Role of endothelium-derived relaxing factors in the renal response to vasoactive agents in hypothyroid rats

This study analyzed the role of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in the abnormal renal vascular reactivity of hypothyroid rats. Renal responses to vasoconstrictors [VC: phenylephrine (PHE) and ANG II] and vasodilators [VD: ACh, sodium nitroprusside (SNP), and papaverine (PV)] were studied in kidneys from control and hypothyroid rats under normal conditions and after NO or EDHF blockade. NO was blocked by the administration of Nomega-nitro-l-arginine methyl ester (l-NAME) and EDHF by the administration of tetraethylammonium (TEA) or by an increased extracellular K+. The response to VC was also evaluated after endothelium removal. Hypothyroid kidneys showed reduced responsiveness to PHE and a normal response to ANG II. l-NAME and TEA administration produced an increased sensitivity to PHE and to ANG II in control preparations. l-NAME also increased the response to PHE in hypothyroid kidneys, but the differences between control and hypothyroid kidneys were maintained. TEA administration did not change the response to either VC in hypothyroid preparations. In endothelium-removed preparations, TEA was unable to increase pressor responsiveness to VC. Hypothyroid kidneys showed reduced responsiveness to ACh and SNP and normal response to PV. The differences between hypothyroid and control preparations in the responses to ACh and SNP were maintained after l-NAME or increased K+. In conclusion, this study shows that 1) the attenuated response to PHE in hypothyroidism is not related to an increased production of endothelium-derived relaxing factors NO and EDHF; 2) the response to VC in hypothyroid preparations is insensitive to EDHF blockade; and 3) hypothyroid preparations have a reduced reactivity to the NO donor, and NO-independent vasodilatation remains unaffected.     

Estrogen modulation of endothelium-derived relaxing factors by human endothelial cells

We report the modulatory effects of estrogen on release of endothelium-derived relaxing factors (EDRFs) in a human endothelial cell line, EA.hy926. Using bioassay, we showed that EA.hy926 released EDRF including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) measured by relaxation of pre-contracted endothelium-denuded rabbit aortic rings. This EDRF production was significantly higher in cells treated for 24 h with 17-beta-estradiol (10(-6)mol/L) than control cells. Addition of L-NAME to the perfusate of cells caused the relaxation induced by the endothelial cell perfusate to become transient and abolished the enhancement of relaxation due to estrogen treatment. Addition of K(Ca) channel blockers to the perfusate abolished the L-NAME-resistant relaxation of the bioassay ring. Using real-time PCR, we demonstrated that eNOS expression in estrogen-treated cells was significantly higher than controls. These results show that estrogen exerts a potentially important vasculo-protective effect by stimulating NO but not EDHF production.     

Gender differences affect blood flow recovery in a mouse model of hindlimb ischemia

Blood flow restoration to ischemic tissue is affected by various risk factors. The aim of this study was to examine gender effects on arteriogenesis and angiogenesis in a mouse ischemic hindlimb model. C57BL/6J mice were subjected to unilateral hindlimb ischemia. Flow recovery was less and hindlimb use impairment was greater in females. No gender difference in vessel number was found at baseline, although 7 days postsurgery females had fewer α-smooth muscle actin-positive vessels in the midpoint of the adductor region. Females had higher hindlimb vascular resistance, were less responsive to vasodilators, and were more sensitive to vasoconstrictors postligation. Western blotting showed that females had higher baseline levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the calf, while 7 days postligation males had higher levels of VEGF, eNOS, and phosphorylated vasodilator stimulated phosphoprotein. Females had less angiogenesis in a Matrigel plug assay and less endothelial cell proliferation in vitro. Females have impaired recovery of flow, a finding presumably caused by multiple factors including decreased collateral remodeling, less angiogenesis, impaired vasodilator response, and increased vasoconstrictor activity; our results also suggest the possibility that new collateral formation, from capillaries, is impaired in females.     

Vascular smooth muscle influences the release of endothelium-derived relaxing factor

Conditioned medium was collected from vascular smooth-muscle cells grown in culture to determine if these cells synthesize vasoactive substances. The medium caused a short-acting endothelium-independent constriction of rat aorta, followed by a prolonged, endothelium-dependent relaxation. This relaxation was mediated through the release of endothelium-derived relaxing factor (EDRF) as it was abolished by the addition of methylene blue (5 x 10(-6) M), haemoglobin (10(-6) M) or methyl arginine, but was not affected by indomethacin (10(-5) M). Smooth-muscle medium stimulated the production of EDRF from both rat and rabbit thoracic aortic rings as well as from cultured bovine pulmonary artery endothelial cells. The prolonged stimulation of EDRF by smooth-muscle medium was not mimicked by known physiological stimuli to EDRF release; EDRF-stimulating activity was not affected when smooth-muscle cells were grown in the presence of indomethacin (10(-5) M), although serum in the medium was required. The EDRF-stimulating substance(s) in the smooth-muscle medium was heat stable and associated with a high molecular mass (30,000 greater than Mr greater than 3500) water-soluble species that is as yet unidentified.     

Stretch may enhance the release of endothelium-derived relaxing factor in rabbit aorta

The effect of vascular stretch on the release of EDRF was studied by measuring tissue cGMP levels of rabbit. Aortic rings of rabbit were quick-frozen in liquid nitrogen during varying resting tensions, and cGMP contents were determined by radio-immunoassay. The tissue cGMP levels significantly elevated with the increase in resting tension in endothelium-intact rings, but not in endothelium-denuded rings. Deprivation of extracellular calcium abolished the stretch-induced elevation of tissue cGMP levels in endothelium-intact segments. These stretch-induced endothelium dependent tissue cGMP elevations were unaffected by Ca2+ channel blockers, nicardipine and diltiazem. Data suggest that vascular stretch may release EDRF via mechanism dependent on extracellular calcium, but probably not through voltage-dependent calcium channel.     

Role of endothelium-derived relaxing factor in active hyperemia of the canine diaphragm.

To assess the effect of endothelium-derived relaxing factor (EDRF) on diaphragmatic vascular resistance at rest and during contractions, we studied an in situ isolated diaphragm preparation in anesthetized and mechanically ventilated dogs. The arterial supply of the left diaphragm (phrenic artery) was catheterized and perfused with arterial blood at a fixed flow rate. Drugs were infused through a side port of the arterial catheter at 1/100th of the phrenic arterial flow. The inferior phrenic vein was catheterized to complete the isolation from the systemic circulation. Three sets of experiments were performed. In set 1 (n = 3), we infused endothelium-dependent (acetylcholine, ACh) and endothelium-independent (sodium nitroprusside, SNP) dilators at increasing concentrations. ACh and SNP infusion elicited a dose-dependent decline in phrenic vascular resistance (Rphr) at concentrations greater than 10(-8) M and 0.50 micrograms/ml, respectively. In set 2 (n = 15), we infused an inhibitor of EDRF synthesis and release, L-argininosuccinic acid (ArgSA), at increasing concentrations (10(-4), 3 x 10(-4), and 6 x 10(-4) M). ArgSA produced a dose-dependent increase in Rphr. Infusion of another EDRF inhibitor (NG-nitro-L-arginine, LNA, 6 x 10(-4) M) elicited increase in Rphr similar to that induced by ArgSA. In set 3 (n = 25), we infused ArgSA or LNA (6 x 10(-4) M) simultaneously with ACh and SNP and during sustained (2-Hz) contractions of the diaphragm. Both ArgSA and LNA completely reversed ACh vasodilation, whereas SNP vasodilation was reversed by 26 and 11%, respectively. ArgSA or LNA infusion during contractions reversed vasodilation by 48 and 52%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hindlimb unweighting on tissue blood flow in the rat.

The purpose of this study was to characterize the distribution of blood flow in the rat during hindlimb unweighting (HU) and post-HU standing and exercise and examine whether the previously reported (Witzmann et al., J. Appl. Physiol. 54: 1242-1248, 1983) elevation in anaerobic metabolism observed with contractile activity in the atrophied soleus muscle was caused by a reduced hindlimb blood flow. After either 15 days of HU or cage control, blood flow was measured with radioactive microspheres during unweighting, normal standing, and running on a treadmill (15 m/min). In another group of control and experimental animals, blood flow was measured during preexercise (PE) treadmill standing and treadmill running (15 m/min). Soleus muscle blood flow was not different between groups during unweighting, PE standing, and running at 15 m/min. Chronic unweighting resulted in the tendency for greater blood flow to muscles composed of predominantly fast-twitch glycolytic fibers. With exercise, blood flow to visceral organs was reduced compared with PE values in the control rats, whereas flow to visceral organs in 15-day HU animals was unaltered by exercise. These higher flows to the viscera and to muscles composed of predominantly fast-twitch glycolytic fibers suggest an apparent reduction in the ability of the sympathetic nervous system to distribute cardiac output after chronic HU. In conclusion, because 15 days of HU did not affect blood flow to the soleus during exercise, the increased dependence of the atrophied soleus on anerobic energy production during contractile activity cannot be explained by a reduced muscle blood flow.     

Fatigability and blood flow in the rat gastrocnemius-plantaris-soleus after hindlimb suspension.

The purpose of this study was to test the hypothesis that hindlimb suspension increases the fatigability of the soleus during intense contractile activity and determine whether the increased fatigue is associated with a reduced muscle blood flow. Cage-control (C) and 15-day hindlimb-suspended (HS) rats were anesthetized, and either the gastrocnemius-plantaris-soleus (G-P-S) muscle group or the soleus was stimulated (100 Hz, 100-ms trains at 120/min) for 10 min in situ. In the G-P-S preparation, blood flow was measured with radiolabeled microspheres before and at 2 and 10 min of contractile activity. The G-P-S fatigued markedly at this stimulation frequency, and the differences between C and HS animals were not significant until the 9th min of contractile activity. In contrast, the stimulation resulted in faster rates and significantly larger amounts of fatigue in the soleus from HS than from C animals. The atrophied soleus showed significant differences by 1 min of stimulation (C = 70 +/- 1% vs. HS = 57 +/- 2% of peak train force) and remained different at 10 min (C = 64 +/- 4% vs. HS = 45 +/- 2% peak train force). Relative blood flow to the soleus was similar between groups before and during contractile activity (rest: C = 20 +/- 3 vs. HS = 12 +/- 3; 2 min: C = 128 +/- 6 vs. HS = 118 +/- 4; 10 min: C = 123 +/- 11 vs. HS = 105 +/- 11 ml.min-1.100 g-1). In conclusion, these results established that 15 days of HS increased the fatigability of the soleus, but the effect was not caused by a reduced muscle blood flow.     

Vascular endothelial growth factor (VEGF) fails to improve blood flow and to promote collateralization in a diabetic mouse ischemic hindlimb model

Background

An insertion/deletion polymorphism in the α_2B-adrenoceptor (AR) has been associated with the risk for acute myocardial infarction (AMI) and sudden cardiac death. In this study we tested whether this polymorphism is associated with the risk for AMI among members of families with type 2 diabetes.

Methods

154 subjects with a history of AMI were matched for age and sex with one of their siblings who did not have a history of AMI. The prevalence of the genotypes of the α_2B-AR insertion/deletion polymorphism was compared between the siblings using McNemar's test. We also explored the data to see whether this genetic variation affects the risk for hypertension by using logistic regression models in the two subpopulations of subjects, with and without a history of AMI.

Results

Among all study subjects, 73 (24%) carried the α_2B-AR deletion/deletion genotype, 103 (33%) carried the insertion/insertion genotype, and 132 (43%) were heterozygous. The distribution of genotypes of the α_2B-AR insertion/deletion variation in the group of subjects with a history of AMI and their phenotype-discordant siblings did not statistically significantly differ from that expected by random distribution (p = 0.52): the deletion/deletion genotype was carried by 34 subjects with AMI (22%), and by 39 subjects without AMI (25%). Neither did we observe any significant difference in deletion allele frequencies of the α_2B-AR insertion/deletion polymorphism between patients with a history of AMI (0.44) and their sib-pair controls (0.46, p = 0.65). In an exploratory analysis, the α_2B-AR deletion/deletion genotype was associated with increased odds for hypertension compared with subjects carrying any of the other genotypes.

Conclusions

The deletion/deletion genotype of the α_2B-AR does not emerge in this study as a risk factor for AMI among members of families with type 2 diabetes; however, it might be involved in the development of hypertension.     

Endothelium-derived relaxing factor participates in the increased blood flow in response to pentagastrin in the rat stomach mucosa

The stimulation of gastric-acid secretion by pentagastrin, a synthetic analogue of the endogenous peptide gastrin, is associated with an increased blood flow to the stomach mucosa, commonly referred to as functional hyperaemia. There are at least two potent vasodilator substances, the local release of which from endothelial cells could contribute to this hyperaemia, endothelium-derived relaxing factor (EDRF) and prostacyclin. EDRF has been identified as nitric oxide, released enzymatically from the guanidino group of L-arginine. In the present studies, the involvement of prostacyclin in the pentagastrin-induced increase in stomach blood flow was eliminated by using the cyclo-oxygenase inhibitor indomethacin. Thus this work was designed to elucidate the participation of EDRF/NO in the pentagastrin-induced hyperaemia and not its relative importance to prostacyclin. The increase in blood flow to the gastric mucosa in response to pentagastrin was measured by laser Doppler flowmetry in situ. Inhibition of EDRF/NO biosynthesis with the L-arginine analogues NG-monomethyl-L-arginine (MeArg) or N omega-nitro-L-arginine (NO2Arg) significantly attenuated (by more than 80%) the increase in mucosal blood flow in response to pentagastrin. However, infusions of the natural substrate L-arginine reversed the inhibitor effect of MeArg on pentagastrin-induced increase in mucosal blood flow. Local intra-arterial injections of the endothelium-independent vasodilator glyceryl trinitrate produced a dose-related increase in blood flow to the rat stomach mucosa that was unaffected by infusion of MeArg. Thus, in the absence of prostacyclin, EDRF/NO participates in the pentagastrin-induced increase in blood flow to the rat stomach mucosa.     

Regulation of canine skeletal muscle and hindlimb blood flow in acute anemia

Redistribution of blood flow away from resting skeletal muscles does not occur during anemic hypoxia even when whole body oxygen uptake is not maintained. In the present study, the effects of sympathetic nerve stimulation on both skeletal muscle and hindlimb blood flow were studied prior to and during anemia in anesthetized, paralyzed, and ventilated dogs. In one series (skeletal muscle group, n = 8) paw blood flow was excluded by placing a tourniquet around the ankle; in a second series (hindlimb group, n = 8) no tourniquet was placed at the ankle. The distal end of the transected left sciatic nerve was stimulated to produce a maximal vasoconstrictor response for 4-min intervals at normal hematocrit (Hct.) and at 30 min of anemia (Hct. = 14%). Arterial blood pressure and hindlimb or muscle blood flow were measured; resistance and vascular hindrance were calculated. Nerve stimulation decreased blood flow (p less than 0.05) in the hindlimb and muscle groups at normal Hct. Blood flow rose (p less than 0.05) during anemia and was decreased (p less than 0.05) in both groups during nerve stimulation. However, the blood flow values in both groups during nerve stimulation in anemic animals were greater (p less than 0.05) than those at normal Hct. Hindlimb and muscle vascular resistance fell significantly during anemia and nerve stimulation produced a greater increase in vascular resistance at normal Hct. Vascular hindrance in muscle, but not hindlimb, was less during nerve stimulation in anemia than at normal Hct.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of non-thermal factors in the control of skin blood flow during exercise

Arguments in favor of the importance of non-thermal factors in the control of skin circulation are presented. Such factors include exercise, posture, water and electrolyte balance, state of training, and acclimatization. The first three factors probably elicit their effects via high- and low-pressure baroreceptors, while the mechanisms involved for the remainder are unknown.     

Effects of sodium nitroprusside-induced hypotension on the cerebral and hindlimb blood flow in dogs

Sodium nitroprusside (SNP) has been commonly used as a vasodilator agent for deliberate hypotension with general anesthesia. The purpose of this study was to observe whether cerebral blood flow (CBF) was significantly reduced when SNP infusion was accomplished to decrease peripheral blood flows with systemic hypotension. We conducted the experiments in 15 pentobarbital-anesthetized dogs. CBF was measured in 7 dogs using a venous outflow method. Hindlimb blood flow (HBF) serving as a representative of the peripheral circulations was obtained by flow measurement in the femoral artery in 8 dogs. The systemic arteral pressure (SAP) was decreased stepwise (approximately 5 mmHg for each step) by adjusting the SNP infusion rate. During the systemic hypotension, the CBF remained fairly constant despite a marked decline in the mean SAP to 40 mmHg. The calculated cerebral vascular resistance was progressively decreased with the systemic hypotension. On the contrary, a reduction in the HBF was observed accompanying the fall in SAP. When the mean SAP was decreased to 50 mmHg, the HBF was only 46.3 +/- 7.6% of the control value. The calculated hindlimb vascular resistance was slightly elevated during the whole course of SNP-induced hypotension. The results reveal the disparity between the brain and hindlimb in the resistance and flow responses to SNP-induced hypotension. The constancy of CBF subserves adequate brain perfusion when deliberate hypotension is conducted for surgery in the peripheral organs.     

Transneuronal tracing of neural pathways that regulate hindlimb muscle blood flow

Despite considerable interest in the neural mechanisms that regulate muscle blood flow, the descending pathways that control sympathetic outflow to skeletal muscles are not adequately understood. The present study mapped these pathways through the transneuronal transport of two recombinant strains of pseudorabies virus (PRV) injected into the gastrocnemius muscles in the left and right hindlimbs of rats: PRV-152 and PRV-BaBlu. To prevent PRV from being transmitted to the brain stem via motor circuitry, a spinal transection was performed just below the L2 level. Infected neurons were observed bilaterally in all of the areas of the brain that have previously been shown to contribute to regulating sympathetic outflow: the medullary raphe nuclei, rostral ventrolateral medulla (RVLM), rostral ventromedial medulla, A5 adrenergic cell group region, locus coeruleus, nucleus subcoeruleus, and the paraventricular nucleus of the hypothalamus. The RVLM, the brain stem region typically considered to play the largest role in regulating muscle blood flow, contained neurons infected following the shortest postinoculation survival times. Approximately half of the infected RVLM neurons were immunopositive for tyrosine hydroxylase, indicating that they were catecholaminergic. Many (47%) of the RVLM neurons were dually infected by the recombinants of PRV injected into the left and right hindlimb, suggesting that the central nervous system has a limited capacity to independently regulate blood flow to left and right hindlimb muscles.     

SK&F 96365 inhibits histamine-induced formation of endothelium-derived relaxing factor in human endothelial cells.

Formation of endothelium-derived relaxing factor (EDRF) strictly correlates with the intracellular free Ca2+ ([Ca2+]i) concentration. We now demonstrate that the histamine-induced rise in [Ca2+]i of human umbilical vein endothelial cells is mostly due to activation of a membrane current which allows Ca2+ entry. This membrane current is sensitive to the novel inhibitor of agonist-induced Ca2+ entry, SK&F 96365, which blocked the histamine-induced sustained rise in [Ca2+]i, as well as 45Ca2+ uptake and membrane currents. Inhibition of the above cellular responses to histamine was accompanied by a considerable reduction of EDRF formation and release. Thus biosynthesis and release of EDRF from human umbilical vein endothelial cells significantly depend on agonist-induced Ca2+ entry involving receptor-operated Ca(2+)-permeable channels which can be blocked by SK&F 96365.     

Effect of hindlimb suspension on VO2 max and regional blood flow responses to exercise

Male rodents were studied before and after undergoing one of three treatment conditions for 9 days: 1) cage control (n = 15, CON), 2) horizontal suspension (n = 15, HOZ), and 3) head-down suspension (n = 18, HDT). Testing included measurements of maximal O2 uptake (VO2 max) and select cardiovascular responses to graded treadmill exercise. VO2 max expressed on an absolute basis (ml/min) was significantly decreased after HOZ (-14.1 +/- 2.5%) and HDT (-14.3 +/- 2.0%), while being essentially unchanged in CON (-1.0 +/- 3.3%). Significant reductions in body weight were observed after both HOZ (-10.1 +/- 4.2 g) and HDT (-22.5 +/- 3.3 g), whereas CON animals exhibited a significant increase in weight (10.4 +/- 3.8 g). As a result, when VO2 max was normalized for body weight, all groups exhibited similar significant reductions of 6-7%. Although no differences in heart rate and blood pressure response to graded exercise were observed, the HDT group exhibited greater increases in mesenteric resistance at the same absolute exercise intensity. Furthermore, both suspended groups had higher iliac resistance values during exercise at similar relative exercise conditions, suggesting that muscle blood flow during treadmill running may have been reduced after suspension. In general, the decrements associated with the HOZ and HDT conditions were similar. It was concluded that reduction in exercise capacity and altered cardiovascular responses to exercise observed after 6-9 days of suspension were attributable to a combination of hypokinesia, lack of hindlimb weight bearing, or restraint, rather than to hydrostatic influences associated with HDT.     

Imbalance between endothelium-derived relaxing and contracting factors in mesenteric arteries from aged OLETF rats, a model of Type 2 diabetes

We investigated whether the balance between endothelium-derived relaxing factors (EDRFs) and endothelium-derived contracting factors (EDCFs) might be altered in mesenteric arteries from aged Otsuka Long-Evans Tokushima Fatty (OLETF) rats (a Type 2 diabetic model) [vs. age-matched control Long-Evans Tokushima Otsuka (LETO) rats]. ACh-induced relaxation was impaired in the OLETF group, and a tendency for the relaxation to reverse at high ACh concentrations was observed in both groups. This tendency was abolished by indomethacin. Nitric oxide- and/or endothelium-derived hypolarizing factor-mediated relaxation and the protein expressions of phospho-endothelial nitric oxide synthase (Ser1177) and extracellular superoxide dismutase were also reduced in OLETF. An ACh-induced contraction was observed at higher ACh concentrations in the presence of N(G)-nitro-L-arginine (L-NNA) but was greater in OLETF rats. This contraction in OLETF rats was reduced by cyclooxygenase (COX) inhibitors and by prostanoid-receptor antagonists. The ACh-induced productions of thromboxane A(2) and PGE(2) were greater in OLETF than LETO rats, as were the mesenteric artery COX-1 and COX-2 protein expressions. Moreover, tert-butyl hydroperoxide (t-BOOH) (membrane-permeant oxidant) induced a concentration-dependent contraction that was greater in OLETF rats. The t-BOOH-mediated contraction was increased both by L-NNA and by endothelium removal in LETO but not OLETF rats, suggesting that a negative modulatory role of the endothelium was lost in OLETF rats. These results suggest that an imbalance between EDRFs and EDCFs may be implicated in the endothelial dysfunction seen in aged OLETF mesenteric arteries, and may be attributable to increased oxidative stress.     

Effect of aminophylline on hindlimb blood flow autoregulation during increased metabolism in dogs

The contribution of adenosine to hindlimb blood flow autoregulation during treadmill exercise or the administration of 2,4-dinitrophenol (DNP) was evaluated in 9 conscious dogs by determining hindlimb vascular bed pressure-flow relationships in the presence and absence of the adenosine receptor site antagonist, aminophylline. Hindlimb pressure-flow relationships were obtained by measuring blood flow during stepwise reductions in perfusion pressure produced with an occlusion cuff located distal to a flow probe on the external iliac artery. The efficiency of autoregulation was quantitated by calculating the closed-loop gain of flow regulation (Gc) at each pressure decrement utilizing the equation Gc = 1 - (% delta flow/% delta pressure). A Gc of one represents perfect autoregulation of flow, and a Gc of zero is indicative of a rigid system. During exercise, Gc averaged 0.44 +/- 0.07. Aminophylline reduced the Gc during exercise to -0.07 +/- 0.06 (P less than 0.05). During DNP administration, Gc averaged 0.54 +/- 0.09 and declined to -0.09 +/- 0.10 in the presence of aminophylline (P less than 0.05). These results support the hypothesis that adenosine is a primary mediator of hindlimb blood flow autoregulation during conditions that increase hindlimb metabolism.