Biomedical subjects

W J Pearce

Publications and source records attributed to W J Pearce.

At least 19 recordsLinked to original sources

PubMed2006

Basic and translational neonatal neuroscience research: whither goest the future of physician-scientists?

OBJECTIVES: In light of declining numbers of physician-scientists, the goal of this project was to identify strategies to invigorate and attract new talent to clinical research in the field of pediatric neurosciences. DESIGN: To develop a broad perspective, a program of direct questions was addressed to both US and non-US physicians at all stages of career development. RESULTS: Respondents identified numerous promising avenues of research but also indicated obstacles to research progress at all stages of career development including medical students, resident physicians, junior medical faculty, mid-career faculty, and senior faculty. At each career stage, ideas were offered to attract resources for, build prestige for, and motivate commitment for participation in clinical research. CONCLUSIONS: Creative promotion of clinical research at all stages of medical education and career development offers great promise to expand current physician-scientist numbers, and thereby stimulate many exciting advances in medicine.

Biomedical Research↗

PubMed2003

ERK inhibition attenuates 5-HT-induced contractions in fetal and adult ovine carotid arteries.

Growth and differentiation-related pathways are much more active in immature than in mature, fully differentiated smooth muscle. Because mitogen-activated protein kinases (MAPK) are intimately involved with growth and differentiation, and the extracellular signal-regulated kinase (ERK) subfamily of MAPKs are involved in some contractile responses, the present studies examined the hypothesis that ERKs play an important and age-dependent role in smooth muscle contraction. The MAPK inhibitors PD098059 and UO126 both inhibited serotonin (5-HT) concentration-response relations more effectively in carotid arteries from term fetal lambs, than in corresponding arteries from mature non-pregnant adult sheep. This inhibition involved significant decreases in both the pD2 (adult: 2-fold; fetus: 4- to 15-fold) and the maximum efficacy (adult: 15-19%; fetus: 34-39%) of 5-HT. Accompanying this age-dependent effect on contraction, quantitative Western blot assays revealed that ERK1 and ERK2 abundances were 39% and 164% greater, respectively, in fetal than in adult carotid arteries. The abundance of the putative ERK target, caldesmon, however, was about 7-fold greater in adult than in fetal arteries. Together, the present results support the view that ERK abundance and activity is upregulated in fetal relative to adult arteries, and that one consequence of this upregulation is that the contribution of ERKs to contraction, at least that initiated by 5-HT2a receptors, is greater in fetal than adult carotid arteries. Whereas the phosphorylation mechanisms through which ERKs augment contraction remain uncertain and controversial, the present results suggest that emphasis should be shifted away from caldesmon and toward other critical contractile proteins, and how these proteins may contribute differently to development of agonist-induced contractile force in immature and mature arteries.

Age Factors↗

PubMed2001

Simultaneous glutamate and gamma-aminobutyric acid release within ventrolateral medulla during skeletal muscle contraction in intact and barodenervated rats.

The purpose of this study was to determine if baroreflex modulates cardiovascular responses and neurotransmitter release within rostral (RVLM) and caudal (CVLM) ventrolateral medulla during static contraction of skeletal muscle using anesthetized rats. We evoked cardiovascular responses by a static muscle contraction and measured simultaneous release of glutamate and gamma-aminobutyric acid (GABA) in both the RVLM and CVLM using microdialysis probes, two inserted bilaterally into the RVLM and two into the CVLM. In intact anesthetized rats, a muscle contraction increased release of glutamate concomitantly in both the RVLM and CVLM along with significant increases in heart rate and arterial blood pressure. In contrast, concentrations of GABA increased within the RVLM, but decreased significantly within the CVLM during the pressor response. These changes were due to contraction-evoked activation of muscle afferents since tibial nerve stimulation following muscle paralysis failed to evoke glutamate, GABA, or any cardiovascular changes. On the other hand, static muscle contractions in baroreceptor denervated rats augmented the increases in heart rate and blood pressure. Furthermore, muscle contraction significantly enhanced the release of glutamate in the RVLM but attenuated its release in the CVLM. In addition, concentrations of GABA within the RVLM were attenuated following a muscle contraction in denervated rats without any changes in GABA within the CVLM. These results demonstrate that the baroreceptors influence cardiovascular responses to static muscle contraction associated with dynamic changes in glutamate and GABA release within the RVLM and CVLM.

Animals↗

PubMed2001

Effects of nitric oxide and GABA interaction within ventrolateral medulla on cardiovascular responses during static muscle contraction.

We hypothesized that nitric oxide (NO) has opposing roles in regulating cardiovascular responses within the rostral (RVLM) and caudal (CVLM) ventrolateral medulla by modulating release of gamma-aminobutyric acid (GABA). We have measured GABA concentrations within the RVLM and CVLM during increases in mean arterial pressure (MAP) and heart rate (HR) following a 2-min tibial nerve stimulation-evoked static muscle contraction before and after microdialysis of the NO precursor, L-arginine (1.0 microM), for 30 min, and after the NO inhibitor, L-NMMA (1.0 microM), for 30 min. In eight anesthetized rats, muscle contraction significantly increased MAP, HR and GABA levels within the RVLM area (from 0.53+/-0.09 to 1.22+/-0.10 ng/10 microl). Following microdialysis of L-arginine, muscle contraction augmented GABA levels (from 0.45+/-0.07 to 2.18+/-0.09 ng/10 microl) and attenuated changes in MAP and HR. Subsequent application of L-NMMA significantly decreased GABA levels (from 0.47+/-0.08 to 0.22+/-0.07 ng/10 microl) but potentiated MAP and HR responses to a muscle contraction. In contrast, muscle contraction significantly increased MAP and HR but decreased GABA concentrations within the CVLM (from 1.20+/-0.20 to 0.78+/-0.17 ng/10 microl). Following microdialysis of L-arginine, muscle contraction significantly attenuated GABA levels (from 1.34+/-0.19 to 0.33+/-0.10 ng/10 microl) and augmented changes in MAP and HR in response to muscle contraction. A subsequent microdialysis of L-NMMA into the CVLM reversed the effects of L-arginine. These results demonstrate that NO within the RVLM and CVLM differentially modulates cardiovascular responses during static muscle contraction and that NO influences exercise-induced cardiovascular responses by modulating GABA release within the ventrolateral medulla.

Afferent Pathways↗

PubMed2001

Modulation of pressor response to muscle contraction via monoamines following AMPA-receptor blockade in the ventrolateral medulla.

We hypothesized that cardiovascular responses to static muscle contraction are mediated via changes in extracellular concentrations of monoamines (norepinephrine, dopamine and serotonin) following the administration of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an AMPA-receptor antagonist) into the rostral (RVLM) or caudal (CVLM) ventrolateral medulla. For the RVLM experiments (n= 8), a 2-min static muscle contraction increased the mean arterial pressure (MAP) and heart rate (HR) by 23 +/- 2 mmHg and 28 +/- 8 bpm, respectively. During this contraction, the concentrations of norepinephrine, dopamine, and serotonin within the RVLM increased by 278 +/- 52%, 213 +/- 23%, and 232 +/- 24%, respectively. Microdialysis of CNQX (1.0 microM) for 30 min into the RVLM attenuated the increases in MAP and HR ( 11 +/- 2 mmHg and 14 +/- 5 bpm) without a change in developed muscle tension. The levels of norepinephrine, dopamine, and serotonin within the RVLM were also attenuated. In contrast, microdialysis of CNQX into the CVLM (n= 8) potentiated the contraction-evoked responses in MAP ( 21 +/- 2 vs 33 +/- 5 mmHg) and HR ( 25 +/- 5 vs 46 +/- 8 bpm) without any effect on the monoamine levels within the CVLM region. These results suggest that AMPA-receptor blockade within the RVLM and CVLM has opposing effects on cardiovascular responses during static muscle contraction. In addition, such receptor blockade modulates extracellular concentrations of monoamines within the RVLM but not in the CVLM. These results provide evidence that AMPA receptors within the ventrolateral medulla play a role in exercise pressor reflex.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2001

Effects of opioid receptor activation on cardiovascular responses and extracellular monoamines within the rostral ventrolateral medulla during static contraction of skeletal muscle.

During static muscle contraction, activation of opioid receptors alters the extracellular glutamate concentrations within the rostral ventrolateral medulla (RVLM). In addition, microdialysis of glutamate in the ventrolateral medulla (VLM) increases the release of norepinephrine (NE), dopamine (DA), and serotonin (5-HT). Therefore, we hypothesized that extracellular concentrations of these monoamines as well as cardiovascular responses during static skeletal muscle contraction would be modulated following administration of [D-Ala(2)]methionine enkephalinamide (DAME), an opioid receptor agonist, into the RVLM. Microdialysis of 100 microM DAME into the RVLM of 10 rats significantly (P<0.01) decreased extracellular levels (in pg/10 microl) of NE (from 3.3+/-0.3 to 1.9+/-0.3), DA (from 5.5+/-0.2 to 3.7+/-0.3), and 5-HT (from 6.1+/-0.8 to 3.6+/-0.2) during static exercise. After microdialysis of DAME, the exercise pressor reflex also significantly (P<0.01) decreased mean arterial pressure (MAP) by 13+/-3 mmHg and heart rate (HR) by 16+/-6 bpm, compared with control (MAP=22+/-4 mmHg and HR=31+/-7 bpm). Subsequently, after 30 min microdialysis of naloxone, an opioid receptor antagonist, muscle contraction increased the extracellular monoamine levels (in pg/10 microl, 3.8+/-0.3 NE; 5.2+/-0.3 DA; and 5.5+/-0.4 5-HT) similar to the control groups and evoked a reversal of cardiovascular responses. Similarly, 30 min of microdialyzing naloxone, added to the perfusing medium containing DAME, reversed the attenuating effects of DAME on monoamines, MAP, and HR during a muscle contraction. Furthermore, microdialysis of 100 microM naloxone alone for 30 min potentiated cardiovascular responses and monoamine levels during a muscle contraction. In summary, the present data demonstrates that microdialysis of DAME into RVLM attenuates the exercise pressor reflex mediated increases in MAP, HR and extracellular levels of biogenic monoamines. A subsequent microdialysis of naloxone reversed the effects suggesting that an opioidergic mechanism within RVLM modulates the exercise pressor reflex. Overall, the present study provides further insights into the opioidergic modulation of the exercise pressor reflex.

Animals↗

PubMed2001

Animal models of neonatal stroke.

Neonatal stroke occurs in approximately 1 in 4,000 to 1 in 10,000 newborns, and more than 80% involve the vascular territory supplied by the middle cerebral artery. Neonatal stroke is associated with many acquired and genetic prothrombotic factors, and follow-up studies indicate that as many as two thirds of neonates develop neurologic deficits. In the past two decades unilateral carotid occlusion with 8% hypoxia has been used to study focal and global ischemia in the newborn, and recently a filament model of middle cerebral artery occlusion has been developed. This review describes the results of studies in these two newborn models covering aspects of the injury cascade that occurs after focal ischemia. A likely requirement is that therapeutic efforts be directed less at using thrombolytic therapy and more toward treatment of events associated with reperfusion injury, the inflammatory cascade, and apoptosis. Additional areas of research that have received attention in the past year include inhibition of nitric oxide and free-radical formation, use of iron chelating agents, the potential role of hypoxia-inducible factors and mediators of caspase activity, use of growth factors, hypothermia, and administration of magnesium sulfate.

Animals↗

PubMed2001

Developmental changes in ryanodine- and IP(3)-sensitive Ca(2+) pools in ovine basilar artery.

To explore the hypothesis that cerebrovascular maturation alters ryanodine- and inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) pool sizes, we measured total intracellular Ca(2+) with (45)Ca and the fractions of intracellular Ca(2+) released by IP(3) and/or caffeine in furaptra-loaded permeabilized basilar arteries from nonpregnant adult and term fetal (139-141 days) sheep. Ca(2+) mass (nmol/mg dry weight) was similar in adult (1.60 +/- 0.18) and fetal (1.71 +/- 0.16) arteries in the pool sensitive to IP(3) alone but was significantly lower for adult (0.11 +/- 0.01) than for fetal (1.22 +/- 0.11) arteries in the pool sensitive to ryanodine alone. The pool sensitive to both ryanodine and IP(3) was also smaller in adult (0.14 +/- 0.01) than in fetal (0.85 +/- 0.08) arteries. Because the Ca(2+) fraction in the ryanodine-IP(3) pool was small in both adult (5 +/- 1%) and fetal (7 +/- 4%) arteries, the IP(3) and ryanodine pools appear to be separate in these arteries. However, the pool sensitive to neither IP(3) nor ryanodine was 10-fold smaller in adult (0.87 +/- 0.10) than in fetal (8.78 +/- 0.81) arteries, where it accounted for 72% of total intracellular membrane-bound Ca(2+). Thus, during basilar artery maturation, intracellular Ca(2+) mass plummets in noncontractile pools, decreases modestly in ryanodine-sensitive pools, and remains constant in IP(3)-sensitive pools. In addition, age-related increases in IP(3) efficacy must involve factors other than IP(3) pool size alone.

Animals↗

PubMed2001

Maturation depresses cGMP-mediated decreases in [Ca2+]i and Ca2+ sensitivity in ovine cranial arteries.

Because cerebrovascular cGMP levels vary significantly during maturation, we examined the hypothesis that the ability of cGMP to relax cerebral arteries also changes during maturation. In concentration-response experiments, potassium-induced tone in basilar arteries was significantly more sensitive to a nonmetabolizable cell-permeant cGMP analogue 8-(p-chlorophenylthio)-cGMP (8-pCPT-cGMP) in term fetal [-log one-half maximal concentration (EC(50)) = 4.4 +/- 0.1 M] than in adult (-log EC(50) = 4.0 +/- 0.1 M) ovine basilar arteries. Serotonin-induced tone also revealed significantly greater sensitivity to the cGMP analogue in fetal (-log EC(50) = 4.9 +/- 0.1 M) than in adult (-log EC(50) = 4.7 +/- 0.1 M) basilars. In fura 2-loaded preparations, 8-pCPT-cGMP had no significant effect on cytosolic calcium concentrations in potassium-contracted arteries but at 6 microM significantly reduced calcium only in fetal basilars (Delta = 33 +/- 8%). Higher 8-pCPT-cGMP concentrations reduced cytosolic calcium in both fetal and adult basilars. Similarly, in both potassium- and 5-hydroxytryptamine (5-HT)-contracted preparations, low concentrations of 8-pCPT-cGMP reduced myofilament calcium sensitivity only in fetal basilars (Delta = 29 +/- 6 and Delta = 42 +/- 10%, respectively), whereas higher concentrations reduced calcium sensitivity in both fetal and adult arteries. In beta-escin-permeabilized arteries, equivalent reductions in basal and agonist-enhanced myofilament calcium sensitivity were produced by much lower 8-pCPT-cGMP concentrations in fetal (172 and 61 microM, respectively) than in adult (410 and 231 microM, respectively) basilars. The mechanisms mediating cGMP-induced vasorelaxation appear similar in fetal and adult arteries, with the exception that they are much more sensitive to cGMP in fetal than adult arteries. These age-related differences in the sensitivity of cytosolic calcium concentration, basal, and agonist-enhanced myofilament calcium sensitivity to cGMP can easily explain why both potassium- and 5-HT-induced tone are more sensitive to cGMP in fetal than adult cerebral arteries.

Animals↗

PubMed2001

Pregnancy enhances endothelium-dependent relaxation of ovine uterine artery: role of NO and intracellular Ca(2+).

The present study tested the hypothesis that the pregnancy-associated increase in endothelium-dependent relaxation of the uterine artery was mediated primarily by an increase in nitric oxide (NO) release, resulting in a reduction in smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)). Uterine arteries obtained from nonpregnant and near-term (140 days gestation) pregnant sheep were used. The Ca(2+) ionophore A23187 induced endothelium-dependent relaxations in both nonpregnant and pregnant uterine arteries, with an increased relaxation in the pregnant tissue. In contrast, endothelium-independent relaxations induced by sodium nitroprusside were the same in nonpregnant and pregnant arteries. In addition, removal of the endothelium significantly increased noradrenaline-induced contractions in pregnant, but not nonpregnant, uterine arteries. In accordance, pregnancy increased both basal and A23187-stimulated NO releases in the uterine artery. Simultaneous measurement of tension and [Ca(2+)](i) in the smooth muscle demonstrated a linear correlation with the slope of unity between A23187-induced relaxation and the reduction of [Ca(2+)](i) in both nonpregnant and pregnant uterine arteries. The A23187-induced reduction of [Ca(2+)](i) was significantly enhanced in pregnant, compared with nonpregnant, uterine arteries. The results indicate that pregnancy increases NO release, which, through decreasing [Ca(2+)](i) in the smooth muscle, accounts for the increased endothelium-dependent relaxation of the uterine artery. Signal transduction pathways distal to NO production are not changed by pregnancy.

Animals↗

PubMed2001

Effects of maturation and acute hypoxia on receptor-IP(3) coupling in ovine common carotid arteries.

Whereas previous studies have established that many mechanisms mediating pharmacomechanical coupling are subject to regulation, evidence of physiological regulation of the coupling efficiency between receptor activation and second-messenger production is scarce. The present studies address the hypothesis that acute hypoxia and maturation can influence the mass of second-messenger production for each activated agonist-bound receptor ("receptor gain"). For this assessment, receptor density and agonist affinity values were used to calculate 5-hydroxytryptamine (5-HT) concentrations that would produce standardized numbers of bound receptors (8.5 fmol/mg protein) in each experimental group and thus minimize effects of age or hypoxia on receptor density or agonist affinity. After 3 min of exposure to these 5-HT concentrations, normoxic magnitudes of contraction were similar (as %potassium maxima) in fetal (50 +/- 14%) and adult (40 +/- 9%) arteries, but hypoxia (PO(2) approximately 9--12 Torr for 30 min) depressed contractile tensions with a significantly different time course and magnitude in fetal (30 +/- 10%) and adult (17 +/- 11%) arteries (P < 0.05). Basal inositol 1,4,5-trisphosphate (IP(3)) values (in pmol/mg protein) were significantly greater in fetal (94 +/- 16) than in adult (44 +/- 6) arteries, and integrated areas above baseline for the IP(3) time courses (in nmol-s/mg protein) were significantly greater in fetal than in adult arteries both in normoxic (14.3 +/- 1.8 vs. 9.1 +/- 1.6) and hypoxic (15.0 +/- 2.1 vs. 8.6 +/- 1.2) conditions (P < 0.05). Hypoxia altered the IP(3) time courses both in the fetus and the adult but had no significant effect on IP(3 )mobilization or receptor gain. These data demonstrate that for the 5-HT(2a) receptor predominant in this preparation, receptor gain can be experimentally determined, is not influenced by acute hypoxia, but is greater in fetal than in adult ovine carotid arteries.

Animals↗

PubMed2001

Effects of maturation on mechanisms of cGMP-induced cerebral vasodilatation.

In light of observations that cerebrovascular levels of cGMP vary during maturation, the present study examines the possibility that the mechanisms mediating cGMP-induced cerebral vasodilatation also change during maturation. Specifically, these experiments explore age-related changes in the ability of cGMP to both: (1) depress cytosolic calcium concentration, and (2) attenuate contractile protein calcium sensitivity in alpha-toxin and beta-escin permeabilized preparations as well as fura-2 loaded arteries. The present data demonstrate that: (1) cGMP attenuates cytosolic calcium concentration at lower concentrations than required to reduce myofilament calcium sensitivity; (2) both potassium-induced and 5HT-induced contractions were more sensitive to cGMP in fetal than adult arteries; (3) all potassium-induced increases in cytosolic calcium were resistant to the effects of cGMP, but those produced by 5HT were sensitive to attenuation by cGMP, and more so in fetal than in adult basilar arteries, and (4) cGMP attenuated both basal and agonist-enhanced myofilament calcium sensitivity. Overall, these data demonstrate that the mechanisms mediating the multiple vasoactive effects of cGMP are more potent in immature than in mature cerebral arteries and are heavily influenced by both the artery type and the method of contraction.

Animals↗

PubMed2000

Maturation attenuates the effects of cGMP on contraction, [Ca2+]i and Ca2+ sensitivity in ovine basilar arteries.

The present study explores the hypothesis that age-related variations in cerebrovascular responses to vasodilators reflect corresponding age-dependent differences in the mechanisms coupling changes in cytosolic cGMP to vasorelaxation. The experiments focused on cGMP's ability to decrease either [Ca2+]i or myofilament Ca2+ sensitivity, because both effects can contribute to cGMP-induced vasodilation. Use of the cGMP analog 8-pCPT-cGMP minimized problems associated with limited cell permeation or cGMP hydrolysis. In fetal basilars contracted with 10 microM serotonin, the EC30 for 8-pCPT-cGMP-induced relaxation was 6 microM. In fura-2 loaded fetal basilars, pretreatment with 6 microM 8-pCPT-cGMP significantly depressed the sensitivity of [Ca2+]i to 5HT, and also myofilament sensitivity to calcium, but only in fetal arteries. In fetal basilar arteries contracted with 120 mM potassium, the EC30 for 8-pCPT-cGMP-induced relaxation was 25 microM. In fura-2 loaded ovine arteries, pretreatment with 25 microM 8-pCPT-cGMP had no effect on the ability of graded concentrations of potassium to elevate [Ca2+]i but reduced potassium's ability to induce contraction and attenuated myofilament calcium sensitivity; these latter effects were significant only in fetal arteries. In alpha-toxin permeabilized preparations, 25 microM 8-pCPT-cGMP significantly depressed both basal- and agonist-stimulated myofilament calcium sensitivity, only in fetal but not in adult basilars. Together, these results demonstrate that: (1) sensitivity to cGMP is greater in fetal than adult sheep arteries independent of method of contraction; (2) cGMP can reduce [Ca2+]i but only in agonist-contracted and not in potassium-contracted arteries; (3) and cGMP attenuates myofilament calcium sensitivity regardless of method of contraction. Overall, the data demonstrate that variations in the ability of cGMP to produce vasodilatation reflect age-, artery-, and agonist-dependent differences in the combination of mechanisms mediating responses to cGMP.

Age Factors↗

PubMed2000

Acute hypoxia modulates 5-HT receptor density and agonist affinity in fetal and adult ovine carotid arteries.

In light of recent observations that receptor-ligand binding and coupling are physiologically regulated, the present study examined the hypothesis that the direct effects of hypoxia on vascular contractility involve modulation of pharmacomechanical coupling via changes in agonist affinity and/or receptor density. Because the direct effects of hypoxia on vascular smooth muscle contractility can vary with age, we carried out these experiments using both fetal and adult arteries. In common carotid arteries from near-term fetal and adult sheep, hypoxia (PO(2) = 9-12 Torr for 30 min) reduced the maximum responses to potassium by 17.8 +/- 3.5% (fetus) and 20.5 +/- 2.2% (adult), significantly reduced the pD(2) for 5-HT in the fetus (7.01 +/- 0.1 to 6.3 +/- 0.2) but not the adult (6.1 +/- 0.1 to 6.0 +/- 0.1), and significantly reduced 5-HT-induced maximum contractions (as % maximum response to 120 mM K(+)) not in the fetus (from 114 +/- 7 to 70 +/- 10%, not significant) but only in the adult (from 83 +/- 15 to 25 +/- 7%, P < 0.05) arteries. Hypoxia significantly attenuated 5-HT binding affinity (pK(A), determined by partial irreversible blockade with phenoxybenzamine) in both fetal (from 6.5 +/- 0.2 to 6.0 +/- 0.2) and adult arteries (from 6.2 +/- 0. 2 to 5.7 +/- 0.1) and also decreased receptor density (fmol/mg protein, determined by competitive binding with ketanserin and mesulergine) in adult (from 18.3 +/- 1.1 to 10.9 +/- 1.0) but not in fetal (21.0 +/- 1.0 to 23.2 +/- 1.4) arteries. These results suggest that acute hypoxia modulates receptor-ligand binding via age-dependent modulation of agonist affinity and receptor density. These effects may contribute to hypoxic vasodilatation and help explain why the effects of hypoxia on vascular contractility differ between fetuses and adults.

Animals↗

PubMed2000

Maturational differences in soluble guanylate cyclase activity in ovine carotid and cerebral arteries.

Basal cGMP concentrations are greater in immature than in mature cranial arteries, which may help explain why cerebrovascular resistance is lower in neonates than in adults. The present studies explore the hypothesis that this difference derives from age-related differences in soluble guanylate cyclase (sGC) activity. Maturation depressed (p < 0.01) maximal sGC activity (pmol cGMP/mg/min) in both carotid (from 11.10 +/- 0.50 to 3.60 +/- 0.20) and cerebral (from 3.10 +/- 0.31 to 1.45 +/- 0.08) arteries. Western blot analysis of relative sGC abundance (relative to sGC expression in adult kidney) found that sGC abundance was significantly greater (p < 0.05) in newborn carotid (0.38 +/- 0.04) and cerebral arteries (0.37 +/- 0.06) than in adult arteries (0.25 +/- 0.05 and 0.17 +/- 0.03, respectively). Basal Km values in carotid and cerebral arteries did not differ significantly between newborns (3- to 7-d old) and adults. Activation of sGC with nitrosylated heme significantly reduced Km values 3- to 5-fold in both types of artery and in both age groups. Within artery type, maturation had no significant effect on activated Km. Between artery types, activated Km values were greater (p < 0.05) in cerebral (200 +/- 40 microM) than in carotid (80 +/- 10 microM) arteries. Together, these data suggest that variations in sGC substrate affinity contribute to observed differences in sGC activity between artery types but not those between age groups. In contrast, variations in enzyme abundance, and possibly also enzyme-specific activity, appear responsible for differences in sGC activity associated with both age and artery type.

Animals↗

PubMed1999

Endothelial nitric oxide release in isolated perfused ovine uterine arteries: effect of pregnancy.

The present study was designed to determine the release of endothelial nitric oxide, measured as combined nitric oxide, nitrite and nitrate (NOx), in isolated perfused uterine arteries obtained from nonpregnant and pregnant sheep. Noradrenaline produced concentration-dependent increases in perfusion pressure in both nonpregnant and pregnant uterine arteries with pD2 values of 5.1+/-0.07 and 4.6+/-0.04, respectively. The maximum responses were 300.8+/-8.8 mmHg for nonpregnant arteries and 86.9+/-1.3 mmHg for pregnant ones. N(G)-nitro-L-arginine increased noradrenaline-mediated maximum response in the pregnant (86.9+/-1.3 to 144.6+/-5.1 mmHg), but not in the nonpregnant, uterine arteries. The basal level of NOx was significantly higher in pregnant than in nonpregnant uterine arteries (346.1+/-63.2 vs. 86.0+/-20.6 pmol/ml). The calcium ionophore A23187 and adenosine triphosphate produced concentration-dependent increases in NOx release in both nonpregnant and pregnant arteries. Compared to the nonpregnant tissue, the agonist-induced increase in NOx release was significantly enhanced in the pregnant uterine artery. In accordance, endothelial NO synthase protein expression in pregnant uterine artery was 197% of that in nonpregnant artery. These data indicate that in the uterine artery, pregnancy increases both basal and agonist-induced release of endothelial nitric oxide, which is likely to play a key role in attenuated vascular reactivity of the uterine artery to vasoconstrictors during the course of pregnancy.

Adenosine Triphosphate↗

PubMed1999

Effects of maturation on adrenergic neurotransmission in ovine cerebral arteries.

The present studies examine the hypothesis that multiple adrenergic neuroeffector mechanisms are not fully developed in fetal, compared with adult, ovine middle cerebral arteries. In arteries denuded of endothelium and pretreated with 1 microM atropine to block involvement of muscarinic receptors, 10 microM capsaicin to deplete sensory peptidergic neurons, and 10 microM nitro-L-arginine methyl ester (L-NAME) to block possible influences from nitric oxidergic innervation, transmural stimulation at 16 Hz increased contractile tensions to 9.5 +/- 3.7% (n = 6) of the potassium maximum in adult arteries. Corresponding values in fetal arteries, however, were significantly less and averaged only 1.1 +/- 0.6% (n =10). However, postsynaptic sensitivity to norepinephrine (NE) was similar in the two age groups; NE pD(2) values (-log EC(50)) averaged 6.11 +/- 0.12 (n = 6) and 6.33 +/- 0.09 M (n = 9) in fetal and adult arteries, respectively. Similarly, NE content measured via HPLC was also similar in the two age groups and averaged 32.4 +/- 5.0 (n = 17) and 32.5 +/- 3.9 ng/ng wet wt (n = 13) in fetal and adult middle cerebral arteries, respectively. In contrast, stimulation-induced NE release was greater in fetal than in adult arteries, whether calculated as total mass released [883 +/- 184 (n = 17) vs. 416 +/- 106 pg NE/mg wet wt (n = 13)] or as fractional release [51.1 +/- 5.3 (n = 17) vs. 22.8 +/- 3.8 pg/pg NE content per pulse x 10(-6)]. Measured as an index of synaptic density, neuronal cocaine-sensitive NE uptake was similar in fetal and adult arteries [1.55 +/- 0.40 (n = 10) and 1.84 +/- 0.51 pmol/mg wet wt (n = 7), respectively]. Overall, age-related differences in postsynaptic sensitivity to NE, NE release, and NE uptake capacity cannot explain the corresponding age-related differences in response to stimulation. The data thus suggest that total synaptic volume and cleft width, in particular, are probably greater and/or that adrenergic corelease of vasoactive substances other than NE is altered in fetal compared with adult middle cerebral arteries.

Adrenergic Fibers↗

PubMed1999

Core and penumbral nitric oxide synthase activity during cerebral ischemia and reperfusion in the rat pup.

Our studies examined the hypothesis that the distribution of cerebral injury after a focal ischemic insult in the immature rat pup is associated with the regional distribution of nitric oxide synthase (NOS) activity and that differences in the vulnerability to ischemia between pup and adult might be related to differences in cofactor availability. We measured NOS activity in well-defined regions prone to become either core or penumbra in controls and at different times (end of occlusion, 0.5 h, and 24 h reperfusion) after middle cerebral artery occlusion (MCAO) from the right and left hemispheres in a 14- to 18-day-old rat pup filament model. Three groups of corresponding isoflurane sham controls were also included. "Core" NOS activity for combined right and left hemispheres ranged from 113% to 217% more than "penumbral" regions in control and sham groups. In the three MCAO groups, marked decreases in ischemic core and penumbral NOS activity were seen; however, core NOS remained higher than penumbral regions bilaterally. The effects of cofactor addition (10 microM tetrahydrobiopterin, 3 microM flavin adenine dinucleotide, and 3 microM flavin mononucleotide) on NOS activity were similar in "core" and "penumbral" regions in control and sham groups. However, after 24 h MCAO, cofactor addition preferentially increased NOS activity in the ischemic hemisphere. Co-factor addition in the pup also had a greater effect on enhancing NOS activity in all regions compared with the adult. Greater NOS activity in core regions in the rat pup, as in the adult, could in part, explain the increased vulnerability of that region to ischemia. NOS activity also can be influenced by the availability of cofactors and this effect may be greater in the immature animal.

Animals↗