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Biomedical subjects

Lawrence D Longo

Publications and source records attributed to Lawrence D Longo.

At least 19 recordsLinked to original sources
PubMed2006

Role of Rho kinases in PKG-mediated relaxation of pulmonary arteries of fetal lambs exposed to chronic high altitude hypoxia.

An increase in Rho kinase (ROCK) activity is implicated in chronic hypoxia-induced pulmonary hypertension. In the present study, we determined the role of ROCKs in cGMP-dependent protein kinase (PKG)-mediated pulmonary vasodilation of fetal lambs exposed to chronic hypoxia. Fourth generation pulmonary arteries were isolated from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days and from control ewes. In vessels constricted to endothelin-1, 8-bromoguanosine-cGMP (8-Br-cGMP) caused a smaller relaxation in chronically hypoxic (CH) vessels compared with controls. Rp-8-Br-PET-cGMPS, a PKG inhibitor, attenuated relaxation to 8-Br-cGMP in control vessels to a greater extent than in CH vessels. Y-27632, a ROCK inhibitor, significantly potentiated 8-Br-cGMP-induced relaxation of CH vessels and had only a minor effect in control vessels. The expression of PKG was increased but was not accompanied with an increase in the activity of the enzyme in CH vessels. The expression of type II ROCK and activity of ROCKs were increased in CH vessels. The phosphorylation of threonine (Thr)696 and Thr850 of the regulatory subunit MYPT1 of myosin light chain phosphatase was inhibited by 8-Br-cGMP to a lesser extent in CH vessels than in controls. The difference was eliminated by Y-27632. These results suggest that chronic hypoxia in utero attenuates PKG-mediated relaxation in pulmonary arteries, partly due to inhibition of PKG activity and partly due to enhanced ROCK activity. Increased ROCK activity may inhibit PKG action through increased phosphorylation of MYPT1 at Thr696 and Thr850.

Amides↗
PubMed2006

Cerebral blood flow and oxygenation in ovine fetus: responses to superimposed hypoxia at both low and high altitude.

For the fetus, although the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (CBF) and cerebral oxygenation, the relation of CBF, cortical tissue P(O2) (tP(O2)), sagittal sinus P(O2), and related indices of cerebral oxygenation to arterial blood gases are not well defined. This is particularly true for that fetus subjected to long-term hypoxia (LTH). In an effort to elucidate these interrelations, we tested the hypothesis that in the fetus acclimatized to high altitude, cerebral oxygenation is not compromised relative to that at low altitude. By use of a laser Doppler flowmeter with a fluorescent O2 probe, in near-term fetal sheep at low altitude (n = 8) and those acclimatized to high altitude hypoxia (3801 m for 90 +/- 5 days; n = 6), we measured laser Doppler CBF (LD-CBF), tP(O2), and related variables in response to 40 min superimposed hypoxia. At both altitudes, fetal LD-CBF, cerebral O2 delivery, tP(O2), and several other variables including sagittal sinus P(O2), correlated highly with arterial P(O2) (P(a,O2)). In response to superimposed hypoxia (P(a,O2) = 11 +/- 1 Torr), LD-CBF was significantly blunted at high altitude, as compared with that at low altitude. In the two altitude groups fetal cerebral oxygenation was similar under both control conditions and with superimposed hypoxia, cortical tP(O2) decreasing from 8 +/- 1 and 6 +/- 1 Torr, respectively, to 2 +/- 1 Torr. Also, for these conditions sagittal sinus P(O2) and [HbO2] values were similar. In response to superimposed hypoxia, cerebral metabolic rate for O(2) decreased approximately 50% in each group (P < 0.05). For both the fetus at low altitude and that acclimatized to high altitude LTH, we present the first dose-response data on the relation of LD-CBF, cortical tP(O2), and sagittal sinus blood gas values to P(a,O2). In addition, despite differences in several variables, the fetus at high altitude showed evidence of successful acclimatization, supporting the hypothesis that such fetuses demonstrate no compromise in cerebral oxygenation.

Altitude↗
PubMed2006

Fetal hypercapnia and cerebral tissue oxygenation: studies in near-term sheep.

The precise role of CO2 in cerebral oxygenation is not as well defined as O2, especially in the immature brain. In the ovine fetus, we tested the hypotheses that arterial Pco2 (Paco2) plays a critical role not only in the regulation of cerebral blood flow but also in the regulation of cerebral tissue oxygenation. By use of a fluorescent O2 probe with a laser Doppler flowmeter and the placement of sagittal sinus catheter in six near-term fetal sheep, we measured values of cortical tissue O2 tension (tPo2), sagittal sinus oxyhemoglobin saturation ([HbO2]), and laser Doppler cerebral blood flow (LD-CBF) in response to 20 min hypercapnia induced by having the ewe breathe CO2. In response to moderate to severe hypercapnia, LD-CBF increased above baseline in a curvilinear fashion, cortical tPo2 increased linearly (1 torr per 3.2 torr Paco2), and sagittal sinus [HbO2] increased significantly in a curvilinear manner. Hypercapnia favored cerebral tissue oxygenation of the fetal brain; and cortical tPo2 and sagittal sinus [HbO2] complement or support one another as indices of cerebral oxygenation under hypercapnic conditions.

Animals↗
PubMed2006

Regulation of alpha1-adrenoceptor-mediated contractions of uterine arteries by PKC: effect of pregnancy.

Protein kinase C (PKC) plays an important role in the regulation of uterine artery contractility and its adaptation to pregnancy. The present study tested the hypothesis that PKC differentially regulates alpha(1)-adrenoceptor-mediated contractions of uterine arteries isolated from nonpregnant (NPUA) and near-term pregnant (PUA) sheep. Phenylephrine-induced contractions of NPUA and PUA sheep were determined in the absence or presence of the PKC activator phorbol 12,13-dibutyrate (PDBu). In NPUA sheep, PDBu produced a concentration-dependent potentiation of phenylephrine-induced contractions and shifted the dose-response curve to the left. In contrast, in PUA sheep, PDBu significantly inhibited phenylephrine-induced contractions and decreased their maximum response. Simultaneous measurement of contractions and intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in the same tissues revealed that PDBu inhibited phenylephrine-induced [Ca(2+)](i) and contractions in PUA sheep. In NPUA sheep, PDBu increased phenylephrine-induced contractions without changing [Ca(2+)](i). Western blot analysis showed six PKC isozymes, alpha, beta(I), beta(II), delta, epsilon, and zeta, in uterine arteries, among which beta(I), beta(II), and zeta isozymes were significantly increased in PUA sheep. In contrast, PKC-alpha was decreased in PUA sheep. In addition, analysis of subcellular distribution revealed a significant decrease in the particulate-to-cytosolic ratio of PKC-epsilon in PUA compared with that in NPUA sheep. The results suggest that pregnancy induces a reversal of PKC regulatory role on alpha(1)-adrenoceptor-mediated contractions from a potentiation in NPUA sheep to an inhibition in PUA sheep. The differential expression of PKC isozymes and their subcellular distribution in uterine arteries appears to play an important role in the regulation of Ca(2+) mobilization and Ca(2+) sensitivity in alpha(1)-adrenoceptor-mediated contractions and their adaptation to pregnancy.

Adrenergic alpha-1 Receptor Agonists↗
PubMed2006

Maternal oxygen administration and fetal cerebral oxygenation: studies on near-term fetal lambs at both low and high altitude.

OBJECTIVE: We tested the hypothesis that O2 administration to the mother would increase arterial O2 tension (PaO2) and cortical tissue O2 tension (tPO2) in both the normoxic control fetus and that acclimatized to high altitude, and that this effect might be greater in the high altitude fetus than that at sea level. STUDY DESIGN: Pregnant ewes were divided into either low-altitude group or high-altitude group (3820 m; maintained from day 30 of gestation to near-term, term = 145 days; n = 6 each). In near-term fetuses we instrumented the cerebral cortex with tissue PO2-laser Doppler flow probes, and placed arterial catheters. With the ewe breathing either air or 50% O2, we measured fetal arterial and brain tissue PO2 and cerebral blood flow (CBF). RESULTS: In response to maternal O2 administration, in both low- and high-altitude fetuses, PaO2, O2 content, and brain tissue PO2 increased significantly. Nonetheless, the fetuses at high altitude showed significantly greater increase in both PaO2 and cerebral tPO2, than that seen in fetuses at low altitude. In neither group was there a change in CBF or cerebral metabolic rate for O2. CONCLUSION: Maternal O2 administration significantly increased PaO2 and cerebral tPO2 of the fetus both at low altitude and at high altitude, with this increase being greater in the high altitude animals. We suggest that maternal O2 administration can have an important effect in ameliorating nonreassuring fetal status, and perhaps be of value in instances of mothers who smoke heavily or with cardiopulmonary disease.

Acclimatization↗
PubMed2006

Regulation of baseline Ca2+ sensitivity in permeabilized uterine arteries: effect of pregnancy.

The adaptation of contractile mechanisms of the uterine artery to pregnancy is not fully understood. The present study examined the effect of pregnancy on the uterine artery baseline Ca2+ sensitivity. In beta-escin-permeabilized arterial preparations, Ca2+ -induced concentration-dependent contractions were significantly decreased in uterine arteries from pregnant animals compared with those of nonpregnant animals. Time-course studies showed that Ca2+ increased phosphorylation of 20-kDa myosin light chain (MLC20), which preceded the tension development in vessels from both pregnant and nonpregnant animals. When compared with vessels from nonpregnant animals, there was a significant increase in the protein level of MLC20 and an accordance increase in the level of Ca2+ -induced phosphorylated MLC20 (MLC20-P) in uterine arteries during pregnancy. Simultaneous measurements of MCL20-P levels and contractions stimulated with Ca2+ in the same tissues demonstrated a significant attenuation in the tension-to-MLC20-P ratio in uterine arteries during pregnancy. Activation of PKC with phorbol 12,13-dibutyrate (PDBu) potentiated Ca2+ -induced contractions in uterine arteries from nonpregnant but not pregnant animals. Accordingly, inhibition of PKC attenuated Ca2+ -induced contractions in uterine arteries from nonpregnant but not pregnant animals. PDBu produced contractions in the presence or absence of Ca2+ in the beta-escin-permeabilized arteries, which were significantly decreased in uterine arteries from pregnant compared with nonpregnant animals. The results suggest that pregnancy upregulates the thick-filament regulatory pathway by increasing MLC20 phosphorylation but downregulates the thin-filament regulatory pathway by decreasing the contractile sensitivity of MLC20-P, resulting in attenuated baseline Ca2+ sensitivity in the uterine artery. In addition, PKC plays an important role in the regulation of basal Ca2+ sensitivity, which is downregulated during pregnancy.

Animals↗
PubMed2006

Gene expression patterns in the developing murine placenta.

OBJECTIVE: Successful placental development is crucial for optimal growth, maturation, and survival of the embryo/fetus. To examine genetic aspects of placental development, we investigated gene expression patterns in the murine placenta at embryonic day 10.5 (E10.5), E12.5, E15.5, and E17.5. METHODS: By use of the Affymetrix MU74A array (Affymetrix, Santa Clara, CA), we measured expression levels for 12,473 probe sets. Using pairwise analysis we selected 622 probe sets, corresponding to 599 genes, that were up- or down-regulated by more than fourfold between time points E10.5 and E12.5, E12.5 and E15.5, E15.5 and E17.5. We analyzed and functionally annotated those genes regulated during development. RESULTS: In comparing E10.5 to E12.5 we found that angiogenesis and fatty acid metabolism and transport related genes were up-regulated at E10.5, while genes involved in hormonal control and ribosomal proteins were up-regulated at E12.5. When comparing E12.5 to E15.5 we noted that genes involved in the cell cycle and RNA metabolism were strongly up-regulated at E12.5, while genes involved in cellular transport were up-regulated at E15.5. Finally, when comparing E15.5 to E17.5, we found genes related to cell cycle control, genes expressed in the nucleus and involved in RNA metabolism were up-regulated at E17.5. CONCLUSION: Microarray analysis has allowed us to describe gene expression patterns and profiles in the developing mouse placenta. Further analysis has demonstrated that several functional classes are up- and down-regulated at specific time points in placental development. These changes may have significant implications for placental development in the human.

Animals↗
PubMed2006

Modulation of BK channel calcium affinity by differential phosphorylation in developing ovine basilar artery myocytes.

Large-conductance Ca2+-sensitive K+ (BK) channel activity is greater in basilar artery smooth muscle cells (SMCs) of the fetus than the adult, and this increased activity is associated with a lower BK channel Ca2+ set point (Ca0). Associated PKG activity is three times greater in BK channels from fetal than adult myocytes, whereas associated PKA activity is three times greater in channels from adult than fetal myocytes. We hypothesized that the change in Ca0 during development results from different levels of channel phosphorylation. In inside-out membrane patch preparations of basilar artery SMCs from adult and fetal sheep, we measured BK channel activity in four states of phosphorylation: native, dephosphorylated, PKA phosphorylated, and PKG phosphorylated. BK channels from adult and fetus exhibited similar voltage-activation curves, Ca0 values, and Ca2+ dissociation constants (Kd) for the dephosphorylated, PKA phosphorylated, and PKG phosphorylated states. However, voltage-activation curves of native fetal BK channels shifted significantly to the left of those of the adult, with Ca0 and Kd values half those of the adult. For the two age groups at each of the phosphorylation states, Ca0 and Kd produced linear relations when plotted against voltage at half-maximal channel activation. We conclude that the Ca0 and Kd values of the BK channel can be modulated by differential channel phosphorylation. Lower Ca0 and Kd values in BK channels of fetal myocytes can be explained by a greater extent of channel phosphorylation of fetal than adult myocytes.

Aging↗
PubMed2006

Beyond the physician charter: reflections on medical professionalism.

The numerous challenges now facing the profession of medicine have led to an intense focus on professionalism by individual physicians and by their professional and academic organizations. In 2002, a distinguished group of leaders in internal medicine created the Physician Charter, which calls on physicians to reaffirm medical professionalism through commitment to three principles and 10 responsibilities. The Charter reflects a duty-based ethic that is chiefly concerned with physician competence. This article offers a critical analysis of the Physician Charter from the perspective of the traditional values of medicine as articulated in medical oaths and championed by leaders of past generations, exemplified by William Osler. The authors argue that medical professionalism should reflect the values of a virtue-based ethic that stresses compassion and beneficence, rather than the values of a duty-based ethic. The challenges that now confront the practice of medicine can be addressed successfully only to the extent that physicians promote virtue-ethics, act collectively in the public interest, and render service that clearly transcends their own self-interests.

Codes of Ethics↗
PubMed2005

Ca2+-activated K+ channel-associated phosphatase and kinase activities during development.

In ovine basilar arterial smooth muscle cells (SMCs), the fetal "big" Ca2+-activated K+ (BK) channel activity is significantly greater and has a lower Ca2+ setpoint than BK channels from adult cells. In the present study, we tested the hypothesis that these differences result from developmentally regulated phosphorylation of these channels. Using the patch-clamp technique and a novel in situ enzymological approach, we measured the rates and extents of changes in BK channel voltage activation from SMC inside-out patch preparations in response to selective activation and inhibition of channel-associated protein phosphatases and kinases (CAPAKs). We show that BK channel activity is modulated during development by differential phosphorylation and that the activities of CAPAKs change substantially during development. In particular, excised membrane patches from adult SMCs exhibited greater protein kinase A activity than those from a fetus. In contrast, fetal SMCs exhibited greater protein kinase G activity and phosphatase activity than adult SMCs. These findings extend our previous observation that the BK channel Ca2+ setpoint differs significantly in adult and fetal cerebrovascular myocytes and suggest a biochemical mechanism for this difference. In addition, these findings suggest that the functional stoichiometry of CAPAKs varies significantly during development and that such variation may be a hitherto unrecognized mechanism of ion channel regulation.

Animals↗
PubMed2005

Alpha1-adrenoceptor-mediated phosphorylation of MYPT-1 and CPI-17 in the uterine artery: role of ERK/PKC.

We previously demonstrated that ERK/PKC signaling pathways play a key role in regulation of Ca(2+) sensitivity and contractility of the uterine artery. The present study tested the hypothesis that ERK and PKC differentially regulated myosin light chain phosphatase activity by phosphorylation of myosin phosphatase target protein-1 (MYPT-1) and CPI-17. Agonist-induced contractions and phosphorylation of MYPT-1/Thr(696), MYPT-1/Thr(850), and CPI-17/Thr(38) were measured simultaneously in the same tissues of isolated near-term pregnant ovine uterine arteries. Phenylephrine produced time-dependent concurrent increases in the phosphorylation of ERK(44/42) and MYPT-1/Thr(850) that preceded contractions. In addition, phenylephrine induced phosphorylation of CPI-17/Thr(38) that was concurrent with the contractions. In contrast, phenylephrine did not induce phosphorylation of MYPT-1/Thr(696) in the uterine artery. PD-098059 inhibited phosphorylation of ERK(44/42) and the initial peak phosphorylation of MYPT-1/Thr(850) but did not affect CPI-17/Thr(38) phosphorylation. Activation of PKC by phorbol 12,13-dibutyrate induced a time-dependent phosphorylation of CPI-17/Thr(38) that preceded contractions of the uterine artery. In addition, phorbol 12,13-dibutyrate activated PKC-alpha and induced a coimmunoprecipitation of PKC-alpha with caldesmon. The results suggest that phosphorylation of MYPT-1/Thr(850) and CPI-17/Thr(38) play important roles in regulation of agonist-mediated Ca(2+) sensitivity in the uterine artery, in part by ERK and PKC, respectively. In addition, phosphorylated CPI-17 may regulate Ca(2+) sensitivity by interacting with caldesmon and reversing its inhibitory effect on myosin ATPase.

Animals↗
PubMed2005

Fetal cerebrovascular acclimatization responses to high-altitude, long-term hypoxia: a model for prenatal programming of adult disease?

During the past several decades, many risk factors for cerebrovascular and cardiovascular disease have been identified. More recently, it has been appreciated that inadequate nutrition and/or other intrauterine factors during fetal development may play an important role in the genesis of these conditions. An additional stress factor that may "program" the fetus for disease later in life is chronic hypoxia. In studies originally designed to examine the function of developing cerebral arterial function in response to long-term hypoxia (LTH), it has become clear that many cellular and subcellular changes may have important implications for later life. Here we review some of the significant alterations in fetal cerebral artery structure and function induced by high-altitude (3,820 m, 12,470 ft) LTH ( approximately 110 days). LTH is associated with augmentation or upregulation of presynaptic functions, including responses to perivascular (i.e., sympathetic) nerve stimulation, and structural maturational changes. In contrast, many postsynaptic functions related to the Ca(2+)-dependent contractile pathway tend to be downregulated, whereas elements of the Ca(2+)-independent contraction pathway are upregulated. The results emphasize the role of high-altitude LTH in modulating many aspects of electromechanical and pharmacomechanical coupling in the developing cerebral vasculature. A complicating factor is that the regulation of cerebrovascular tone by Ca(2+)-dependent and Ca(2+)-independent pathways changes significantly as a function of maturational age. In addition to highlighting independent regulation of various elements of the signal transduction cascade, the studies demonstrate the potential for LTH to program the fetus for cerebrovascular and other disease as an adult.

Acclimatization↗
PubMed2005

PKC-induced ERK1/2 interactions and downstream effectors in ovine cerebral arteries.

Both protein kinase C (PKC) and extracellular signal-regulated kinases (ERK1/2) are involved in mediating vascular smooth muscle contraction. We tested the hypotheses that in addition to PKC activation of ERK1/2, by negative feedback ERKs modulate PKC-induced contraction, and that their interactions modulate both thick and thin myofilament pathways. In ovine middle cerebral arteries (MCA), we measured isometric tension and intracellular free calcium concentration ([Ca(2+)](i)) responses to PKC stimulation [phorbol 12,13-dibutyrate (PDBu), 3 x 10(-6) M] in the absence or presence of ERK1/2 inhibition (U-0126, 10(-5) M). After PDBu +/- ERK1/2 inhibition, we also examined by Western immunoblot the levels of total and phosphorylated ERK1/2, caldesmon(Ser789), myosin light chain(20) (MLC(20)), and CPI-17. PDBu induced significant increase in tension in the absence of increased [Ca(2+)](i). PDBu also increased phosphorylated ERK1/2 levels, a response blocked by U-0126. In turn, U-0126 augmented PDBu-induced contractions. PDBu also was associated with significant increases in phosphorylated caldesmon(Ser789) and MLC(20) levels, each of which peaked at 5 to 10 min. PDBu also increased phosphorylated CPI-17 levels, which peaked at 2 to 3 min. Rho kinase inhibition (Y-27632, 3 x 10(-7) M) did not alter PDBu-induced contraction. These results support the idea that PKC activation can increase CPI-17 phosphorylation to decrease myosin light chain phosphatase activity. In turn, this increases MLC(20) phosphorylation in the thick filament pathway and increases Ca(2+) sensitivity. In addition, ERK1/2-dependent phosphorylation of caldesmon(Ser789) was not necessary for PDBu-induced contraction and appears not to be involved in the reversal of caldesmon's inhibitory effect on actin-myosin ATPase.

Animals↗
PubMed2004

Expression of several cytoskeletal proteins in ovine cerebral arteries: developmental and functional considerations.

Cytoskeleton proteins play important roles in regulating vascular smooth muscle (VSM) contraction and relaxation. We tested the hypotheses that the expression levels of several of these proteins change significantly during the course of development, and that these changes contribute to age-related changes in contractile responses. In cerebral arteries from 95-day (d) gestation and 140-d fetus, newborn lambs, and adult sheep, by Western immunoblot (n= 5 for each age) we quantified the relative expression of alpha-actin, alpha-tubulin, cyclophilin A, and proliferating cell nuclear antigen (PCNA). In addition, we examined middle cerebral artery tension responses to phenylephrine (PHE) stimulation in the absence or presence of cytochalasin D (3 x 10(-7)m) and nocodazole (3 x 10(-6)m), inhibitors of alpha-actin and alpha-tubulin polymerization, respectively. The expression levels of alpha-actin and cyclophilin A varied little during the course of development. In contrast, alpha-tubulin expression was approximately 2.5-fold greater in both fetal age groups as compared to adult. Also, as compared to adult and as expected, expression of PCNA was several-fold greater in cerebral arteries of the 95-d fetus (x8), 140-d fetus (x 5), and newborn (x 3). In both adult and fetal middle cerebral artery, cytochalasin D-induced inhibition of actin polymerization decreased PHE-induced contraction, to approximately 60 and approximately 40% of control, respectively (despite no significant change in expression level). In contrast, alpha-tubulin inhibition by nocodazole showed little effect on PHE-induced tension (in spite of the age-related decrease in expression). In conclusion, expression levels of alpha-actin, a thin filament protein involved in contraction, remained relatively constant during the course of development, as did the effects of inhibition of its polymerization on contractility. In contrast, alpha-tubulin, important in intracellular protein trafficking, showed a significant age-related decrease in expression and played a relatively minor role in contractility. The present studies suggest that other cytoskeletal structural proteins and/or elements of pharmaco-mechanical coupling are important to developmental differences in cerebrovascular contractility. In addition, the relatively constant expression levels of alpha-actin and cyclophilin A with development, suggest that these are useful internal standards for studies of cytosolic protein expression.

Actin Cytoskeleton↗
PubMed2004

Development affects in vitro vascular tone and calcium sensitivity in ovine cerebral arteries.

We have shown recently that development from neonatal to adult life affects cerebrovascular tone of mouse cerebral arteries through endothelium-derived vasodilatory mechanisms. The current study tested the hypothesis that development from fetal to adult life affects cerebral artery vascular smooth muscle (VSM) [Ca(2+)](i) sensitivity and tone through a mechanism partially dependent upon endothelium-dependent signalling. In pressurized resistance sized cerebral arteries ( approximately 150 microm) from preterm (95 +/- 2 days gestation (95 d)) and near-term (140 +/- 2 days gestation (140 d)) fetuses, and non-pregnant adults, we measured vascular diameter (microm) and [Ca(2+)](i) (nm) as a function of intravascular pressure. We repeated these studies in the presence of inhibition of nitric oxide synthase (NOS; with l-NAME), cyclo-oxygenase (COX; with indomethacin) and endothelium removal (E-). Cerebrovasculature tone (E+) was greater in arteries from 95 d fetuses and adults compared to 140 d sheep. Ca(2+) sensitivity was similar in 95 d fetuses and adults, but much lower in 140 d fetuses. Removal of endothelium resulted in a reduction in lumen diameter as a function of pressure (greater tone) in all treatment groups. [Ca(2+)](i) sensitivity differences among groups were magnified after E-. NOS inhibition decreased diameter as a function of pressure in each age group, with a significant increase in [Ca(2+)](i) to pressure ratio only in the 140 d fetuses. Indomethacin increased tone and increased [Ca(2+)](i) in the 140 d fetuses, but not the other age groups. Development from near-term to adulthood uncovered an interaction between NOS- and COX-sensitive substances that functioned to modulate artery diameter but not [Ca(2+)](i). This study suggests that development is associated with significant alterations in cerebral vascular smooth muscle (VSM), endothelium, NOS and COX responses to intravascular pressure. We speculate that these changes have important implications in the regulation of cerebral blood flow in the developing organism.

Animals↗
PubMed2004

Absence of robust ischemic preconditioning by five 1-minute total umbilical cord occlusions in fetal sheep.

OBJECTIVE: To determine to what extent a series of five 1-minute total umbilical cord occlusions, intended to induce ischemic preconditioning (IP), affects the physiologic responses to a 10-minute total umbilical cord occlusion (damaging insult [DI]) 1 hour later and provides cardio- and neuroprotection. METHODS: In 14 chronically catheterized late gestation fetal sheep (127-131 days' gestation), we performed a 10-minute total umbilical cord occlusion (DI), preceded by a series of five 1-minute total cord occlusions with 2-minute intervals (5CO, n = 7) or sham occlusions (n = 7) 1 hour prior to DI. RESULTS: The 5CO induced a reduction in the arterial partial pressure of oxygen (Po(2)) from 21 +/-1 to 14 +/-3 Torr, arterial O(2) content from 6.9 +/- 0.4 to 3.1 +/- 0.7 vol%, and increases in the partial pressure of carbon dioxide (Pco(2)) from 46 +/- 2 to 58 +/- 3 Torr, and [H(+)] from 43 +/- 1 to 54 +/- 2 nM. 5CO reduced fetal heart rate from 178 +/- 6 to 151 +/- 6 beats per minute (bpm), and increased arterial pressure from 45 +/- 1 to 57 +/- 2 mmHg, cerebral blood flow (CBF) from 100 +/- 3 to 129 +/- 10%, and cerebral heat production (H(brain)) from 25 +/- 2 to 29 +/- 1% degrees C. The responses to DI were not significantly different between the groups without and with 5CO; values for Po(2) were 5.6 +/- 1.5 and 5.8 +/- 1.9 Torr, O(2) content 0.6 +/- 0.1 and 0.8 +/- 0.1 vol%, lactate 10.7 +/- 0.7 and 10.8 +/- 0.7 mM, fetal heart rate 97 +/- 5 and 87 +/- 8 bpm, mean arterial pressure 22 +/- 3 and 21 +/- 2 mmHg, CBF 50 +/- 10 and 36 +/- 5%, and H(brain) 7.0 +/- 1.4 and 5.9 +/- 1.1% degrees C, respectively, except for Pco(2) (126 +/- 4 and 112 +/- 2 Torr) and [H(+)] (126 +/- 3 and 114 +/- 3 nM). Histologic proof of cardio- or neuroprotection by 5CO could not be obtained because five fetuses died before they were to be killed at day 3 after the experiment; two fetuses in the 5CO group demonstrated major histologic damage of myocardium and brain. CONCLUSION: In the late gestation fetal sheep, a series of five 1-minute total umbilical cord occlusions did not result in major changes in physiologic responses to a hypoxic-ischemic DI 1 hour later. In addition, the procedure did not result in robust cardio- and neuroprotection, in contrast to IP reported in adults.

Animals↗
PubMed2004

ERK-mediated uterine artery contraction: role of thick and thin filament regulatory pathways.

We have demonstrated that extracellular signal-regulated kinase (ERK) plays an important role in the regulation of uterine artery contraction. The present study tested the hypothesis that ERK regulates thick and thin filament regulatory pathways in the uterine artery. Isometric tension, intracellular free Ca2+ concentration ([Ca2+]i), and 20-kDa myosin light chain (LC20) phosphorylation were measured simultaneously in uterine arteries isolated from near-term (140 days gestation) pregnant sheep. Phenylephrine produced time-dependent increases in [Ca2+]i and LC20 phosphorylation that preceded the contraction, which were inhibited by the MEK (ERK) inhibitor PD-098059. In addition, PD-098059 decreased the intercept of the regression line of LC20 phosphorylation vs. [Ca2+]i but increased the rate of tension development vs. LC20 phosphorylation. In contrast to phenylephrine, phorbol 12,13-bibutyrate (PDBu) produced contractions without changing [Ca2+]i or LC20 phosphorylation. PD-098059 potentiated PDBu-induced contractions without affecting [Ca2+]i and LC20 phosphorylation. PDBu produced time-dependent increases in phosphorylation of p42 and p44 ERK and ERK-dependent phosphorylation of caldesmon at Ser789 in the uterine artery. PD-098059 blocked PDBu-mediated phosphorylation of p42 and p44 ERK and caldesmon. The results indicate that ERK may regulate force by a dual regulation of thick and thin filaments in uterine artery smooth muscle. ERK potentiates the thick filament regulatory pathway by enhancing LC20 phosphorylation via increases in [Ca2+]i and Ca2+ sensitivity of LC20 phosphorylation. In contrast, ERK attenuates the thin filament regulatory pathway and suppresses contractions independent of changes in LC20 phosphorylation in the uterine artery.

Animals↗