Parathyroid hormone-related protein-mediated responses in pulmonary arteries and veins of newborn lambs

PTHrP has important roles in lung development and function. Here we determined the vasomotor responses of isolated pulmonary arteries and veins of newborn and adult sheep to PTHrP. In vessels constricted with endothelin-1, PTHrP (PTHrP 1-34) caused greater relaxation of veins than of arteries. In both vessel types, relaxation to the peptide was less in adult than in newborn vessels. In newborn lambs, PTHrP-induced relaxation was not affected by endothelium removal, inhibition of eNOS, or inhibition of adenylyl cyclases by SQ-22536. However, relaxation was attenuated by 4-aminopyridine, inhibitor of voltage-dependent potassium channels, in both arteries and veins, and by charybdotoxin, inhibitor of calcium-activated potassium channels, in veins. When vessels were saturated with 8-BrcAMP (3 x 10(-4) M), to eliminate relaxation mediated by endogenous cAMP, PTHrP-induced relaxation was partially attenuated. In vessels treated with 8-BrcAMP (3 x 10(-4) M), 4-aminopyridine but not charybdotoxin inhibited relaxation induced by PTHrP 1-34 in both arteries and veins. Radioimmunoassay showed that, in the presence of a general phosphodiesterase inhibitor, PTHrP caused a concentration-dependent increase in intracellular cAMP content in arteries and veins, which was largely abolished by SQ-22536. Our results demonstrate that PTHrP is a potent vasodilator of pulmonary vessels, with a greater effect in veins than in arteries. Relaxation induced by the peptide contains both cAMP-dependent and -independent components. In both arteries and veins, voltage-dependent potassium channels mediate the response to PTHrP, at least in part, in a cAMP-independent fashion; and in veins, calcium-activated potassium channels may be stimulated by elevated cAMP levels.     

Developmental change in isoproterenol-mediated relaxation of pulmonary veins of fetal and newborn lambs

-Adrenergic agonists are important regulatorsof perinatal pulmonary circulation. They cause vasodilation primarilyvia the adenyl cyclase-adenosine 3',5'-cyclic monophosphate(cAMP) pathway. We examined the responses of isolated fourth-generationpulmonary veins of term fetal (145 ± 2 days gestation)and newborn (10 ± 1 days) lambs to isoproterenol, a -adrenergicagonist. In vessels preconstricted with U-46619 (a thromboxaneA₂ analog), isoproterenol inducedgreater relaxation in pulmonary veins of newborn lambs than in those offetal lambs. The relaxation was eliminated by propranolol, a-adrenergic antagonist. Forskolin, an activator of adenyl cyclase,also caused greater relaxation of veins of newborn than those of fetallambs. 8-Bromoadenosine 3',5'-cyclic monophosphate, a cellmembrane-permeable analog of cAMP, induced a similar relaxation of allvessels. Biochemical studies show that isoproterenol and forskolininduced a greater increase in cAMP content and in adenyl cyclaseactivity of pulmonary veins in the newborn than in the fetal lamb.These results demonstrate that -adrenergic-agonist-mediatedrelaxation of pulmonary veins increases with maturation. An increase inthe activity of adenyl cyclase may contribute to the change.

     

Prostaglandin E2 causes hypoventilation and apnea in newborn lambs

To test the hypothesis that prostaglandin (PG) E2 is a respiratory depressant in the newborn lamb, 12 chronically catheterized, unanesthetized lambs (age 2-6 days) were infused with progressively increasing doses of PGE2 (0.1, 0.5, 1.0, and 5.0 micrograms.kg-1.min-1; 30 min for each dose) into the ascending aorta. PGE2 caused significant progressive decreases in ventilation (due to decreased tidal volume and breathing rate), heart rate, blood pressure, and percent of the time spent in low-voltage electrocortical activity (LVA). PGE2 also caused respiratory acidosis, hypoxemia, and increased frequency and duration of apneic events (greater than 3 s). During the infusion there was a dose-related increase in plasma concentration of PGE2. At 30 min postinfusion, all measured variables showed recovery, although arterial pH, CO2 tension, and plasma PGE2 remained significantly different from control values, and the percent time in LVA was even higher than during control. Infusion of the vehicle alone (n = 5) caused no significant changes in any of the measured variables. The results, taken in combination with previous fetal studies, indicate that PGE2 has marked inhibitory effects on breathing movements both before and after birth.     

Altered endothelium-dependent relaxations in lambs with high pulmonary blood flow and pulmonary hypertension

Congenital heart disease associated with increased pulmonary blood flow produces pulmonary hypertension. To characterize vascular alterations in the nitric oxide (NO)-cGMP cascade induced by increased pulmonary blood flow and pulmonary hypertension, 10 fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). When the lambs were 4-6 wk of age, we assessed responses of pulmonary arteries (PAs) and pulmonary veins (PVs) isolated from lungs of control and shunted lambs. PVs from control and shunted lambs relaxed similarly to exogenous NO (S-nitrosyl-acetyl-penicillamine), to NO produced endogenously (zaprinast and A-23187), and to cGMP (atrial natriuretic peptide). In contrast, relaxations to A-23187 and zaprinast were blunted in PAs isolated from shunted lambs relative to controls. Inhibitors of NO synthase (NOS) and soluble guanylate cyclase constricted control but not shunt PAs, indicating reduced basal NOS activity in shunt PAs. Pretreatment of shunt PAs with the substrates L-arginine and sepiapterin, a precursor for tetrahydrobiopterin synthesis, did not augment A-23187 relaxations. However, pretreatment with superoxide dismutase and catalase significantly enhanced A-23187 relaxations in shunt PAs. We conclude that increased pulmonary blood flow induces an impairment of endothelium-dependent relaxation that is selective to PAs. The impaired relaxation may be mediated in part by excess superoxide production.     

Role of cGMP-dependent protein kinase in development of tolerance to nitric oxide in pulmonary veins of newborn lambs

Continuous exposure to nitrovasodilators and nitric oxide induces tolerance to their vasodilator effects in vascular smooth muscle. This study was done to determine the role of cGMP-dependent protein kinase (PKG) in the development of tolerance to nitric oxide. Isolated fourth-generation pulmonary veins of newborn lambs were studied. Incubation of veins for 20 h with DETA NONOate (DETA NO; a stable nitric oxide donor) significantly reduced their relaxation response to the nitric oxide donor and to beta-phenyl-1,N2-etheno-8-bromo-cGMP (8-Br-PET-cGMP, a cell-permeable cGMP analog). Incubation with DETA NO significantly reduced PKG activity and protein and mRNA levels in the vessels. These effects were prevented by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) and Rp-8-Br-PET-cGMPS (an inhibitor of PKG). A decrease in PKG protein and mRNA levels was also observed after continuous exposure to cGMP analogs. The PKG inhibitor abrogated these effects. The decrease in cGMP-mediated relaxation and in PKG activity caused by continuous exposure to DETA NO was not affected by KT-5720, an inhibitor of cAMP-dependent protein kinase. Prolonged exposure to 8-Br-cAMP (a cell-permeable cAMP analog) did not affect PKG protein level in the veins. These results suggest that continuous exposure to nitric oxide or cGMP downregulates PKG by a PKG-dependent mechanism. Such a negative feedback mechanism may contribute to the development of tolerance to nitric oxide in pulmonary veins of newborn lambs.     

Zero-stress states of human pulmonary arteries and veins

The zero-stressstates of the pulmonary arteries and veins fromorder3 toorder9 were determined in six normal humanlungs within 15 h postmortem. The zero-stress state of each vessel was obtained by cutting the vessel transversely into a series of short rings, then cutting each ring radially, which caused the ring to springopen into a sector. Each sector was characterized by its opening angle.The mean opening angle varied between 92 and 163° in the arterialtree and between 89 and 128° in the venous tree. There was atendency for opening angles to increase as the sizes of the arteriesand veins increased. We computed the residual strains based on theexperimental measurements and estimated the residual stresses accordingto Hooke's law. We found that the inner wall of a vessel at the statein which the internal pressure, external pressure, and longitudinalstress are all zero was under compression and the outer wall was intension, and that the magnitude of compressive stress was greater thanthe magnitude of tensile stress.

     

Prostaglandins E1 and E2 stimulate the proliferation of pulmonary artery smooth muscle cells.

Prostaglandins E1 and E2 are thought to be inhibitors of the growth of systemic vascular smooth muscle cells (SMC). However, their effect on the proliferation of SMC from the pulmonary artery (PA) has not been described and was the subject of this investigation. Cultures of bovine PA SMC were exposed to PGE1 and PGE2 under various conditions and their growth was assessed. PGE1 and PGE2 did not inhibit the growth of PA SMC in 10% fetal calf serum (FCS), but instead caused a dose dependent (10 ng - 1 microgram/ml) increase in [3H]-thymidine incorporation when added to cultures containing 0.5% FCS; the highest doses resulted in 95% and 75% increases in [3H]-thymidine uptake at 24 hours with PGE1 and PGE2 respectively. This was accompanied by a modest increase in actual cell numbers (e.g., 20% with 1 microgram/ml PGE1). Furthermore, PGE1 could mimic insulin-like growth factor (IGF-1) by potentiating the stimulation of SMC growth by fibroblast growth factor, suggesting that PGE1 may act as a progression factor in the growth cycle of these cells. There was, however, no effect of PGE1 on the proliferation of bovine aortic SMC. We conclude that, contrary to most reported effects on systemic SMC, PGE1 and PGE2 do not inhibit the proliferation of PA SMC but rather stimulate it.     

Development of pulmonary intravascular macrophage function in newborn lambs.

We sought to determine whether pulmonary intravascular macrophages are involved in pulmonary vascular sensitivity to intravenously injected particles in sheep. We estimated that newborn lambs have few of these macrophages at birth but develop a 10-fold greater density within 2 wk. Awake, chronically instrumented newborn lambs showed no change in pulmonary vascular driving pressure (pulmonary arterial minus left atrial pressure) after injection of either liposomes [2 +/- 3 (SD) cmH2O; n = 5] or Monastral blue particles (3 +/- 2 cmH2O; n = 6) and showed no net pulmonary production of thromboxane B2, the stable metabolite of the vasoconstrictor thromboxane A2. In contrast, five of those lambs 2 wk later showed both an increase in pulmonary vascular driving pressure after injection of liposomes and Monastral blue (20 +/- 16 and 25 +/- 15 cmH2O, respectively; P < 0.05) and net pulmonary production of thromboxane B2 (171 +/- 103 and 429 +/- 419 pg/ml plasma, respectively; P < 0.05). Older lambs (n = 5) had higher pulmonary uptakes than newborn lambs (n = 6) of radioactive liposomes (47 +/- 13 vs. 12 +/- 10%; P < 0.01) and Monastral blue (53 +/- 6 vs. 21 +/- 10%; P < 0.05). We conclude that pulmonary intravascular macrophages are responsible for the sensitivity of sheep to intravenous foreign particles and are essential for a cascade of processes leading to microvascular injury.     

Development of the pulmonary vasculature in newborn lambs: structure-function relationships

Our objectives were 1) to describe the quantitative light microscopy and ultrastructure of newborn lamb lungs and 2) to correlate hemodynamic changes during normoxia and hypoxia with the morphology. By light microscopy, we measured the percent muscle thickness (%MT) and peripheral muscularization of pulmonary arteries and veins from 25 lambs aged less than 24 h, 2-4 days, 2 wk, and 1 mo. At the same ages, lungs were isolated and perfused in situ and, after cyclooxygenase blockade with indomethacin, total, arterial (delta Pa), middle (delta Pm), and venous pressure gradients at inspired O2 fractions of 0.28 (mild hyperoxia) and 0.04 (hypoxia) were determined with inflow-outflow occlusion. During mild hyperoxia, delta Pa and delta Pm fell significantly between 2-4 days and 2 wk, whereas during hypoxia, only delta Pm fell. The %MT of all arteries (less than 50 to greater than 1,000 microns diam) decreased, and peripheral muscularization of less than 100-microns-diam arteries fell between less than 4 days and greater than 2 wk. Our data suggest that 1) the %MT of arteries determines normoxic pulmonary vascular resistance, because only arterial and middle segment resistance fell, 2) peripheral muscularization is a major determinant of hypoxic pulmonary vasoconstriction, because we observed a fall with age in peripheral muscularization of less than 100-micron-diam arteries and in delta Pm with hypoxia, and 3) the arterial limit of the middle segment defined by inflow-outflow occlusion lies in 100- to 1,000-microns-diam arteries.     

Prostaglandins E1, E2 and I2: evidence for three distinct actions in vascular smooth muscle.

In the perfused mesenteric artery of the rat prostaglandins (PGs) E₁, E₂ and I₂ had distinct actions. PGE₂ potentiated pressor responses to noradrenaline, angiotensin II and potassium ions. PGE₁ potentiated responses to noradrenaline and angiotensin at low concentrations and inhibited them at high concentrations: no concentrations had any effect on potassium responses. PGI₂ inhibited responses to noradrenaline and angiotensin but had no effect on potassium responses. These three distinct actions suggest that the binding sites for the three PGs in this vascular muscle must be distinct.     

TxA2 production by human arteries and veins

Human arterial and venous segments from patients under-going operations when incubated in Tris buffer both alone and with arachidonic acid were able to produce thromboxane B2 (assessed by radioimmunoassay). Thromboxane B2 (TxB2) production was progressive in time (till 40 min.) and was enhanced by the addition of 1mM norepinephrine. Contamination of tissues by platelet was checked and platelets did not contribute to thromboxane formation. The investigation of the conversion of 1-14C arachidonic acid by vascular tissue indicated that human vascular tissues produce the metabolites of the cyclooxygenase dependent pathway and that prostacyclin is the main metabolite with a PGI2/TxA2 ratio of 4:1. The arterial wall was found to possess an active lipoxygenase dependent pathway. Thromboxane production by intimal cells was negligible and the main source of thromboxane was the media. The production of thromboxane did not change in relation to age, but arterial segments from men produced significantly larger amounts of thromboxane than those from women.     

cagA+ Helicobacter pylori induces greater levels of prostaglandin E2 than cagA- strains

cagA+ Helicobacter pylori (HP) infection is associated with an increased risk of distal gastric cancer. Previous studies investigating the effect of HP infection on prostaglandin E2 (PGE2) levels have not differentiated between cagA+ and cagA- strains and consequently have produced contradictory results. The aim was to investigate the effect of cagA+ strains on PGE2 and enhance the understanding of the mechanisms leading to gastric diseases. Hundred patients without peptic ulcers and not on medication were recruited (one later excluded) from endoscopy clinics: six biopsies were obtained from each patient. PGE2, colonization density and histology were determined. In addition, HP status was assessed by histology, CLOtest and culture with cagA+ being determined by PCR. Sixty-nine patients were HP- and 30 HP+ (10 cagA+, 18 cagA-, 2 undetermined). In age and sex-matched patients, PGE2 was significantly greater (P = 0.04) in HP+ (37.2 +/- 1.2 pg/mg per 20 min) than in HP- (22.6 +/- 1.2). In patients without atrophy, those infected with cagA+ had significantly higher (P = 0.03) PGE2 levels (53 +/- 1.1) than HP- patients (22.6 +/- 1.1) and greater levels (P = 0.29) than cagA- patients (35 +/- 1.3). In conclusion, the increased levels of PGE2 in the presence of cagA+ infection could be an important factor by which cagA+ strains enhance the gastric mucus layer protective functions leading to established colonization, gastritis and increased risk of gastric cancer. However, further evaluation with a large-scale multi-centre study is required to substantiate this hypothesis.     

Prostaglandins and control of breathing in newborn piglets

To investigate the possible role of prostaglandins in regulation of postnatal breathing, phrenic neural activity (PMO) was recorded as an index of breathing in 42 anesthetized, paralyzed piglets less than 30 days of age (weight 2.4 +/- 0.2 kg, age 9.9 +/- 1.5 days) who were mechanically ventilated with 100% O2 at a fixed tidal volume (8-10 ml/kg). End-tidal CO2 was held constant by an electronic servocontroller which adjusted ventilator rate; ventilator rate was monitored as an index of CO2 production. Rectal temperature was maintained at 39.0 +/- 0.2 degrees C. The effects on PMO of intravenous and brain ventricular injections of NaCl and agents active in the prostaglandin cascade were compared. Intravenous (0.25-1.0 mg/kg, n = 9) and brain (5-33 micrograms/kg, n = 6) indomethacin, a cyclooxygenase inhibitor, doubled PMO within 30 min. Intravenous (1-10 micrograms/kg, n = 6) and brain (1-40 micrograms/kg, n = 6) prostaglandin E1 inhibited PMO by one-half at 10 and 30 min.     

Differential relaxant responses of pulmonary arteries and veins in lung explants of guinea pigs

Shi, Weibin, David H. Eidelman, and René P. Michel.Differential relaxant responses of pulmonary arteries and veins inlung explants of guinea pigs. J. Appl.Physiol. 83(5): 1476-1481, 1997.The endotheliumregulates vascular tone through release of relaxing or contractingfactors, with nitric oxide (NO) being a major endothelium-derivedrelaxing factor. In the present study, we used a lung explant techniqueto determine the differential abilities and mechanisms of pulmonaryarteries and veins of normal guinea pigs to relax after precontraction.Excised lungs of 15 guinea pigs were filled through the airways with1% agarose, cut into 1-mm-thick slices, and cultured overnight.Luminal areas of vascular cross sections were measured with animage-analysis system. Vessels were precontracted with U-46619, andresponses to histamine, acetylcholine (ACh), sodium nitroprusside, andpapaverine were examined. We also determined the effects ofN-nitro-L-arginineand of indomethacin on ACh-induced responses. We found that histaminerelaxed arteries more than veins and that ACh relaxed only arteries.N-nitro-L-arginine pretreatmentabolished ACh-induced relaxation of arteries and caused ACh-inducedcontraction of veins, whereas indomethacin markedly augmentedACh-induced relaxation of arteries (maximal relaxation: 48.5 ± 4.7 vs. 19.2 ± 5.1% without it) and induced a dose-dependentrelaxation of veins (maximal relaxation: 17.0 ± 4.1%). Sodiumnitroprusside induced a significantly greater relaxation of arteriesthan veins, whereas papaverine relaxed them equally. We conclude thatin guinea pigs endothelial NO-mediated relaxation is greater inpulmonary arteries than in veins and that ACh-induced NO-mediatedrelaxation is reduced by the simultaneous production ofcyclooxygenase-derived vasoconstrictors.

     

Cardiovascular agents affect the tone of pulmonary arteries and veins in precision-cut lung slices

Introduction

Cardiovascular agents are pivotal in the therapy of heart failure. Apart from their action on ventricular contractility and systemic afterload, they affect pulmonary arteries and veins. Although these effects are crucial in heart failure with coexisting pulmonary hypertension or lung oedema, they are poorly defined, especially in pulmonary veins. Therefore, we investigated the pulmonary vascular effects of adrenoceptor agonists, vasopressin and angiotensin II in the model of precision-cut lung slices that allows simultaneous studies of pulmonary arteries and veins.

Materials and Methods

Precision-cut lung slices were prepared from guinea pigs and imaged by videomicroscopy. Concentration-response curves of cardiovascular drugs were analysed in pulmonary arteries and veins.

Results

Pulmonary veins responded stronger than arteries to α₁-agonists (contraction) and β₂-agonists (relaxation). Notably, inhibition of β₂-adrenoceptors unmasked the α₁-mimetic effect of norepinephrine and epinephrine in pulmonary veins. Vasopressin and angiotensin II contracted pulmonary veins via V_1a and AT₁ receptors, respectively, without affecting pulmonary arteries.

Discussion

Vasopressin and (nor)epinephrine in combination with β₂-inhibition caused pulmonary venoconstriction. If applicable in humans, these treatments would enhance capillary hydrostatic pressures and lung oedema, suggesting their cautious use in left heart failure. Vice versa, the prevention of pulmonary venoconstriction by AT₁ receptor antagonists might contribute to their beneficial effects seen in left heart failure. Further, α₁-mimetic agents might exacerbate pulmonary hypertension and right ventricular failure by contracting pulmonary arteries, whereas vasopressin might not.