague-Dawley rats during 7 days of normoxia or hypobaric hypoxia (380 mmHg), and alpha-CGRP ASODN was used as a technical control. CGRP, RAMP1, and RDC-1 ASODNs significantly elevated pulmonary artery pressure (PPA) in chronic hypoxic rats compared with hypoxic mismatched ASODN (MMODN) and saline vehicle controls. CGRP and RAMP1 ASODNs raised PPA in normoxic rats briefly exposed to 10% O2 above MMODN and saline controls. Moreover, normoxic rats treated with CGRP ASODN had higher basal pulmonary vascular tone compared with controls. These data confirm the protective role of CGRP in the pulmonary circulation and suggest that endogenous RAMP1 and RDC-1 are essential in regulation of PPA in hypoxia. This is the first in vivo evidence supporting RDC-1 and RAMP1 as functional CGRP receptor and receptor component.     

Distribution of the pulmonary artery and vein in the chimpanzee lung

Lungs of two chimpanzees (Pan troglodytes) were examined. The right pulmonary artery runs across the ventral side of the right upper lobe bronchiole and, then across the dorsal side of the right middle lobe bronchiole. Thereafter, it runs between the dorsal bronchiole system and the lateral bronchiole system, along the right bronchus. During its course, it gives off arterial branches which run along each bronchiole. The left pulmonary artery runs across the dorsal side of the left middle lobe bronchiole and then between the dorsal bronchiole system and the lateral bronchiole system. The branches of the pulmonary artery run mainly along the dorsal or lateral side of the bronchiole. The pulmonary veins run mainly along the ventral or medial side of the bronchioles, and between them. Finally, they enter the left atrium with four large veins, i.e. the common trunk of the right upper lobe vein and the right middle lobe vein, right lower lobe pulmonary venous trunk, left middle lobe vein, and left lower lobe pulmonary venous trunk.     

NO responsiveness in pulmonary artery and airway smooth muscle: the role of cGMP regulation

The purpose of this study was to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Canine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used to perform concentration response studies to an NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM exhibited a greater NO responsiveness whether PASM and TSM were contracted with receptor agonists, phenylephrine and acetylcholine, respectively, or with KCl. The >10-fold difference in NO sensitivity in PASM was observed with both submaximal and maximal contractions. This difference in NO responsiveness was not due to differences in endothelial or epithelial barriers, since these were removed, nor was it due to the presence of cGMP-independent NO-mediated relaxation in either tissue. At equal concentrations of NO, the intracellular cGMP concentration ([cGMP]i) was also greater in PASM than in TSM. Phosphodiesterase (PDE) inhibition using isobutylmethylxanthine indicated that the greater [cGMP]i in PASM was not due to greater PDE activity in TSM. Expression of soluble guanylate cyclase (sGC) subunit mRNA (2 +/- 0.2 and 1.3 +/- 0.2 attomol/microg total RNA, respectively) and protein (47.4 +/- 2 and 27.8 +/- 3.9 ng/mg soluble homogenate protein, respectively) was greater in PASM than in TSM. sGCalpha1 and sGCbeta1 mRNA expression was equal in PASM but was significantly different in TSM, suggesting independent regulation of their expression. An intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is greater sGC activity.