Calcitonin gene-related peptide antagonism attenuates the haemodynamic and glycaemic responses to acute hypoxaemia in the late gestation sheep fetus

The fetal defence to acute hypoxaemia involves cardiovascular and metabolic responses, which include peripheral vasoconstriction and hyperglycaemia. Both these responses are mediated via neuroendocrine mechanisms, which require the stimulation of the sympathetic nervous system. In the adult, accumulating evidence supports a role for calcitonin gene-related peptide (CGRP) in the activation of sympathetic outflow. However, the role of CGRP in stimulated cardiovascular and metabolic functions before birth is completely unknown. This study tested the hypothesis that CGRP plays a role in the fetal cardiovascular and metabolic defence responses to acute hypoxaemia by affecting sympathetic outflow. Under anaesthesia, five sheep fetuses at 0.8 of gestation were surgically instrumented with catheters and a femoral arterial Transonic flow-probe. Five days later, fetuses were subjected to 0.5 h hypoxaemia during either i.v. saline or a selective CGRP antagonist in randomised order. Treatment started 30 min before hypoxaemia and ran continuously until the end of the challenge. Arterial samples were taken for blood gases, metabolic status and hormone analyses. CGRP antagonism did not alter basal arterial blood gas, metabolic, cardiovascular or endocrine status. During hypoxaemia, similar falls in P _a,O2 occurred in all fetuses. During saline infusion, hypoxaemia induced hypertension, bradycardia, femoral vasoconstriction, hyperglycaemia and an increase in haemoglobin, catecholamines and neuropeptide Y (NPY). In contrast, CGRP antagonism markedly diminished the femoral vasoconstrictor and glycaemic responses to hypoxaemia, and attenuated the increases in haemoglobin, catecholamines and NPY. Combined, these results strongly support the hypothesis that CGRP plays a role in the fetal cardiovascular and metabolic defence to hypoxaemia by affecting sympathetic outflow.     

Blunted Sympathetic Vasoconstriction in Contracting Skeletal Muscle of Healthy Humans: is Nitric Oxide Obligatory?

We tested the hypothesis that nitric oxide (NO) is responsible for blunting sympathetic α-adrenergic vasoconstriction in the active muscles of humans (functional sympatholysis). We measured forearm blood flow (Doppler ultrasound) and calculated the reductions in forearm vascular conductance (FVC) in response to α-adrenergic receptor stimulation during rhythmic handgrip exercise and during a control non-exercise vasodilator condition (intra-arterial adenosine), before and after local NO synthase (NOS) inhibition in healthy men. The forearm vasoconstrictor responses to endogenous noradrenaline release (intra-arterial tyramine) were significantly blunted during moderate exercise compared with adenosine, and these vasoconstrictor responses were not restored by NOS inhibition with N ^G-monomethyl- l -arginine ( l -NMMA;   n = 6  ) or N ^G-nitro- l -arginine methyl ester ( l -NAME;   n = 8  ). Similarly, l -NAME did not restore the vasoconstrictor responses to tyramine in contracting muscle during heavy rhythmic handgrip exercise (   n = 4  ). In four additional subjects, we also found that the vasoconstrictor responses evoked by tyramine during exercise or adenosine were repeatable in the absence of NOS inhibition (i.e. time control). Finally, in five subjects the forearm vasoconstrictor responses to direct α₁-adrenergic (phenylephrine) and α₂-adrenergic (clonidine) receptor stimulation were blunted during moderate exercise compared with adenosine; these responses were also unaffected by l -NAME. Taken together, our results demonstrate that NO is not obligatory for functional sympatholysis in contracting skeletal muscles of healthy men.     

Influence of endogenous nitric oxide on sympathetic vasoconstriction in normoxia, acute and chronic systemic hypoxia in the rat

We studied the role of nitric oxide (NO) in blunting sympathetically evoked muscle vasoconstriction during acute and chronic systemic hypoxia. Experiments were performed on anaesthetized normoxic (N) and chronically hypoxic (CH) rats that had been acclimated to 12% O₂ for 3–4 weeks. The lumbar sympathetic chain was stimulated for 1 min with bursts at 20 or 40 Hz and continuously at 2 Hz. In N rats, acute hypoxia (breathing 8% O₂) reduced baseline femoral vascular resistance (FVR) and depressed increases in FVR evoked by all three patterns of stimulation, but infusion of the NO donor sodium nitroprusside (SNP), so as to similarly reduce baseline FVR, did not affect sympathetically evoked responses. Blockade of NO synthase (NOS) with l -NAME increased baseline FVR and facilitated the sympathetically evoked increases in FVR, but when baseline FVR was restored by SNP infusion, these evoked responses were restored. Acute hypoxia after l -NAME still reduced baseline FVR and depressed evoked responses. In CH rats breathing 12% O₂, baseline FVR was lower than in N rats breathing air, but l -NAME had qualitatively similar effects on baseline FVR and sympathetically evoked increases in FVR. SNP similarly restored baseline FVR and evoked responses. Inhibition of neuronal NOS or inducible NOS did not affect baselines, or evoked responses. We propose that in N and CH rats sympathetically evoked muscle vasoconstriction is modulated by tonically released NO, but not depressed by additional NO released on sympathetic activation. The present results suggest that hypoxia-induced blunting of sympathetic vasoconstriction in skeletal muscle is not mediated by NO.     

Role of Nitric Oxide in Hypoxic Cerebral Vasodilatation in the Ovine Fetus

To investigate the role of nitric oxide (NO) in fetal cerebral circulatory responses to acute hypoxia, near-term fetal sheep were instrumented with laser Doppler probes placed in the parasagittal parietal cortices and vascular catheters in the sagittal sinus and brachiocephalic artery. After a 3 day recovery period, responses of cortical blood flow (CBF) to hypoxia were compared with and without inhibition of nitric oxide synthase (NOS). After an initial 30 min baseline period, fetuses were given a bolus followed by a continuous infusion of N ^ω-nitro- l -arginine methyl ester ( l -NAME), or saline vehicle as control. After administration of l -NAME, CBF decreased by 14 ± 6 % (   P < 0.01  ) despite increases in arterial blood pressure of 15 mmHg, resulting in an ∼60 % increase in cerebrovascular resistance. Thirty minutes following initiation of l -NAME or vehicle infusion, fetal systemic hypoxia was induced by allowing the ewes to breathe 10–11 % oxygen. In control fetuses CBF increased progressively to 145 ± 9 % of baseline (   P < 0.01  ) after 30 min, while cortical release of cyclic guanylate cyclase (cGMP), an index of NOS activity, increased 26 ± 8 % (   P < 0.05  ). In contrast, CBF in l -NAME-treated fetuses increased to only 115 % of the reduced CBF baseline, whereas cortical release of cGMP did not change significantly. In summary, basal levels of NO lower resting cortical vascular resistance by ∼15 % in the fetal sheep. Inhibition of NO synthesis attenuates hypoxic cerebral relaxation but does not completely prevent the characteristic increases in CBF. Hypoxic increases in NO directly increase cortical production of cGMP and inhibition of NO synthesis ablates these changes in cGMP.     

Effect of maternal nutrient restriction in early gestation on responses of the hypothalamic-pituitary-adrenal axis to acute isocapnic hypoxaemia in late gestation fetal sheep

Epidemiological and experimental evidence suggests that maternal undernutrition during pregnancy may alter development of fetal organ systems. We have demonstrated previously that fetal hypothalamic-pituitary-adrenal (HPA) axis responses to exogenous corticotropin-releasing hormone (CRH) + arginine vasopressin (AVP), or adrenocorticotrophin hormone (ACTH), are reduced in fetuses of mildly undernourished ewes. To examine these effects further we tested HPA axis responses to acute isocapnic hypoxaemia in fetal sheep at 114-129 days gestation (dGA), following 15% reduction in maternal nutritional intake between 0 and 70 dGA. Fetuses from control (C) and nutrient-restricted (R) ewes were chronically catheterised and plasma ACTH and cortisol responses were determined at 114-115, 120-123 and 126-129 dGA during hypoxaemia (1 h) induced by lowering the maternal inspired O2 fraction (FI,O2). Basal plasma cortisol concentrations and HPA axis responses at 114-115 and 120-123 dGA did not differ between C and R fetuses. At 126-129 dGA, both plasma ACTH (P < 0.01) and cortisol (P < 0.05) responses were smaller in R fetuses compared to C fetuses. Fetal blood gas status, fetal body weight, body proportions and organ weights did not differ between the groups. We conclude that mild maternal undernutrition alters development of the fetal HPA axis producing a reduction in pituitary and adrenal responsiveness to endogenous stimuli.     

Chemoreflexes – physiology and clinical implications

The chemoreflexes are important modulators of sympathetic activation. The peripheral chemoreceptors located in the carotid bodies respond primarily to hypoxaemia. Central chemoreceptors located in the region of the brainstem respond to hypercapnia. Activation of either the hypoxic or hypercapnic chemoreflex elicits both hyperventilation and sympathetic activation. During apnoea, when the inhibitory influence of stretch of the pulmonary afferents is eliminated, there is a potentiation of the sympathetic response to both hypoxia and hypercapnia. This inhibitory influence of the pulmonary afferents is more marked on the sympathetic response to peripheral compared with central chemoreceptor activation. The arterial baroreflexes also have a powerful inhibitory influence on the chemoreflexes. This inhibition is again more marked with respect to the peripheral compared with central chemoreflexes. In patients with hypertension, there is a marked increase in the sympathetic and ventilatory response to hypoxaemia. During apnoea, with elimination of the inhibitory influence of breathing, the sympathetic response in untreated mild hypertensive patients is strikingly greater than that seen in matched normotensive controls. This potentiated peripheral chemoreflex sensitivity in hypertension may be explained in part by impaired baroreflex function in these patients. Enhanced peripheral chemoreflex sensitivity is also evident in patients with obstructive sleep apnoea. This peripheral chemoreflex enhancement is not explained by obesity, as obese individuals have a selective potentiation of the central chemoreceptors with peripheral chemoreflex responses similar to those seen in lean controls. Increased sensitivity to hypoxaemia has important implications in patients with obstructive sleep apnoea who experience repetitive and severe hypoxaemic stress. Tonic activation of the chemoreflex may also contribute to the high levels of sympathetic activity evident even during normoxic daytime wakefulness in sleep apnoea patients. Administration of 100% oxygen in patients with sleep apnoea results in reductions in heart rate, blood pressure and central sympathetic outflow. In patients with heart failure, the central chemoreflex response to hypercapnia is markedly and selectively enhanced. This increased central chemoreflex sensitivity may contribute to the development of central sleep apnoea in heart failure patients. Administration of 100% oxygen does not lower sympathetic activity in patients with heart failure, providing further evidence against any peripheral chemoreflex potentiation. The peripheral and central chemoreflexes have powerful effects on sympathetic activity in both health and disease and may contribute importantly to disease pathophysiology, particularly in conditions such as hypertension, obstructive sleep apnoea and heart failure.     

Chemoreflexes--physiology and clinical implications

The chemoreflexes are important modulators of sympathetic activation. The peripheral chemoreceptors located in the carotid bodies respond primarily to hypoxaemia. Central chemoreceptors located in the region of the brainstem respond to hypercapnia. Activation of either the hypoxic or hypercapnic chemoreflex elicits both hyperventilation and sympathetic activation. During apnoea, when the inhibitory influence of stretch of the pulmonary afferents is eliminated, there is a potentiation of the sympathetic response to both hypoxia and hypercapnia. This inhibitory influence of the pulmonary afferents is more marked on the sympathetic response to peripheral compared with central chemoreceptor activation. The arterial baroreflexes also have a powerful inhibitory influence on the chemoreflexes. This inhibition is again more marked with respect to the peripheral compared with central chemoreflexes. In patients with hypertension, there is a marked increase in the sympathetic and ventilatory response to hypoxaemia. During apnoea, with elimination of the inhibitory influence of breathing, the sympathetic response in untreated mild hypertensive patients is strikingly greater than that seen in matched normotensive controls. This potentiated peripheral chemoreflex sensitivity in hypertension may be explained in part by impaired baroreflex function in these patients. Enhanced peripheral chemoreflex sensitivity is also evident in patients with obstructive sleep apnoea. This peripheral chemoreflex enhancement is not explained by obesity, as obese individuals have a selective potentiation of the central chemoreceptors with peripheral chemoreflex responses similar to those seen in lean controls. Increased sensitivity to hypoxaemia has important implications in patients with obstructive sleep apnoea who experience repetitive and severe hypoxaemic stress. Tonic activation of the chemoreflex may also contribute to the high levels of sympathetic activity evident even during normoxic daytime wakefulness in sleep apnoea patients. Administration of 100% oxygen in patients with sleep apnoea results in reductions in heart rate, blood pressure and central sympathetic outflow. In patients with heart failure, the central chemoreflex response to hypercapnia is markedly and selectively enhanced. This increased central chemoreflex sensitivity may contribute to the development of central sleep apnoea in heart failure patients. Administration of 100% oxygen does not lower sympathetic activity in patients with heart failure, providing further evidence against any peripheral chemoreflex potentiation. The peripheral and central chemoreflexes have powerful effects on sympathetic activity in both health and disease and may contribute importantly to disease pathophysiology, particularly in conditions such as hypertension, obstructive sleep apnoea and heart failure.     

Angiotensin II regulation of ovine fetoplacental artery endothelial functions: interactions with nitric oxide

During normal pregnancy, elevated angiotensin II (Ang II) concentrations in the maternal and fetal circulations are associated with dramatic increases in placental angiogenesis and blood flow. Much is known about a local renin–angiotensin system within the uteroplacental vasculature. However, the roles of Ang II in regulating fetoplacental vascular functions are less well defined. In the fetal placenta, the overall in vivo vasoconstrictor responses of the blood vessels to Ang II infusion is thought to be less than that in its maternal counterpart, even though infused Ang II induces vasoconstriction. Recent data from our laboratories suggest that Ang II stimulates cell proliferation and increases endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) in ovine fetoplacental artery endothelial cells. These data imply that elevations of the known vasoconstrictor Ang II in the fetal circulation may indeed play a role in the marked increases in fetoplacental angiogenesis and that Ang II-elevated endothelial NO production may partly attenuate Ang II-induced vasoconstriction on vascular smooth muscle. Together with both of these processes, the high levels of Ang II in the fetal circulation may serve to modulate overall fetoplacental vascular resistance. In this article, we review currently available data on the expression of Ang II receptors in the ovine fetal placenta with particular emphasis on the effects of Ang II on ovine fetoplacental endothelium. The potential cellular mechanisms underlying the regulation of Ang II on endothelial growth and vasodilator production are discussed.     

Does nitric oxide allow endothelial cells to sense hypoxia and mediate hypoxic vasodilatation?in vivo and in vitro studies

Hypoxia-evoked vasodilatation is a fundamental regulatory mechanism that is often attributed to adenosine. The identity of the O₂ sensor is unknown. Nitric oxide (NO) inhibits endothelial mitochondrial respiration and ATP generation by competing with O₂ for its binding site on cytochrome oxidase. We proposed that in vivo this interaction allows endothelial cells to release adenosine when O₂ tension falls or NO concentration increases. Using anaesthetised rats, we confirmed that the increase in femoral vascular conductance (FVC, hindlimb vasodilatation) evoked by systemic hypoxia is attenuated by NO synthesis blockade with l -NAME, but restored when baseline FVC is restored by infusion of NO donor. This 'restored' hypoxic response, like the control hypoxic response, is inhibited by the adenosine A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Similarly, the FVC increase evoked by adenosine infusion was attenuated by l -NAME but restored by infusion of NO donor. However, when baseline FVC was restored after l -NAME with 8-bromo-cGMP, the FVC increase evoked by adenosine infusion was restored, but not in response to systemic hypoxia, suggesting that adenosine was no longer released by hypoxia. Infusion of NO donor at a given rate after treatment with l -NAME evoked a greater FVC increase during systemic hypoxia than during normoxia, both responses being reduced by 8-cyclopentyl-1,3-dipropylxanthine. Finally, both bradykinin and NO donor released adenosine from superfused endothelial cells in vitro ; l -NAME attenuated only the former response. We propose that in vivo , shear-released NO increases the apparent K _m of endothelial cytochrome oxidase for O₂, allowing the endothelium to act as an O₂ sensor, releasing adenosine in response to moderate falls in O₂.     

Calcitonin gene-related peptide contributes to the umbilical haemodynamic defence response to acute hypoxaemia

Despite clinical advances in obstetric practice, undiagnosed fetal hypoxaemia still contributes to a high incidence of perinatal morbidity. The fetal defence to hypoxaemia involves a redistribution of blood flow away from peripheral circulations towards essential vascular beds, such as the umbilical, cerebral, myocardial and adrenal circulations. In marked contrast to other essential vascular beds, the mechanisms mediating maintained perfusion of the umbilical circulation during hypoxaemia remain unknown. This study determined the role of calcitonin gene-related peptide (CGRP) in the maintenance of umbilical blood flow during basal and hypoxaemic conditions. Under anaesthesia, five sheep fetuses were instrumented with catheters and a Transonic probe around an umbilical artery, inside the fetal abdomen, at 0.8 of gestation. Five days later, fetuses were subjected to 0.5 h hypoxaemia during either i.v. saline or a selective CGRP antagonist in randomised order. Treatment started 30 min before hypoxaemia and ran continuously until the end of the challenge. The CGRP antagonist did not alter basal blood gas or cardiovascular status in the fetus. A similar fall in P _a,O2 occurred in fetuses during either saline (21 ± 0.8 to 9 ± 0.9 mmHg) or antagonist treatment (20 ± 0.9 to 9 ± 1.2 mmHg). Hypoxaemia during saline led to significant increases in arterial blood pressure, umbilical blood flow and umbilical vascular conductance. In marked contrast, hypoxaemia during CGRP antagonist treatment led to pronounced falls in both umbilical blood flow and umbilical vascular conductance without affecting the magnitude of the hypertensive response. In conclusion, CGRP plays an important role in the umbilical haemodynamic defence response to hypoxaemia in the late gestation fetus.     

The late gestation fetal cardiovascular response to hypoglycaemia is modified by prior peri-implantation undernutrition in sheep

Undernourished late gestation fetuses display asymmetric growth restriction, suggestive of a redistribution of nutritional resources. The modification of fetal organ blood supply in response to acute hypoxia is well characterized, but it is not known whether similar responses occur in response to acute reductions in nutrition, or if such late gestation responses can be influenced by early gestation nutrition. In pregnant sheep, total nutrient requirements were restricted during the peri-implantation period (PI40, 40%; PI50, 50% of total, days 1–31) or in late gestation (L, 50% total, days 104–postmortem). Control animals were fed 100% nutrient requirements. Fetal organ blood flows were measured at baseline, and during acute fetal hypoglycaemia induced by maternal insulin infusion at 125 dGA. Baseline heart rate was increased in PI40 fetuses. During hypoglycaemia, an initial rise in fetal heart rate was followed by a slower fall. Fetal femoral artery blood flow decreased, and adrenal blood flow and femoral vascular resistance increased in all fetuses during hypoglycaemia. These changes were accompanied by increased fetal plasma adrenaline and cortisol, and reduced plasma insulin levels. The maximum femoral artery blood flow response to hypoglycaemia occurred earlier in PI50 and PI40 compared with control fetuses. The late gestation fetal cardiovascular response to acute hypoglycaemia was consistent with a redistribution of combined ventricular output away from the periphery and towards central organs. One element of the peripheral vascular response was modified by peri-implantation nutrient restriction, indicating that nutritional challenges early in gestation can have an enduring impact on cardiovascular control.     

Haemodynamic responses to angiotensin II in conscious lambs: role of nitric oxide and prostaglandins

It was hypothesized that nitric oxide (NO) and prostaglandins (PGs) play a synergistic role in modulating haemodynamic responses to angiotensin II (ANG II) in an age-dependent manner. To this end, experiments were carried out in conscious, chronically instrumented lambs aged ∼1 week (N = 9) and ∼6 weeks (N = 10) to evaluate the haemodynamic responses to ANG II, before and after treatment with the l-arginine analogue, N-nitro-l-arginine methyl ester (l-NAME), as well as the cyclooxygenase inhibitor, indomethacin (INDO). Pressor and renal blood flow responses to ANG II were measured before (control) and after administration of l-NAME (20 mg kg⁻¹), following pretreatment with either vehicle (VEH) (experiment 1) or INDO (1 mg kg⁻¹, experiment 2). The two experiments were carried out at minimum intervals of 48 h. In both age groups, the pressor and renal vasoconstrictor responses to ANG II were augmented by pretreatment with INDO, the effects being similar at 1 and 6 weeks. The haemodynamic responses to ANG II were, however, not altered after l-NAME following pretreatment with either VEH or INDO. These data provide new evidence that soon after birth, endogenously produced PGs, but not endogenously produced NO, balance the vasoconstrictor actions of ANG II. There is, however, no apparent interaction between PGs and NO in modulating the responses to ANG II postnatally.     

Endogenous nitric oxide attenuates neutrally mediated cutaneous vasoconstriction

Cutaneous vasoconstrictor responsiveness may be impaired by substance(s) directly or indirectly responsible for cutaneous active vasodilatation. In this study, we tested the hypothesis that endogenous nitric oxide (NO) attenuates the reduction in cutaneous vascular conductance (CVC) during an orthostatic challenge combined with whole-body heating, as well as during whole-body cooling. In protocol 1, healthy subjects were pretreated with an intradermal injection of botulinum toxin A (BTX) to block the release of neurotransmitters from nerves responsible for cutaneous active vasodilatation. On the experimental day, a microdialysis probe was placed at the BTX-treated site as well as at two adjacent untreated sites. N^G -nitro- l -arginine methyl ester ( l -NAME, 10 m m ) was perfused through the probe placed at the BTX-treated site and at one untreated site. After confirmation of the absence of cutaneous vasodilatation at the BTX site during whole-body heating, adenosine was infused through the microdialysis probe at this site to increase skin blood flow to a level similar to that at the untreated site. Subsequently, 30 and 40 mmHg lower-body negative pressures (LBNPs) were applied. The reduction in CVC to LBNP was greatest at the BTX-treated site (15.0 ± 2.4% of the maximum level (% max)), followed by the l -NAME-treated site (11.3 ± 2.6% max), and then the untreated site (3.8 ± 3.0% max; P < 0.05 for all comparisons). In protocol 2, two microdialysis membranes were inserted in the dermal space of one forearm. Adenosine alone was infused at one site while the other site received adenosine and l -NAME. The reduction in CVC in response to whole-body cooling was significantly greater at the l -NAME-treated site than at the adjacent adenosine alone site. These results suggest that endogenous NO is capable of attenuating cutaneous vasoconstrictor responsiveness.     

Impaired vasomodulation is associated with reduced neuronal nitric oxide synthase in skeletal muscle of ovariectomized rats

In exercising skeletal muscle, vasoconstrictor responses to α-adrenoceptor activation are attenuated in part by nitric oxide (NO) produced by the neuronal isoform of NO synthase (nNOS), which is expressed constitutively in skeletal muscle cells. In skeletal muscle of pregnant animals, nNOS mRNA is upregulated, suggesting that muscle nNOS expression is modulated by the steroid hormone oestrogen. Whether oestrogen-induced changes in nNOS expression have measurable effects on vasoregulation in skeletal muscle is unknown. In this study, we hypothesized that oestrogen deficiency would reduce muscle nNOS expression, resulting in impaired modulation of sympathetic vasoconstriction in exercising skeletal muscle. Compared to gonadally intact rats, we found that ovariectomized (OVX) rats were characterized by greater sympathetic vasoconstriction in contracting hindlimb and reduced nNOS, but not eNOS, in skeletal muscle. In addition, NOS inhibition resulted in a greater enhancement of sympathetic vasoconstriction in contracting hindlimbs of intact compared to OVX rats. These effects of oestrogen deficiency were prevented by chronic treatment of OVX rats with 17β-oestradiol, but not with chronic progesterone or acute oestradiol. Further analysis revealed that skeletal muscle nNOS correlated directly with plasma 17β-oestradiol and inversely with the magnitude of sympathetic vasoconstrictor responses in contracting hindlimbs. These data indicate that NO-dependent attenuation of sympathetic vasoconstriction in contracting skeletal muscle is impaired in oestrogen-deficient female rats, and suggest that this impairment may be mediated by reduced skeletal muscle nNOS expression.     

Is estradiol cardioprotection a nitric oxide-mediated effect?

BACKGROUND: Estradiol exerts a number of biological effects that support extensive observational data suggesting a protective role for estrogen in cardiovascular disease prevention. These include effects on lipid and carbohydrate metabolism, coagulation/fibrinolysis as well as a possible effect on vascular reactivity. It has been proposed that this might be mediated by vascular endothelial nitric oxide (NO) production. Accordingly, we designed complementary in-vivo and in-vitro studies to investigate this hypothesis further. METHODS: Firstly, in a group of 10 healthy post-menopausal women, bilateral venous occlusion plethysmography was used to examine forearm vasoconstrictor responses to intrabrachial N(G)-monomethyl-l-arginine (l-NMMA; a substrate inhibitor of nitric oxide synthase) both before and after 4 weeks of treatment with transdermal 17beta-estradiol (E(2)) (80 microg/day). Secondly, we examined the direct effects of acute (24 h) and chronic (7 days) treatment with E(2) (10 pmol/l and 10 nmol/l) on endothelial nitric oxide synthase (eNOS) gene expression in cultured human aortic endothelial cells. RESULTS: No significant differences were observed between the vasoconstrictor responses to l-NMMA (2, 4, 8 micromol/min) before and after E(2) treatment. Comparison of E(2)-treated endothelial cells with control cells showed no significant increase in eNOS mRNA expression following either acute or chronic estradiol treatment. CONCLUSIONS: The present studies do not provide evidence for an eNOS-mediated cardioprotective response to estrogen and therefore suggest that additional mechanisms other than the endothelial NO system may have an important role in the cardiovascular effects of estrogen.     

Effects of maternal hypoxia or nutrient restriction during pregnancy on endothelial function in adult male rat offspring

Compromised fetal growth impairs vascular function; however, it is unclear whether chronic hypoxia in utero affects adult endothelial function. We hypothesized that maternal hypoxia (H, 12% O₂, n = 9) or nutrient restriction (NR, 40% of control, n = 7) imposed from day 15–21 pregnancy in rats would impair endothelial function in adult male offspring (relative to control, C, n = 10). Using a wire myograph, endothelium-dependent relaxation in response to methacholine was assessed in small mesenteric arteries from 4- and 7-month-old (mo) male offspring. Nitric oxide (NO) mediation of endothelium-dependent relaxation was evaluated using N ^ω-nitro- l -arginine methyl ester ( l -NAME; NO synthase inhibitor). Observed differences in the NO pathway at 7 months were investigated using exogenous superoxide dismutase (SOD) to reduce NO scavenging, and sodium nitroprusside (SNP; NO donor) to assess smooth muscle sensitivity to NO. Sensitivity to methacholine-induced endothelium-dependent relaxation was reduced in H offspring at 4 months ( P < 0.05), but was not different among groups at 7 months. l -NAME reduced methacholine sensitivity in C ( P < 0.01), H ( P < 0.01) and NR ( P < 0.05) offspring at 4 months, but at 7 months l -NAME reduced sensitivity in C ( P < 0.05), tended to in NR ( P = 0.055) but had no effect in H offspring. SOD did not alter sensitivity to methacholine in C, but increased sensitivity in H offspring ( P < 0.01). SNP responses did not differ among groups. In summary, prenatal hypoxia, but not nutrient restriction impaired endothelium-dependent relaxation at 4 months, and reduced NO mediation of endothelial function at 7 months, in part through reduced NO bio-availability. Distinct effects following reduced maternal oxygen versus nutrition suggest that decreased oxygen supply during fetal life may specifically impact adult vascular function.     

Lack of effect of moderate hypoxaemia on human postural reflexes to skeletal muscle

The effect of moderate hypoxemia upon postural sympathetic vasoconstrictor reflexes in skeletal muscle was studied in five healthy young students, aged 20-30 years. The vasoconstrictor response to head-up tilt was studied in brachio radial muscle kept at heart level and in the anterior tibial muscle. The local sympathetic veno-arteriolar axon reflex was studied in the anterior tibial muscle placed at heart level and lowered 30-50 cm below heart level. Muscle blood flow was measured by the local 133Xe wash-out technique. The measurements were carried out with the subject breathing atmospheric air and with the subject breathing 10-11% oxygen. No sign of orthostatic intolerance was seen during hypoxaemia as arterial blood pressure remained constant in the tilted position. Hypoxaemia did not alter the head-up tilt induced vasoconstriction in brachio-radial and anterior tibial muscles. The vasoconstriction elicited by the local veno-arteriolar reflex was slightly reduced during hypoxaemia. The results suggest that central orthostatic vasoconstrictor reflexes to muscle are essentially normal during moderate hypoxaemia, but local veno-arteriolar reflexes are slightly attenuated. This does not, however, significantly alter blood pressure control during head-up tilt.     

Desensitization of menthol-activated cold receptors in lower extremities during local cooling in young women with a cold constitution

To test the hypothesis that topical menthol-induced reactivity of cold sensation and cutaneous vasoconstriction to local cooling is augmented in individuals with a cold constitution, we examined thermal sensation and cutaneous vasoconstrictor responses at menthol-treated and untreated sites in the legs during local skin cooling in young women complaining of chilliness (C group) and young women with no complaint as a normal control group (N group). During local skin cooling, the sensitivity to cold sensation was greater in the C group than in the N group. The application of menthol enhanced the cold sensation at a low temperature in the N group, but not in the C group. Cutaneous vasoconstrictor responses to local skin cooling were not altered by menthol treatment in either of the two groups. These findings suggest the desensitization of menthol-activated cold receptors in the legs of C group subjects, and a minor role of cold receptor activity in cutaneous vasoconstrictor response to local cooling.     

The cardiovascular and renal effects of acute and chronic inhibition of nitric oxide production in fetal sheep

The acute and long-term effects of blockade of nitric oxide (NO) production were studied in six chronically catheterised fetal sheep aged from 116 and 118 days; six untreated fetal sheep received injections of saline. Injection of 10 mg (kg maternal body wt)(-1) of the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (NOLA) to the fetus, caused an immediate rise in fetal mean arterial pressure (MAP, P < 0.005) and a reflex fall in fetal heart rate (FHR, P < 0.001). Plasma renin concentration (PRC) fell from 8.4 +/- 3.3 to 1.5 +/- 0.3 ng ml(-1) h(-1) (P < 0.001) and was dependent on MAP (P = 0.001). Glomerular filtration rate (GFR) tended to increase, but renal blood flow (RBF) velocity decreased (P < 0.001). Thus filtration fraction (FF) increased (P < 0.025). Urine flow and sodium excretion increased (P < 0.001 for both). Fractional sodium reabsorption decreased (P < 0.05). In fetuses treated with NOLA, arterial pressure was found to affect glomerular haemodynamics and renal tubular handling of sodium. No such relationships were observed in untreated fetuses. The vascular responses to acetylcholine tended to be less (P = 0.07) and the responses to noradrenaline were enhanced in NOLA-treated fetuses. There were no changes in untreated fetuses. Fetuses were then injected twice daily with either 5 mg kg(-1) NOLA or saline for the next 2 days. On the 4th day, injection of 10 mg kg(-1) NOLA did not have any effects on MAP, FHR or renal function. However, the pressor responses to angiotensin II (Ang II) were enhanced (P < 0.005), as was the response to noradrenaline but to a lesser extent. It is concluded that endothelial production of NO maintains normal fetal blood pressure, renal vascular resistance and fetal renal function. When NO production was blocked by repeated injections of NOLA, other vasodilator pathways took over the maintenance of cardiovascular and renal vascular tone. However, alterations in both cardiovascular and renal function were still present. That is, there was increased pressor sensitivity to exogenous Ang II and unmasking of effects of arterial pressure on glomerular and tubular function.     

Evolving in thin air--lessons from the llama fetus in the altiplano

Compared with lowland species, fetal life for mammalian species whose mothers live in high altitude is demanding. For instance, fetal llamas have to cope with the low fetal arterial PO2 of all species, but also the likely superimposition of hypoxia as a result of the decreased oxygen environment in which the mother lives in the Andean altiplano. When subjected to acute hypoxia the llama fetus responds with an intense peripheral vasoconstriction mediated by alpha-adrenergic mechanisms plus high plasma concentrations of catecholamines and neuropeptide Y (NPY). Endothelial factors such as NO and endothelin-1 also play a role in the regulation of local blood flows. Unlike fetuses of lowland species such as the sheep, the llama fetus shows a profound cerebral hypometabolic response to hypoxia, decreasing cerebral oxygen consumption, Na-K-ATPase activity and temperature, and resulting in an absence of seizures and apoptosis in neural cells. These strategies may have evolved to prevent hypoxic injury to the brain or other organs in the face of the persistent hypobaric hypoxia of life in the Andean altiplano.