Maturation of the contractile response of the Emu ductus arteriosus

The avian embryo has a pair of ductus arteriosi that allow the blood to bypass the pulmonary circulation prior to the initiation of lung ventilation. Our objective was to characterize the factors regulating DA tone during the later stages of development in the emu embryo. We examined in vitro the reactivity of the emu ductus from day 39 through 49 of a 50-day incubation. Steady state tension was not altered by the COX inhibitor indomethacin or the nitric oxide synthase inhibitor l-NAME. However, prostaglandin E₂ (PGE₂) produced a significant relaxation. Norephinephrine and U-46619 produced strong significant contractions in the emu DA and the adrenergic response matured with development. The contractile response to oxygen matured as the embryo developed with significant oxygen-induced contraction on days 45 and 49, but not on day 39 of incubation. The Kv channel inhibitor 4-aminopyridine induced the contraction of the day 48–49 ductus of similar magnitude as the oxygen-induced contraction. The oxygen-induced contraction was reversed by the reducing agent DTT and the electron transport chain inhibitor rotenone. These results suggest that while the emu DA responds to PGE₂, locally produced PGE₂ are not the important regulators of vessel tone. Additionally, relaxation upon addition of the mitochondria electron transport chain inhibitor rotenone suggests that the mitochondria might be acting as vascular oxygen sensors in this system through the production of reactive oxygen species to stimulate the oxygen-induced contraction in a similar fashion to mammals.     

Interaction of prostaglandins and the myogenic factor in the mechanism of closure of the ductus arteriosus

Although the role of prostaglandins (PG) in the mechanism of dilatation of the ductus arteriosus (DA) has received considerable attention, no data on their possible interaction with the pressure-induced myogenic reaction are available. There is also a lack of information on PG production by the foetal blood vessels of the guinea-pig, in which the DA closes rapidly. Use of the RIA method showed relatively low PG production by isolated foetal guinea pig blood vessels, as represented by the main products, PGI1 and PGE2. When computed in pmol per mg wet weight, the production of 6-keto-PGF1 alpha (an equivalent of PGI2) was statistically significantly higher in the foetal DA (4.06 +/- 1.13) than in the foetal aorta (2.04 +/- 0.33). The isolated DA reacts to a sudden increase in perfusion pressure by marked constriction, which in some cases leads to functional closure of the DA. In 10(-7) to 10(-5) mol.l-1 concentration, PGE2 reversibly inhibits pressure-induced myogenic constriction, while under the same conditions the contractility of the DA in response to oxygen is unaffected. In concentrations of 10(-6)-10(-5) mol.l-1, indomethacin, a blocker of PG biosynthesis, also induces pressure constriction and it raises the basal flow resistance of isolated DA preparations. The results indicate that PGs play a modulator role in the pressure myogenic response of the DA of guinea-pig and rabbit foetuses.     

Prostacyclin (PGI2) mediates hypoxic relaxation of bovine coronary arterial strips

Bovine coronary arterial strips (BCA) exhibiting spontaneous tone, relax in response to a decrease in the PO₂ of the batching medium. Experiments were performed to determine if prostaglandins (PGs) mediate the oxygen-induced changes in tension. BCA were equilibrated in Krebs-bicarbonate solution at 37 °C gassed with 95% O₂, 5% CO₂ and tension was measured isometrically. When the PO₂ of the bathing medium was decreased, BCA exhibited reversible reductions in tension. Switching from 95% O₂, 5% CO₂ to 95% N₂, 5% CO₂ (anoxia) elicited an initial relaxation followed by a contraction. In contrast, a change to 5% O₂, 5% CO₂, 90% N₂ (hypoxia) was followed by a sustained relaxation. Re-introduction of O₂ to anoxic strips produced a biphasic response: relaxation followed by contraction. Indomethacin or eicosatetraynoic acid (EYA) increased tone and inhibited the relaxation produced by anoxia or hypoxia. Indomethacin or EYA did not inhibit the relaxation of anoxic strips during re-introduction of O₂, but did inhibit the contraction partially. Relaxation of arterial strips to arachidonic acid (AA) was similar to relaxation to prostacyclin (PGI₂). Anoxia limited the relaxation to AA but not to PGI₂. We conclude that PG synthesis contributes to the basal tone and the hypoxia-induced relaxation of CBA. In addition, hypoxia, unless severe, does not prevent the conversion of AA to PGI₁.     

Factors that increase the contractile tone of the ductus arteriosus also regulate its anatomic remodeling

Permanent closure of the full-term newborn ductus arteriosus (DA) occurs only if profound hypoxia develops within the vessel wall during luminal obliteration. We used fetal and newborn baboons and lambs to determine why the immature DA fails to remodel after birth. When preterm newborns were kept in a normoxic range (Pa(O(2)): 50-90 mmHg), 86% still had a small patent DA on the sixth day after birth; in addition, the preterm DA wall was only mildly hypoxic and had only minimal remodeling. The postnatal increase in Pa(O(2)) normally induces isometric contractile responses in rings of DA; however, the excessive inhibitory effects of endogenous prostaglandins and nitric oxide, coupled with a weaker intrinsic DA tone, make the preterm DA appear to have a smaller increment in tension in response to oxygen than the DA near term. We found that oxygen concentrations, beyond the normoxic range, produce an additional increase in tension in the preterm DA that is similar to the contractile response normally seen at term. We predicted that preterm newborns, kept at a higher Pa(O(2)), would have increased DA tone and would be more likely to obliterate their lumen. We found that preterm newborns, maintained at a Pa(O(2)) >200 mmHg, had only a 14% incidence of patent DA. Even though DA constriction was due to elevated Pa(O(2)), obliteration of the lumen produced profound hypoxia of the DA wall and the same features of remodeling that were observed at term. DA wall hypoxia appears to be both necessary and sufficient to produce anatomic remodeling in preterm newborns.     

Patency of the preterm fetal ductus arteriosus is regulated by endothelial nitric oxide synthase and is independent of vasa vasorum in the mouse

Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17-19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.     

Postnatal constriction, ATP depletion, and cell death in the mature and immature ductus arteriosus

After birth, constriction of the full-term ductus arteriosus induces oxygen, glucose and ATP depletion, cell death, and anatomic remodeling of the ductus wall. The immature ductus frequently fails to develop the same degree of constriction or anatomic remodeling after birth. In addition, the immature ductus loses its ability to respond to vasoconstrictive agents, like oxygen or indomethacin, with increasing postnatal age. We examined the effects of premature delivery and postnatal constriction on the immature baboon ductus arteriosus. By 6 days after birth, surrogate markers of hypoxia (HIF1alpha/VEGF mRNA) and cell death [dUTP nick-end labeling (TUNEL)-staining] increased, while glucose and ATP concentrations (bioluminescence imaging) decreased in the immature ductus. TUNEL-staining was significantly related to the degree of glucose and ATP depletion. Glucose and ATP depletion were directly related to the degree of ductus constriction; while TUNEL-staining was logarithmically related to the degree of ductus constriction. Extensive cell death (>15% TUNEL-positive cells) occurred only when there was no Doppler flow through the ductus lumen. In contrast, HIF1alpha/VEGF expression and ATP concentrations were significantly altered even when the immature ductus remained open after birth. Decreased ATP concentrations produced decreased oxygen-induced contractile responses in the immature ductus. We hypothesize that ATP depletion in the persistently patent immature newborn ductus is insufficient to induce cell death and remodeling but sufficient to decrease its ability to constrict after birth. This may explain its decreasing contractile response to oxygen, indomethacin, and other contractile agents with increasing postnatal age.     

Nitric oxide-endothelin-1 interactions after acute ductal constriction in fetal lambs

Acute partial compression of the fetal ductus arteriosus (DA) results in an initial increase in pulmonary blood flow (PBF) that is followed by acute vasoconstriction. The objective of the present study was to determine the role of nitric oxide (NO)-endothelin-1 (ET-1) interactions in the acute changes in pulmonary vascular tone after in utero partial constriction of the DA. Twelve late-gestation fetal lambs (132-140 days) were instrumented to measure vascular pressures and left PBF. After a 24-h recovery period, acute constriction of the DA was performed by partially inflating a vascular occluder, and the hemodynamic variables were observed for 4 h. In control lambs (n = 7), acute ductal constriction initially increased PBF by 627% (P < 0.05). However, this was followed by active vasoconstriction, such that PBF was restored to preconstriction values by 4 h. This was associated with a 43% decrease in total NO synthase (NOS) activity (P < 0.05) and a 106% increase in plasma ET-1 levels (P < 0.05). Western blot analysis demonstrated no changes in lung tissue endothelial NOS, preproET-1, endothelin-converting enzyme-1, or ET(B) receptor protein levels. The infusion of PD-156707 (an ET(A) receptor antagonist, n = 5) completely blocked the vasoconstriction and preserved NOS activity. These data suggest that the fetal pulmonary vasoconstriction after acute constriction of the DA is mediated by NO-ET-1 interactions. These include an increase in ET(A) receptor-mediated vasoconstriction and an ET(A) receptor-mediated decrease in NOS activity. The mechanisms of these NO-ET-1 interactions, and their role in mediating acute changes in PBF, warrant further studies.     

Age-dependent changes in the response of the lamb ductus arteriosus to oxygen and ibuprofen.

Circular strips of ductus arteriosus from lambs of gestational age between 90 and 144 days (term 147 days) were studied in vitro at low (8--16 torr (1 torr = 133.322 Pa)) and high (426--622 torr) PO2. Potassium- and oxygen-induced contractions increased with the gestational age and attained a maximum at term. At low PO2, ibuprofen, a blocker of prostaglandin synthesis, produced a dose-dependent contraction of the ductus at all ages and enhanced the potassium-induced contraction of the immature ductus (90--124 days). Both effects were relatively greater in the 103- to 107-day gestational group. At that age, ibuprofen also potentiated the oxygen-induced contraction. These findings, while confirming that a prostaglandin is involved in ductus patency, indicate that the prostaglandin-relaxing mechanism becomes functional at an early stage of gestation and reaches maximal activity before term. The existence of an active, prostaglandin-mediated relaxation in the preterm ductus may account, in part, for the reduced responsiveness of the vessel to oxygen. It is confirmed that ibuprofen and other nonsteroidal antiinflammatory drugs are well suited for the management of the premature infant with patent ductus arteriosus.     

Calcium-dependent and calcium-sensitizing pathways in the mature and immature ductus arteriosus

Studies performed in sheep and baboons have shown that after birth, the normoxic muscle media of ductus arteriosus (DA) becomes profoundly hypoxic as it constricts and undergoes anatomic remodeling. We used isolated fetal lamb DA (pretreated with inhibitors of prostaglandin and nitric oxide production) to determine why the immature DA fails to remain tightly constricted during the hypoxic phase of remodeling. Under normoxic conditions, mature DA constricts to 70% of its maximal active tension (MAT). Half of its normoxic tension is due to Ca(2+) entry through calcium L-channels and store-operated calcium (SOC) channels. The other half is independent of extracellular Ca(2+) and is unaffected by inhibitors of sarcoplasmic reticulum (SR) Ca(2+) release (ryanodine) or reuptake [cyclopiazonic acid (CPA)]. The mature DA relaxes slightly during hypoxia (to 60% MAT) due to decreases in calcium L-channel-mediated Ca(2+) entry. Inhibitors of Rho kinase and tyrosine kinase inhibit both Ca(2+)-dependent and Ca(2+)-independent DA tension. Although Rho kinase activity may increase during gestation, immature DA develop lower tensions than mature DA, primarily because of differences in the way they process Ca(2+). Calcium L-channel expression increases with advancing gestation. Under normoxic conditions, differences in calcium L-channel-mediated Ca(2+) entry account for differences in tension between immature (60% MAT) and mature (70% MAT) DA. Under hypoxic conditions, differences in both calcium L-channel-dependent and calcium L-channel-independent Ca(2+) entry, account for differences in tension between immature (33% MAT) and mature (60% MAT) DA. Stimulation of Ca(2+) entry through reverse-mode Na(+)/Ca(2+) exchange or CPA-induced SOC channel activity constrict the DA and eliminate differences between immature and mature DA during both hypoxia and normoxia.     

Oxygen increases ductus arteriosus smooth muscle cytosolic calcium via release of calcium from inositol triphosphate-sensitive stores

In utero, blood shunts away from the lungs via the ductus arteriosus (DA) and the foramen ovale. After birth, the DA closes concomitant with increased oxygen tension. The present experimental series tests the hypothesis that oxygen directly increases DA smooth muscle cell (SMC) cytosolic calcium ([Ca(2+)](i)) through inactivation of a K(+) channel, membrane depolarization, and entry of extracellular calcium. To test the hypothesis, DA SMC were isolated from late-gestation fetal lambs and grown to subconfluence in primary culture in low oxygen tension (25 Torr). DA SMC were loaded with the calcium-sensitive fluorophore fura-2 under low oxygen tension conditions and studied using microfluorimetry while oxygen tension was acutely increased (120 Torr). An acute increase in oxygen tension progressively increased DA SMC [Ca(2+)](i) by 11.7 +/- 1.4% over 40 min. The effect of acute normoxia on DA SMC [Ca(2+)](i) was mimicked by pharmacological blockade of the voltage-sensitive K(+) channel. Neither removal of extracellular calcium nor voltage-operated calcium channel blockade prevented the initial increase in DA SMC [Ca(2+)](i). Manganese quenching experiments demonstrated that acute normoxia initially decreases the rate of extracellular calcium entry. Pharmacological blockade of inositol triphosphate-sensitive, but not ryanodine-sensitive, intracellular calcium stores prevented the oxygen-induced increase in [Ca(2+)](i). Endothelin increased [Ca(2+)](i) in acutely normoxic, but not hypoxic, DA SMC. Thus acute normoxia 1) increases DA SMC [Ca(2+)](i) via release of calcium from intracellular calcium stores, and subsequent entry of extracellular calcium, and 2) potentiates the effect of contractile agonists. Prolonged patency of the DA may result from disordered intracellular calcium homeostasis.     

Vasoactivity of the gasotransmitters hydrogen sulfide and carbon monoxide in the chicken ductus arteriosus

Besides nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide (H(2)S) is a third gaseous messenger that may play a role in controlling vascular tone and has been proposed to serve as an O(2) sensor. However, whether H(2)S is vasoactive in the ductus arteriosus (DA) has not yet been studied. We investigated, using wire myography, the mechanical responses induced by Na(2)S (1 μM-1 mM), which forms H(2)S and HS(-) in solution, and by authentic CO (0.1 μM-0.1 mM) in DA rings from 19-day chicken embryos. Na(2)S elicited a 100% relaxation (pD(2) 4.02) of 21% O(2)-contracted and a 50.3% relaxation of 62.5 mM KCl-contracted DA rings. Na(2)S-induced relaxation was not affected by presence of the NO synthase inhibitor l-NAME, the soluble guanylate cyclase (sGC) inhibitor ODQ, or the K(+) channel inhibitors tetraethylammonium (TEA; nonselective), 4-aminopyridine (4-AP, K(V)), glibenclamide (K(ATP)), iberiotoxin (BK(Ca)), TRAM-34 (IK(Ca)), and apamin (SK(Ca)). CO also relaxed O(2)-contracted (60.8% relaxation) and KCl-contracted (18.6% relaxation) DA rings. CO-induced relaxation was impaired by ODQ, TEA, and 4-AP (but not by L-NAME, glibenclamide, iberiotoxin, TRAM-34 or apamin), suggesting the involvement of sGC and K(V) channel stimulation. The presence of inhibitors of H(2)S or CO synthesis as well as the H(2)S precursor L-cysteine or the CO precursor hemin did not significantly affect the response of the DA to changes in O(2) tension. Endothelium-dependent and -independent relaxations were also unaffected. In conclusion, our results indicate that the gasotransmitters H(2)S and CO are vasoactive in the chicken DA but they do not suggest an important role for endogenous H(2)S or CO in the control of chicken ductal reactivity.     

High dietary salt reduces the contribution of 20-HETE to arteriolar oxygen responsiveness in skeletal muscle

The coupling of tissue blood flow to cellular metabolic demand involves oxygen-dependent adjustments in arteriolar tone, and arteriolar responses to oxygen can be mediated, in part, by changes in local production of 20-HETE. In this study, we examined the long-term effect of dietary salt on arteriolar oxygen responsiveness in the exteriorized, superfused rat spinotrapezius muscle and the role of 20-HETE in this responsiveness. Rats were fed either a normal-salt (NS, 0.45%) or high-salt (HS, 4%) diet for 4-5 wk. There was no difference in steady-state tissue Po(2) between NS and HS rats, and elevation of superfusate oxygen content from 0% to 10% caused tissue Po(2) to increase by the same amount in both groups. However, the resulting reductions in arteriolar diameter and blood flow were less in HS rats than NS rats. Inhibition of 20-HETE formation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or 17-octadecynoic acid (17-ODYA) attenuated oxygen-induced constriction in NS rats but not HS rats. Exogenous 20-HETE elicited arteriolar constriction that was greatly reduced by the large-conductance Ca(2+)-activated potassium (K(Ca)) channel inhibitors tetraethylammonium chloride (TEA) and iberiotoxin (IbTx) in NS rats and a smaller constriction that was less sensitive to TEA or IbTx in HS rats. Arteriolar responses to exogenous angiotensin II were similar in both groups but more sensitive to inhibition with DDMS in NS rats. Norepinephrine-induced arteriolar constriction was similar and insensitive to DDMS in both groups. We conclude that 20-HETE contributes to oxygen-induced constriction of skeletal muscle arterioles via inhibition of K(Ca) channels and that a high-salt diet impairs arteriolar responses to increased oxygen availability due to a reduction in vascular smooth muscle responsiveness to 20-HETE.     

Cyclooxygenase-2 inhibitors constrict the fetal lamb ductus arteriosus both in vitro and in vivo

Nonselective cyclooxygenase (COX) inhibitors are potent tocolytic agents; however, they also have adverse fetal effects such as constriction of the fetal ductus arteriosus. Recently, selective COX-2 inhibitors have been used in the management of preterm labor in the hope of avoiding fetal complications. However, both COX-1 and -2 are expressed by cells of the ductus arteriosus. We used fetal lambs (0.88 gestation) to assess the ability of selective COX-2 inhibitors celecoxib and NS398 to affect the ductus arteriosus. Both selective COX-2 inhibitors decreased PGE(2) and 6ketoPGF(1alpha) production in vitro; both inhibitors constricted the isolated ductus in vitro. The nonselective COX-1/COX-2 inhibitor indomethacin produced a further reduction in PG release and an additional increase in ductus tension in vitro. We used a prodrug of celecoxib to achieve 1.4 +/- 0.6 microg/ml, mean +/- standard deviation, of the active drug in vivo. This concentration of celecoxib produced both an increase in pressure gradient and resistance across the ductus; celecoxib also decreased fetal plasma concentrations of PGE(2) and 6ketoPGF(1alpha). Indomethacin (0.7 +/- 0.2 microg/ml) produced a significantly greater fall in ductus blood flow than celecoxib and tended to have a greater effect on ductus resistence in vivo. We conclude that caution should be used when recommending COX-2 inhibitors for use in pregnant women, because COX-2 appears to play a significant role in maintaining patency of the fetal ductus arteriosus.     

Opposite effects of redox status on membrane potential,cytosolic calcium,and tone in pulmonary arteries and ductus arteriosus

At birth, associated with the rise in oxygen tension, the pulmonary arteries (PA) dilate and the ductus arteriosus (DA) constricts. Both PA and DA constrict with vasoconstrictors and dilate with vasodilators. They respond in a contrary manner only to changes in oxygen tension. We hypothesized that the effects of changes in oxygen are mediated by changes in redox status. Consequently, we tested whether a reducing agent, DTT, and an oxidizing agent, dithionitrobenzoic acid (DTNB), would have opposite effects on a major oxygen signaling pathway in the PA and DA smooth muscle cells (SMCs), the sequence of change in potassium current (IK), membrane potential (Em), cytosolic calcium, and vessel tone. Under normoxic conditions, DTT constricted adult and fetal resistance PA rings, whereas in DA rings DTT acted as a potent vasodilator. In normoxia, voltage-clamp measurements showed inhibition of IK by DTT in PASMCs and, in contrast, activation in DASMCs. Consequently, DTT depolarized fetal and adult PASMCs and hyperpolarized DASMCs. [Ca2+]i was increased by DTT in fetal and adult PASMCs and decreased in DASMCs. Under hypoxic conditions, DTNB constricted DA rings and caused vasodilatation in fetal PA rings. DTNB inhibited IK and depolarized the cell membrane in DASMCs. In contrast, activation of IK and hyperpolarization was seen in PASMCs. Thus the same redox signal can elicit opposite effects on IK, Em, cytosolic calcium, and vascular tone in resistance PA and the DA. These observations support the concept that redox changes could signal the opposite effects of oxygen in the PA and DA.     

Role of neuronal nitric oxide synthase in regulation of vascular and ductus arteriosus tone in the ovine fetus

Nitric oxide (NO) is produced by NO synthase (NOS) and contributes to the regulation of vascular tone in the perinatal lung. Although the neuronal or type I NOS (NOS I) isoform has been identified in the fetal lung, it is not known whether NO produced by the NOS I isoform plays a role in fetal pulmonary vasoregulation. To study the potential contribution of NOS I in the regulation of basal fetal pulmonary vascular resistance (PVR), we studied the hemodynamic effects of a selective NOS I antagonist, 7-nitroindazole (7-NINA), and a nonselective NOS antagonist, N-nitro-L-arginine (L-NNA), in chronically prepared fetal lambs (mean age 128 +/- 3 days, term 147 days). Brief intrapulmonary infusions of 7-NINA (1 mg) increased basal PVR by 37% (P < 0.05). The maximum increase in PVR occurred within 20 min after infusion, and PVR remained elevated for up to 60 min. Treatment with 7-NINA also increased the pressure gradient between the pulmonary artery and aorta, suggesting constriction of the ductus arteriosus (DA). To test whether 7-NINA treatment selectively inhibits the NOS I isoform, we studied the effects of 7-NINA and L-NNA on acetylcholine-induced pulmonary vasodilation. The vasodilator response to acetylcholine remained intact after treatment with 7-NINA but was completely inhibited after L-NNA, suggesting minimal effects on endothelial or type III NOS after 7-NINA infusion. Western blot analysis detected NOS I protein in the fetal lung and great vessels including the DA. NOS I protein was detected in intact and endothelium-denuded vessels, suggesting that NOS I is present in the medial or adventitial layer. We conclude that 7-NINA, a selective NOS I antagonist, increases basal PVR, systemic arterial pressure, and DA tone in the late-gestation fetus and that NOS I protein is present in the fetal lung and great vessels. We speculate that NOS I may contribute to NO production in the regulation of basal vascular tone in the pulmonary and systemic circulations and the DA.     

Intracellular and extracellular calcium utilization during hypoxic vasoconstriction of cyclostome aortas

Hypoxic vasoconstriction (HV) is an intrinsic response of mammalian pulmonary and cyclostome aortic vascular smooth muscle. The present study examined the utilization of calcium during HV in dorsal aortas (DA) from sea lamprey and New Zealand hagfish. HV was temporally correlated with increased free cytosolic calcium (Ca2+c) in lamprey DA. Extracellular calcium (Ca2+o) did not contribute significantly to HV in lamprey DA, but it accounted for 38.1 +/- 5.3% of HV in hagfish DA. Treatment of lamprey DA with ionomycin, ryanodine, or caffeine added to thapsigargin-reduced HV, whereas HV was augmented by BAY K 8644. Methoxyverapamil (D600) in zero Ca2+o did not affect HV in lamprey DA, nor did it prevent further constriction when Ca2+o was restored during hypoxia in hagfish DA. Removal of extracellular sodium (Na+o) caused a constriction in both species. Lamprey DA relaxed to prehypoxic tension following return to normoxia in zero Na+o, whereas relaxation was inhibited in hagfish DA. Relaxation following HV was inhibited in lamprey DA when Na+o and Ca2+o were removed. These results show that HV is correlated with [Ca2+]c in lamprey DA and that Na+/Ca2+ exchange is used during HV in hagfish but not lamprey DA. Multiple receptor types appear to mediate stored intracellular calcium release in lamprey DA, and L-type calcium channels do not contribute significantly to constriction in either cyclostome.     

Endogenous AA metabolites and their possible role in tracheal smooth muscle tone in guinea pigs

The effects of endogenous arachidonic acid (AA) metabolites on inherent tone and histamine-induced constriction were studied in guinea pig tracheal smooth muscle. Inhibitors of either cyclooxygenase (indomethacin) or lipoxygenase (AA 861) significantly diminished the inherent tone of the muscle. Antagonists of prostaglandins (SC 19220) or leukotrienes (FPL 55712) also diminished the inherent tone, whereas an inhibitor of thromboxane synthase (OKY 046) had no significant effect. These results show that the metabolites of the lipoxygenase pathway as well as prostaglandins also participate in the maintenance of inherent tone. To reexamine the previously reported augmentation of histamine constriction induced by the inhibitors and the antagonists, we compared the active tension of the muscle measured from the maximum relaxed level as the base line to eliminate the fluctuation of inherent tone. Such comparison revealed that the inhibitors and the antagonists have no augmentative effect on either the maximum response to histamine or the concentration required to produce 50% of maximum active tension and that there is functional synergism between the exogenously added histamine and the endogenously produced AA metabolites. Therefore the zero active tension is useful as a base line to compare the contractile response of a drug-treated preparation with that of a nontreated preparation.     

Regulation of the fetal mouse ductus arteriosus is dependent on interaction of nitric oxide and COX enzymes in the ductal wall

Nitric oxide (NO) and cyclooxygenase (COX)-derived prostaglandins are critical regulators of the fetal ductus arteriosus. To examine the interaction of these pathways within the ductus wall, the ductus arteriosus of term and preterm fetal mice was evaluated by pressurized myography. The isolated preterm ductus was more sensitive to NOS inhibition than at term. Sequential NOS and COX inhibition caused 36% constriction of the preterm ductus regardless of drug order. In contrast, constriction of the term ductus was dependent on the sequence of inhibition; NOS inhibition prior to COX inhibition produced greater constriction than when inhibitors were given in reverse order (36 ± 6% versus 23 ± 5%). Selective COX-1 or COX-2 inhibition prior to N(G)-nitro-l-arginine methyl ester (l-NAME) induced the expected degree of constriction. However, NOS inhibition followed by selective COX-2 inhibition caused unexpected ductal dilation. These findings are consistent with NO-induced activation of COX in the ductus arteriosus wall and the production of a COX-2-derived constrictor prostanoid that contributes to the balance of vasoactive forces that maintain fetal ductus arteriosus tone.     

Effect of Nitroprusside (Nitric Oxide) on Endogenous Dopamine Release from Rat Striatal Slices

It is becoming apparent that the synthesis of nitric oxide (NO) from L-arginine not only explains endothelium-dependent vascular relaxation, but is a widespread mechanism for the regulation of cell function and communication. We examined the role of NO on the endogenous dopamine (DA) release from rat striatum. Nitroprusside, in the concentration range of 3-100 microM, induced a dose-dependent increase in the endogenous DA release from rat striatal slices. The maximal response was 330% over the baseline release. A higher concentration of nitroprusside (300 microM) produced an inhibitory effect on the spontaneous release of DA. L-Arginine (10 and 100 microM), a substrate in the NO-forming enzyme system, also produced an elevation of DA release. L-Arginine-induced DA release was attenuated by NG-monomethyl-L-arginine, an inhibitor of NO synthase. NADPH (1 microM), a cofactor of NO synthase, enhanced L-arginine-induced DA release. These results suggest a possible involvement of NO in the DA release process in rat striatum.