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Rationale : Although an age-related decrease in NO bioavailability contributes to vascular stiffness, the underlying molecular mechanisms remain incompletely understood. We hypothesize that NO constrains the activity of the matrix crosslinking enzyme tissue transglutaminase (TG2) via S -nitrosylation in young vessels, a process that is reversed in aging. Objective : We sought to determine whether endothelium-dependent NO regulates TG2 activity by S -nitrosylation and whether this contributes to age-related vascular stiffness. Methods and Results : We first demonstrate that NO suppresses activity and increases S -nitrosylation of TG2 in cellular models. Next, we show that nitric oxide synthase (NOS) inhibition leads to increased surface and extracellular matrix–associated TG2. We then demonstrate that endothelium-derived bioactive NO primarily mediates its effects through TG2, using TG2 −/− mice chronically treated with the NOS inhibitor l - N G -nitroarginine methyl ester (L-NAME). We confirm that TG2 activity is modulated by endothelium-derived bioactive NO in young rat aorta. In aging rat aorta, although TG2 expression remains unaltered, its activity increases and S -nitrosylation decreases. Furthermore, TG2 inhibition decreases vascular stiffness in aging rats. Finally, TG2 activity and matrix crosslinks are augmented with age in human aorta, whereas abundance remains unchanged. Conclusions : Decreased S -nitrosylation of TG2 and increased TG activity lead to enhanced matrix crosslinking and contribute to vascular stiffening in aging. TG2 appears to be the member of the transglutaminase family primarily contributing to this phenotype. Inhibition of TG2 could thus represent a therapeutic target for age-associated vascular stiffness and isolated systolic hypertension.

Extracorporeal circulation provides critical life support in the face of cardiopulmonary or renal failure, but it also introduces a host of unique morbidities characterized by edema formation, cardiac insufficiency, autonomic dysfunction, and altered vasomotor function. We tested the hypothesis that cyclohexanone (CHX), a solvent used in production of extracorporeal circuits and intravenous (IV) bags, leaches into the contained fluids and can replicate these clinical morbidities. Crystalloid fluid samples from circuits and IV bags were analyzed by gas chromatography-mass spectrometry to provide a range of clinical CHX exposure levels, revealing CHX contamination of sampled fluids (9.63–3,694 μg/l). In vivo rat studies were conducted ( n = 49) to investigate the effects of a bolus IV infusion of CHX vs. saline alone on cardiovascular function, baroreflex responsiveness, and edema formation. Cardiovascular function was evaluated by cardiac output, heart rate, stroke volume, vascular resistance, arterial pressure, and ventricular contractility. Baroreflex function was assessed by mean femoral arterial pressure responses to bilateral carotid occlusion. Edema formation was assessed by the ratio of wet to dry organ weights for lungs, liver, kidneys, and skin. CHX infusion led to systemic hypotension; pulmonary hypertension; depressed contractility, heart rate, stroke volume, and cardiac output; and elevated vascular resistance ( P < 0.05). Mean arterial pressure responsiveness to carotid occlusion was dampened after CHX infusion (from +17.25 ± 1.8 to +5.61 ± 3.2 mmHg; P < 0.05). CHX infusion led to significantly higher wet-to-dry weight ratios vs. saline only (3.8 ± 0.06 vs. 3.5 ± 0.05; P < 0.05). CHX can reproduce clinical cardiovascular, neurological, and edema morbidities associated with extracorporeal circulatory treatment.

Oxidized low-density lipoproteins increase arginase activity and reciprocally decrease endothelial NO in human aortic endothelial cells. Here, we demonstrate that vascular endothelial arginase activity is increased in atherogenic-prone apolipoprotein E–null (ApoE −/− ) and wild-type mice fed a high cholesterol diet. In ApoE −/− mice, selective arginase II inhibition or deletion of the arginase II gene (Arg II −/− mice) prevents high-cholesterol diet–dependent decreases in vascular NO production, decreases endothelial reactive oxygen species production, restores endothelial function, and prevents oxidized low-density lipoprotein–dependent increases in vascular stiffness. Furthermore, arginase inhibition significantly decreases plaque burden. These data indicate that arginase II plays a critical role in the pathophysiology of cholesterol-mediated endothelial dysfunction and represents a novel target for therapy in atherosclerosis.

Arginase, expressed in endothelial cells and upregulated in aging blood vessels, competes with NO synthase (NOS) for l -arginine, thus modulating vasoreactivity and attenuating NO signaling. Moreover, arginase inhibition restores endothelial NOS signaling and l -arginine responsiveness in old rat aorta. The arginase isoform responsible for modulating NOS, however, remains unknown. Because isoform-specific arginase inhibitors are unavailable, we used an antisense (AS) oligonucleotide approach to knockdown arginase I (Arg I). Western blot and quantitative PCR confirmed that Arg I is the predominant isoform expressed in endothelialized aortic rings and is upregulated in old rats compared with young. Aortic rings from 22-month-old rats were incubated for 24 hours with sense (S), AS oligonucleotides, or medium alone (C). Immunohistochemistry, immunoblotting, and enzyme assay confirmed a significant knockdown of Arg I protein and arginase activity in AS but not S or C rings. Conversely, calcium-dependent NOS activity and vascular metabolites of NO was increased in AS versus S or C rings. Acetylcholine (endothelial-dependent) vasorelaxant responses were enhanced in AS versus S or C treated rings. In addition, 1H-oxadiazolo quinoxalin-1-one (10 μmol/L), a soluble guanylyl cyclase inhibitor, increased the phenylephrine response in AS compared with S and C rings suggesting increased NO bioavailability. Finally, l -arginine (0.1 mmol/L)-induced relaxation was increased in AS versus C rings. These data support our hypothesis that Arg I plays a critical role in the pathobiology of age-related endothelial dysfunction. AS oligonucleotides may, therefore, represent a novel therapeutic strategy against age-related vascular endothelial dysfunction.

The α-1 adrenergic receptors (α 1 ARs) are critical in sympathetically mediated vasoconstriction. The specific role of each α 1 AR subtype in regulating vasoconstriction remains highly controversial. Limited pharmacological studies suggest that differential α 1 AR responses may be the result of differential activation of junctional versus extrajunctional receptors. We tested the hypothesis that the α 1B AR subtype is critical in mediating sympathetic junctional neurotransmission. We measured in vivo integrated cardiovascular responses to a hypotensive stimulus (induced via transient bilateral carotid occlusion [TBCO]) in α 1B AR knockout (KO) mice and their wild-type (WT) littermates. In WT mice, after dissection of the carotid arteries and denervation of aortic baroreceptor buffering nerves, TBCO produced significant pressor and positive inotropic effects. Both responses were markedly attenuated in α 1B AR KO mice (change systolic blood pressure 46±8 versus 11±2 mm Hg; percentage change in the end-systolic pressure-volume relationship [ESPVR] 36±7% versus 12±2%; WT versus KO; P 1 AR mesenteric microvascular contractile responses to endogenous norepinephrine (NE; elicited by electrical field stimulation 10 Hz) was markedly depressed in α 1B AR KO mice compared with WT (12.4±1.7% versus 21.5±1.2%; P 1B AR KO and WT mice (22.4±7.3% versus 33.4±4.3%; NS). Collectively, these results demonstrate a critical role for the α 1B AR in baroreceptor-mediated adrenergic signaling at the vascular neuroeffector junction. Moreover, α 1B ARs modulate inotropic responses to baroreceptor activation. The critical role for α 1B AR in neuroeffector regulation of vascular tone and myocardial contractility has profound clinical implications for designing therapies for orthostatic intolerance.

Vascular contractile hyporesponsiveness is an important mechanism underlying orthostatic intolerance after microgravity. Baroreceptor reflexes can modulate both pulmonary resistance and capacitance function and thus cardiac output. We hypothesized, therefore, that pulmonary vasoreactivity is impaired in the hindlimb-unweighted (HLU) rat model of microgravity. Pulmonary artery (PA) contractile responses to phenylephrine (PE) and U-46619 (U4) were significantly decreased in the PAs from HLU vs. control (C) animals. N G-nitro-l-arginine methyl ester (10−5 M) enhanced the contractile responses in the PA rings from both C and HLU animals and completely abolished the differential responses to PE and U4 in HLU vs. C animals. Vasorelaxant responses to ACh were significantly enhanced in PA rings from HLU rats compared with C. Moreover, vasorelaxant responses to sodium nitroprusside were also significantly enhanced. Endothelial nitric oxide synthase (eNOS) and soluble guanlyl cyclase expression were significantly enhanced in PA and lung tissue from HLU rats. In marked contrast, the expression of inducible nitric oxide synthase was unchanged in lung tissue. These data support the hypothesis that vascular contractile responsiveness is attenuated in PAs from HLU rats and that this hyporesponsiveness is due at least in part to increased nitric oxide synthase activity resulting from enhanced eNOS expression. These findings may have important implications for blood volume distribution and attenuated stroke volume responses to orthostatic stress after microgravity exposure.

Hydrogen sulfide (H2S) has emerged as an important gasotransmitter in the vasculature. In this study, we tested the hypothesis that H2S contributes to coronary vasoregulation and evaluated the physiological relevance of two sources of H2S, namely, cystathionine-γ-lyase (CSE) and 3-mercaptypyruvate sulfertransferase (MPST). MPST was detected in human coronary artery endothelial cells as well as rat and mouse coronary artery; CSE was not detected in the coronary vasculature. Rat coronary artery homogenates produced H2S through the MPST pathway but not the CSE pathway in vitro. In vivo coronary vasorelaxation response was similar in CSE knockout mice, wild-type mice (WT), and WT mice treated with the CSE inhibitor propargylglycine, suggesting that CSE-produced H2S does not have a significant role in coronary vasoregulation in vivo. Ex vivo, the MPST substrate 3-mercaptopyruvate (3-MP) and H2S donor sodium hydrosulfide (NaHS) elicited similar coronary vasoreactivity responses. Pyruvate did not have any effects on vasoreactivity. The vasoactive effect of H2S appeared to be nitric oxide (NO) dependent: H2S induced coronary vasoconstriction in the presence of NO and vasorelaxation in its absence. Maximal endothelial-dependent relaxation was intact after 3-MP and NaHS induced an increase in preconstriction tone, suggesting that endothelial NO synthase activity was not significantly inhibited. In vitro, H2S reacted with NO, which may, in part explain the vasoconstrictive effects of 3-MP and NaHS. Taken together, these data show that MPST rather than CSE generates H2S in coronary artery, mediating its effects through direct modulation of NO. This has important implications for H2S-based therapy in healthy and diseased coronary arteries.

Microgravity is associated with an impaired stroke volume and, therefore, cardiac output response to orthostatic stress. We hypothesized that a decreased venous filling pressure due to increased venous compliance may be an important contributing factor in this response. We used a constant flow, constant right atrial pressure cardiopulmonary bypass procedure to measure total systemic vascular compliance (CT), arterial compliance (CA), and venous compliance (CV) in seven control and seven 21-day hindlimb unweighted (HLU) rats. These compliance values were calculated under baseline conditions and during an infusion of 0.2 μg · kg−1 · min−1norepinephrine (NE). The change in reservoir volume, which reflects changes in unstressed vascular volume (ΔV0) that occurred upon infusion of NE, was also measured. CT and CV were larger in HLU rats both at baseline and during the NE infusion ( P < 0.05). Infusion of NE decreased CT and CV by ∼20% in both HLU and control rats ( P < 0.01). CA was also significantly decreased in both groups of rats by NE ( P < 0.01), but values of CA were similar between HLU and control rats both at baseline and during the NE infusion. Additionally, the NE-induced ΔV0 was attenuated by 53% in HLU rats compared with control rats ( P < 0.05). The larger CV and attenuated ΔV0 in HLU rats could contribute to a decreased filling pressure during orthostasis and thus may partially underlie the mechanism leading to the exaggerated fall in stroke volume and cardiac output seen in astronauts during an orthostatic stress after exposure to microgravity.

Microgravity is associated with an impaired cardiac output response to orthostatic stress. Mesenteric veins are critical in modulating cardiac filling through venoconstriction. The purpose of this study was to determine the effects of simulated microgravity on the capacitance of rat mesenteric small veins. We constructed pressure-diameter relationships from vessels of 21-day hindlimb-unweighted (HLU) rats and control rats by changing the internal pressure and measuring the external diameter. Pressure-diameter relationships were obtained both before and after stimulation with norepinephrine (NE). The pressure-diameter curves of HLU vessels were shifted to larger diameters than control vessels. NE (10−4 M) constricted veins from control animals such that the pressure-diameter relationship was significantly shifted downward (i.e., to smaller diameters at equal pressure). NE had no effect on vessels from HLU animals. These results indicate that, after HLU, unstressed vascular volume may be increased and can no longer decrease in response to sympathetic stimulation. This may partially underlie the mechanism leading to the exaggerated fall in cardiac output and stroke volume seen in astronauts during an orthostatic stress after exposure to microgravity.

We have developed a chronic technique to isolate the carotid sinus baroreceptor region in the conscious rat model. Our technique, when used in conjunction with other methods, allows for the study of the control of arterial pressure, heart rate, and cardiac output by the carotid sinus baroreceptor reflex in conscious, unrestrained rats. The performance of our technique was evaluated in two strains: normotensive Sprague-Dawley (SD) rats and spontaneously hypertensive rats (SHR). Each rat was instrumented with an aortic flow probe and a catheter placed in the right femoral artery to monitor cardiac output and arterial pressure, respectively. The cervical sympathetic trunk and aortic depressor nerve were ligated and cut bilaterally, leaving vagus nerves intact. The right and left carotid sinuses were isolated using our new technique. We tested the open-loop function of the carotid sinus baroreceptor reflex system in the conscious rat after recovery from the isolation surgery. We found that changes in nonpulsatile carotid sinus pressure caused significant changes in arterial pressure, heart rate, and total peripheral resistance in both rat strains. However, the cardiac output responses differed dramatically between strains. Significant changes were seen in the cardiac output response of SHR, whereas no significant changes were observed in normotensive SD rats. We have found this technique to be a highly reliable tool for the study of the carotid sinus baroreceptor reflex system in the conscious rat.

Isovolemic hemodilution and subsequent anemia increase cerebral blood flow (CBF). We hypothesized that pial microvascular pressure also increases with hemodilution and that arteriolar diameter varies concurrently as a myogenic autoregulatory response. First- and second-order arterioles (31–92 microns, n = 29) and large venules (65–215 microns, n = 17) were studied in thiopental-anesthetized rats. Microvascular pressure was determined using the servo-null technique, and vessel diameters were obtained directly from a video monitoring system. We measured the increase in CBF (radiolabeled microspheres) that accompanies hemodilution in a separate group of animals (n = 20). Hematocrit was reduced to 16-36% with homologous plasma (hemodilution group, n = 13) or held constant with homologous whole blood (control group, n = 4). In control animals, arteriolar and venular diameter varied +/- 1-2 microns from baseline values, and microvascular pressure remained unchanged from baseline. In the hemodilution group, CBF increased, but there was no systematic pial vasodilation. Furthermore, intraluminal pressure did not increase in pial microvessels, suggesting that proximal vasodilation was negligible even at the lowest hematocrit studied. Vascular resistance fell proportionately in both large vessel and microvascular segments. We conclude that experimental anemia does not produce alterations in microvascular pressure in rats, and the hyperemia accompanying hemodilution is largely viscosity mediated.

Emerging evidence suggests that nitric oxide (NO) plays a pivotal role in the mechanism of vascular hyporesponsiveness contributing to microgravity-induced orthostatic intolerance. The cellular and enzymatic source of the NO, however, remains controversial. In addition, the time course of the endothelial-dependent contribution remains unstudied. We tested the hypotheses that the change in vasoresponsiveness seen in acute (3-day) hindlimb unweighted (HLU) animals is due to an endothelium-dependent mechanism and that endothelial-dependent attenuation in vasoreactivity is due to endothelial nitric oxide synthase (NOS-3) dependent activation. Vasoreactivity was investigated in rat aortic rings following acute HLU treatment. Dose responsiveness to norepinepherine (NE) was depressed after 3-day HLU [1,338 +/- 54 vs. 2,325 +/- 58 mg at max (NE), HLU vs. C, P < 0.001]. However, removal of the endothelium restored the vascular contractility to that of C. In addition, 1H-oxadiazole quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, restored the reduced vasoconstrictor responses to phenylephrine (PE) seen in 3-day HLU rings (1.30 +/- 0.10 vs. 0.53 +/- 0.07 g, HLU + ODQ vs. HLU, P = 0.0001). Ca(+) dependent nitric oxide synthase (NOS) activity was increased, as was vascular NO products as a result of HLU. While NOS-3 expression was not increased in HLU rats, phosphorylation of NOS-3 at serine-1177 (an activator of NOS-3) was increased while phosphorylation of serine-495 (an inactivator of NOS-3) was decreased. These findings demonstrate that changes in vasoresponsiveness in the acute HLU model of microgravity are due to an upregulation of the endothelial-dependent NO/cGMP pathway through NOS phosphorylation.

Radiation exposure is associated with the development of various cardiovascular diseases. Although irradiation is known to cause elevated oxidant stress and chronic inflammation, both of which are detrimental to vascular function, the molecular mechanisms remain incompletely understood. We previously demonstrated that radiation causes endothelial dysfunction and increased vascular stiffness by xanthine oxidase (XO) activation. In this study, we investigated whether dietary inhibition of XO protects against radiation-induced vascular injury. We exposed 4-mo-old rats to a single dose of 0 or 5 Gy gamma radiation. These rats received normal drinking water or water containing 1 mM oxypurinol, an XO inhibitor. We measured XO activity and superoxide production in rat aorta and demonstrated that both were significantly elevated 2 wk after radiation exposure. However, oxypurinol treatment in irradiated rats prevented aortic XO activation and superoxide elevation. We next investigated endothelial function through fluorescent measurement of nitric oxide (NO) and vascular tension dose responses. Radiation reduced endothelium-dependent NO production in rat aorta. Similarly, endothelium-dependent vasorelaxation in the aorta of irradiated rats was significantly attenuated compared with the control group. Dietary XO inhibition maintained NO production at control levels and prevented the development of endothelial dysfunction. Furthermore, pulse wave velocity, a measure of vascular stiffness, increased by 1 day postirradiation and remained elevated 2 wk after irradiation, despite unchanged blood pressures. In oxypurinol-treated rats, pulse wave velocities remained unchanged from baseline throughout the experiment, signifying preserved vascular health. These findings demonstrate that XO inhibition can offer protection from radiation-induced endothelial dysfunction and cardiovascular complications.

This series of experiments quantified the role of the rat small intestinal arterioles in capacity changes during bilateral carotid occlusion (BCO) and compared them with previous venular studies. We also determined the role of autoregulation in arteriolar constriction during BCO. First-order, second-order, and third-order arteriolar diameter changes were measured during changes in arteriolar pressure and/or sympathetic activity (via BCO). The results indicated that an arteriolar pressure drop caused an immediate significant transient diameter decrease of 14% (P < 0.001), followed by an average steady-state diameter increase of 11% (P < 0.001) over control because of autoregulation. During BCO, the arterioles demonstrated an initial 8–22% decrease in diameter (P < 0.05). The largest first-order vessels were the least responsive to changes in pressure and BCO, while the smallest third-order vessels were the most responsive. Apparently autoregulation, and not sympathetic activity, was responsible for most of the arteriolar constriction during BCO. We also developed an anatomic model of the rat intestinal vasculature which revealed that venules hold 70% of the microcirculatory volume and are responsible for 80% of the total blood shift during BCO. Venular constriction and, to a minor degree, arteriolar constriction result in a 14% decrease in microcirculatory intestinal blood volume during BCO.

We tested the hypothesis that the venules of the small intestinal muscle are responsible for decreases in vascular capacitance during bilateral carotid artery occlusion. We measured microvascular venular pressure and diameter relations in 135 vessels during both control and baroreflexive conditions (bilateral carotid occlusion). Microvascular pressure was measured using a servo-null pressure system, and diameters were obtained from a video-monitoring system with a total magnification of X1,000. First-, second-, and fourth-order microvenules were studied in rat small intestinal muscle. The vessels showed an average diameter decrease of 11-12% and an increased stiffness or pressure-diameter slope of 31-53% during bilateral occlusion. We also tested whether the observed constriction during bilateral occlusion was caused by an increase in the sympathetic nerve activity to the venules and/or increased hormonal release via the baroreflex system. We studied an additional 22 microvenules before and after denervation of the preparation. Denervation eliminated any significant change in diameter or stiffness during bilateral occlusion. Based on our data, we conclude that the changes in the venular properties observed during bilateral occlusion are due to the increased sympathetic nerve activity resulting from decreased carotid sinus pressure. Intestinal venules can actively constrict to change vascular capacitance during bilateral carotid occlusion.

We developed a method by which we can completely isolate the carotid sinus baroreceptor regions in the rat. The carotid sinus baroreceptor region is exposed and, with the use of extra-fine forceps, a human hair is placed around and tied at the root of the bifurcation. This procedure occludes the external carotid artery and blood flow to the carotid body. An injector is then attached to a catheter in the common carotid artery. We introduce a cylindrical rubber plug into either the palentine or internal carotid artery. A second plug is introduced to occlude the other artery. In six of the eight rats studied, these procedures completely isolated the carotid sinus region. In those cases where a small leak persisted at a carotid sinus pressure of 180 mmHg, we introduced a small particle of the animal's own previously clotted blood. Carotid sinus pressure was either randomly changed between 40 and 180 mmHg in 20-mmHg increments or in sequential 20-mmHg steps from 40 to 180 mmHg while measuring the animal's pulsatile and mean blood pressures. Arterial pressure-carotid sinus pressure relationship indicates that there is a highly sigmoidal relationship between the two pressures. The peak gain of the carotid sinus reflex system had a range from 1.5 to 4.0 and a mean value of 2.07 +/- 0.08. Our data indicate that the rat exhibits a significant carotid sinus baroreceptor reflex response. This technique combined with other techniques will allow for the study of neural control of cardiovascular function in the rat.

Absolute left ventricular volume measurement by the conductance (volume) catheter requires subtraction of the conductance contribution from structures extrinsic to the cavity blood pool. Previously, this parallel conductance volume (Vp) has been assumed constant throughout the cardiac cycle, and the technique described for its estimation in situ yields a single value. We present a new method for parallel conductance determination that yields multiple estimates during systole, enabling an assessment of Vp variability [Vp(t)]. For isolated blood-perfused ejecting canine left ventricles with empty (vented) right ventricles, Vp(t) displayed virtually no variation throughout systole. For in situ hearts, despite the presence of other cardiac chambers, Vp(t) also displayed little variation, with no statistically significant deviation from its mean value throughout systole. Volume signal simulations found the new technique to be less sensitive to signal noise and thus more robust than the one previously published. The isolated and in situ heart data indicate that for the left ventricle, the parallel conductance is relatively constant throughout normal ejection.

Emerging evidence supports the idea that arginase, expressed in the vascular endothelial cells of humans and other species, modulates endothelial nitric oxide (NO) synthase-3 (NOS-3) activity by regulating intracellular l-arginine bioavailability. Arginase II is thought to be expressed in the mitochondria of a variety of nonendothelial cells, whereas arginase I is known to be confined to the cytosol of hepatic and other cells. The isoforms that regulate NOS-3 and their subcellular distribution, however, remain incompletely characterized. We therefore tested the hypothesis that arginase II is confined to the mitochondria and that mitochondrial arginase II reciprocally regulates vascular endothelial NO production. Western blot analysis, immunocytochemistry with MitoTracker, and immunoelectron microscopy confirmed that arginase II is confined predominantly but not exclusively to the mitochondria. Arginase activity was significantly decreased, whereas NO production was significantly increased in the aorta and isolated endothelial cells from arginase II knockout (ArgII−/−) mice compared with wild-type (WT) mice. The vasorelaxation response to acetylcholine (ACh) was markedly enhanced and the vasoconstrictor response to phenylephrine (PE) attenuated in ArgII−/− in pressurized mouse carotid arteries. Furthermore, inhibition of NOS-3 by NG-nitro-l-arginine methyl ester (l-NAME) impaired ACh response and restored the PE response to that observed in WT vessels. Vascular stiffness, as assessed by pulse wave velocity (PWV), was significantly decreased in ArgII−/− compared with WT mice. On the other hand, 14 days of oral l-NAME treatment significantly increased PWV in both WT and ArgII−/− mice, such that they were not significantly different from one another. These data suggest that arginase II is predominantly confined to the mitochondria and that this mitochondrial arginase II regulates NO production, vascular endothelial function, and vascular stiffness by modulating NOS-3 activity.

Endothelial function is impaired in aging because of a decrease in NO bioavailability. This may be, in part, attributable to increased arginase activity, which reciprocally regulates NO synthase (NOS) by competing for the common substrate, l -arginine. However, the high K m of arginase (>1 mmol/L) compared with NOS (2 to 20 μmol/L) seemingly makes direct competition for substrate unlikely. One of the mechanisms by which NO exerts its effects is by posttranslational modification through S -nitrosylation of protein cysteines. We tested the hypothesis that arginase1 activity is modulated by this mechanism, which serves to alter its substrate affinity, allowing competition with NOS for l -arginine. We demonstrate that arginase1 activity is altered by S -nitrosylation, both in vitro and ex vivo. Furthermore, using site-directed mutagenesis we demonstrate that 2 cysteine residues (C168 and C303) are able to undergo nitrosylation. S -Nitrosylation of C303 stabilizes the arginase1 trimer and reduces its K m value 6-fold. Finally, arginase1 nitrosylation is increased (and thus its K m decreased) in blood vessels from aging rats, likely contributing to impaired NO bioavailability and endothelial dysfunction. This is mediated by inducible NOS, which is expressed in the aging endothelium. These findings suggest that S -nitrosylated arginase1 can compete with NOS for l -arginine and contribute to endothelial dysfunction in the aging cardiovascular system.

Oxidized low-density lipoprotein (OxLDL) impairs NO signaling and endothelial function, and contributes to the pathogenesis of atherosclerosis. Arginase reciprocally regulates NO levels in endothelial cells by competing with NO synthase for the substratel-arginine. In human aortic endothelial cells, OxLDL stimulation increased arginase enzyme activity in a time- and dose-dependent manner. Arginase activity reached its maximum as early as 5 minutes, was maintained for a period of more than 48 hours, and was associated with a reciprocal decrease in NO metabolite (NOx [nitrite and nitrate]) production. Furthermore, OxLDL induced arginase II mRNA expression after 4 hours. Small interfering RNA targeted to arginase II decreased both the quantity and the activity of arginase from baseline, prevented OxLDL-dependent increases in arginase activity, and induced an increase in NOx production. Immunofluorescence analysis revealed an association of arginase II with the microtubule cytoskeleton. Microtubule disruption with nocodazole caused a dramatic redistribution of arginase II to a diffuse cytosolic pattern, increased arginase activity, and decreased NOx production, which was restored in the presence of the specific arginase inhibitor (S)-(2-boronoethyl)-l-cysteine (BEC). On the other hand, epothilone B prevented microtubule disruption and inhibited OxLDL-dependent increases in arginase activity and attenuated OxLDL-dependent decreases in NOx. Preincubation of rat aortic rings with OxLDL resulted in an increase in arginase activity and a decrease in NOx production. This was reversed by arginase inhibition with the BEC. Thus, OxLDLs increase arginase activity by a sequence of regulatory events that involve early activation through decreased association with microtubules and a later increase in transcription. Furthermore, increased arginase activity contributes to OxLDL-dependent impairment of NOx production. Arginase, therefore, represents a novel target for therapy in atherosclerosis.

Background— Although abnormal l -arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. l -arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating l -arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. Methods and Results— Inhibition of arginase with (S)-(2-boronoethyl)- l -cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4±9.4% at 10 −5 mol/L, P N -hydroxy-nor- l -arginine (nor-NOHA) and difluoromethylornithine (DFMO). This effect required intact endothelium and was prevented by 1H-oxadiazole quinoxalin-1-one ( P P P l -arginine (10 −4 mol/L)–dependent vasorelaxant responses in O rings to those of Y. Arginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. Conclusions— These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular l -arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

Obesity is a major health care problem and is associated with significant cardiovascular morbidity. Leptin, a neuroendocrine hormone released by adipose tissue, is important in modulating obesity by signaling satiety and increasing metabolism. Moreover, leptin receptors are expressed on vascular endothelial cells (ECs) and mediate angiogenesis. We hypothesized that leptin may also play an important role in vasoregulation. We investigated vasoregulatory mechanisms in the leptin-deficient obese ( ob/ob) mouse model and determined the influence of leptin replacement on endothelial-dependent vasorelaxant responses. The direct effect of leptin on EC nitric oxide (NO) production was also tested by using 4,5-diaminofluorescein-2 diacetate staining and measurement of nitrate and nitrite concentrations. Vasoconstrictor responses to phenylephrine, norepinephrine, and U-46619 were markedly enhanced in aortic rings from ob/ob mice and were modulated by NO synthase inhibition. Vasorelaxant responses to ACh were markedly attenuated in mesenteric microvessels from ob/ob mice. Leptin replacement resulted in significant weight loss and reversal of the impaired endothelial-dependent vasorelaxant responses observed in ob/ob mice. Preincubation of ECs with leptin enhanced the release of NO production. Thus leptin-deficient ob/ob mice demonstrate marked abnormalities in vasoregulation, including impaired endothelial-dependent vasodilation, which is reversed by leptin replacement. These findings may be partially explained by the direct effect of leptin on endothelial NO production. These vascular abnormalities are similar to those observed in obese, diabetic, leptin-resistant humans. The ob/ob mouse may, therefore, be an excellent new model for the study of the cardiovascular effects of obesity.

Despite the well-established fact that the carotid sinus baroreflex system has profound control over the physical properties of the systemic circulation, the resistance to venous return (RVR) seems to be invariant of such control. We hypothesized that this apparent paradox may be explained from the baroreflex changes in systemic arterial compliance. In 12 pentobarbital-anesthetized mongrel dogs, RVR was measured at controlled carotid sinus pressures (CSP) of 50 and 200 mmHg with normal and artificially increased arterial compliance. Arterial compliance was determined from the arterial pressure decay when systemic blood flow was stopped with total vena caval occlusion. Changing CSP between 50 and 200 mmHg changed RVR significantly only under the condition of artificially increased arterial compliance. A four-parameter lumped model of the systemic circulation revealed that the baroreflex changes in arterial compliance and arterial resistance, which occurred in opposite directions, prevented a change in RVR when CSP was changed. The data also suggested that approximately 75% of RVR was attributed to large and conduit veins, the resistances along which were insensitive to baroreflex control. We concluded that the invariance of RVR results from a combination of 1) baroreflex change in the arterial compliance, 2) baroreflex insensitivity of the resistance along large and conduit veins, and 3) spatially distinct location between the major site of reflex change in capacitance and the major site of compliance.

We examined the active and passive contributions of the alpha- and beta-adrenergic receptor mechanisms to the changes in systemic vascular capacitance caused by the carotid sinus baroreflex system in anesthetized, vagotomized dogs. The carotid sinuses were isolated from the systemic circulation and perfused with controlled pressures. To determine the changes in vascular capacitance, a constant flow, constant venous pressure cardiopulmonary bypass was used. The changes in unstressed vascular volume were calculated when carotid sinus pressure was reduced from 200 to 50 mmHg without any adrenergic receptor antagonist, with either an alpha- (phentolamine) or a beta- (propranolol) antagonist and then with both. The reflex change in unstressed vascular volume in the systemic circulation (22.6 +/- 9.0 ml/kg without any antagonist) was reduced by 72% with phentolamine, by 35% with propranolol, and by 73% with both antagonists. Our results suggest that the alpha-adrenergic mechanisms contribute significantly to active changes in systemic venous capacity. In addition, the beta-adrenergic system has very little effect on active changes in venous vessels but does contribute to the overall capacity changes by dilating the hepatic outflow resistance when the carotid sinus baroreflex system is activated.

Hemodynamic properties of the systemic vasculature were measured in eight anesthetized dogs using two-port impedance analysis. Blood pressures and flows were measured at the aortic root and the caval-atrial junction. Impedances were computed from 0.05 to 20 Hz to characterize the systemic vasculature. Pseudorandom variations in flow were produced with an extracorporeal perfusion system. Impedance measurements were made at carotid baroreceptor pressures of 50, 125, and 200 mmHg. A six-parameter lumped-element model best fitted the measured impedance spectra. At 125 mmHg, the mean parameter values were venous inertance, 13.5 g.kg.cm-4; venous and arterial compliances, 0.769 and 0.0214 ml.mmHg-1.kg-1; venous and arterial characteristic impedances, 0.028 and 0.084 mmHg.kg.min.ml-1; and arterial-to-venous small-vessel resistance, 0.706 mmHg.kg.min.ml-1. Regression analysis showed significant dependence of small-vessel resistance on baroreceptor pressure. The other parameters were not dependent on carotid sinus pressure, which is consistent with baroreflex control of venous unstressed volume but not compliance. We conclude that two-port impedance analysis is a useful tool for studying venous hemodynamics and the dynamic coupling between the veins and the right heart.

To determine whether microvessels in resting or contracting skeletal muscle constrict during baroreceptor activation, vascular diameters were measured in the spinotrapezius muscle of adult rats (n = 12) during occlusion of the common carotid arteries. Neural and myogenic components were distinguished using two types of occlusion: 1) "normal" (arterial pressure was allowed to increase with baroreceptor activation) and 2) "isobaric" (arterial pressure was maintained constant by decreasing blood volume). During normal occlusions, intermediate and small arteriolar diameters decreased in resting and contracting muscle (10-15% and 25-30%, respectively). Large arterioles and all-sized venules distended slightly (approximately 5%) in resting muscle, but diameters were maintained or decreased in contracting muscle. When arterial pressure was maintained constant (isobaric), the microvascular responses to baroreceptor activation in both resting and contracting muscle were essentially eliminated. We conclude that nearly all the arteriolar constriction observed in the spinotrapezius muscle during normal carotid artery occlusion is myogenic in origin, secondary to increased arterial pressure. This pressure-dependent constriction is augmented during skeletal muscle contraction and functional vasodilation.

The hypothesis that the pressure-diameter relationship of intestinal venules in rats is primarily determined by sympathetic nervous system activity was tested. The pressure-diameter relationship of the smallest to largest diameter (20-100 microns) intestinal venules of the rat was measured at rest, during hemorrhage to increase sympathetic neural activity, and during saline volume expansion to decrease sympathetic activity. During hemorrhage, the diameter of all venules decreased approximately 10% at 10 mmHg venous pressure, and the slope of the pressure-diameter relationship increased approximately 50% above control. Blood volume expansion led to an approximately 10% increase in venule diameter at 10 mmHg and a 25% decrease in slope. Denervation of the vessels causes concomitant vasodilation, which was greater than the vasodilation caused by blood volume expansion. Hemorrhage after denervation caused no significant changes in the relationship when compared with denervated control. Nitroprusside caused an even greater vasodilation when compared with the pressure-diameter relationship after denervation. The results suggest that the slope and 10-mmHg intercept of the pressure-diameter relationship for the largest through smallest intestinal venules and, therefore, their vascular compliance and capacitance characteristics are primarily determined by sympathetic activity.

The contribution of the splanchnic vascular bed in the carotid sinus baroreceptor reflex control of vascular resistance and capacity was studied in nine pentobarbital-anesthetized dogs. The splanchnic circulation was vascularly isolated in an unopened abdomen and perfused at constant flow and venous pressure. Decreasing carotid sinus pressure from 200 to 50 mmHg resulted in a 72% increase in splanchnic vascular resistance and a decrease in splanchnic blood volume of 4.7 ml/kg. Changes in splanchnic inflow from 0 to 70 ml.min-1.kg-1 resulted in linear changes in splanchnic arterial pressure. Increasing carotid sinus pressure significantly decreased the slope (P less than 0.005) and intercept (P less than 0.025) of the splanchnic pressure-flow relationship. It is concluded that in the dog, the splanchnic vascular bed contributes a major portion of blood volume mobilized by the carotid sinus reflex.

Venous return curves and cardiac function relationships were simultaneously determined in 10 pentobarbital-anesthetized dogs at three different isolated carotid sinus pressures. Changing carotid sinus pressure (CSP) between 50 and 200 mmHg produced large changes in the zero flow intercept of the venous return curves from 15.37 +/- 0.97 to 11.94 +/- 1.36 mmHg (P less than 0.001) but no change in slope. These changes in the intercept of the venous return curve were due to alterations in systemic vascular capacity caused by the carotid sinus baroreceptor reflex. Changes in the cardiac function curve with baroreceptor pressure were masked by concomitant changes in arterial pressure afterload; however, when arterial pressure was controlled, there was a significant change in the slope of the cardiac function curve from 60.32 +/- 26.9 to 37.06 +/- 13.31 ml X min-1 X kg-1 X mmHg-1 as CSP was changed from 50 to 200 mmHg. We conclude that changes in vascular capacity are the primary mechanism responsible for changes in cardiac output during activation of the carotid sinus baroreflex.

The purpose of this study was to determine the mechanism of the decrease in left ventricular stroke volume during spontaneous inspiration. We determined the transmural pressures of the left heart by measuring left atrial and diastolic left ventricular pressures relative to esophageal pressure. We estimated the directional changes in end-systolic and end-diastolic volumes of the left ventricle by determining the transit time of sound transmission between two ultrasonic crystals facing each other across the minor axis of the left ventricle. Left ventricular stroke volume decreased with spontaneous inspiratory effort as pleural pressure fell, regardless of whether lung volume increased or remained constant. The stroke volume was decreased during the fall in pleural pressure because of an increase in end-systolic volume with an essentially unchanged diastolic volume. Thus, the decrease in stroke volume was due to a decrease in ejection, rather than a decrease in filling of the left ventricle. We believe that left ventricular ejection was impeded by the fall in pressure around the heart relative to the pressure in the aorta. In spite of the essentially constant diastolic volume, diastolic left ventricular transmural pressure rose, suggesting that spontaneous inspiration decreases the diastolic compliance of the left ventricle. The change in diastolic compliance contributed to the decrease in stroke volume but was not the primary cause.

To quantify the importance of the carotid sinus baroreceptor reflex and the interaction with epinephrine infusion on total pulmonary vascular capacity and resistance, I have simultaneously measured total pulmonary vascular compliance, changes in pulmonary blood volumes, and changes in resistances in seven sodium pentobarbital-anesthetized dogs. A preparation was used that bypasses the right and left hearts allowing for the simultaneous measurement of the pulmonary as well as the systemic vascular bed parameters. At intrasinus pressures of 50, 125, and 200 mm Hg, without epinephrine infusion, the pulmonary vascular resistance was 0.134, 0.121, and 0.109 mm Hg/(ml per min per kg) and the systemic vascular resistance was 1.21, 0.87, and 0.63 mm Hg/(ml per min per kg). Epinephrine infusion of 1 microgram/min per kg at each intrasinus pressure caused the resistances of both vascular beds to increase. Pulmonary vascular resistance increased to 0.157 mm Hg/(ml per min per kg) and showed no further changes with changes in ISP. However, systemic vascular resistance did decrease from 1.49 to 1.41 and 1.25 at intrasinus pressures of 50, 125, and 200 mm Hg. During control runs, pulmonary vascular capacity changed 1.06 ml/kg and systemic capacity changed 7.63 ml/kg for SP changes between 50 and 200 mm Hg. Both responses were greatly attenuated after epinephrine infusion and amounted to only 0.17 ml/kg for the pulmonary and vascular bed and 2.26 ml/kg for the systemic vascular bed. The differences in capacity changes between control and epinephrine runs for the two vascular beds were nearly equal to the change in capacities brought about by epinephrine when it was infused at a fixed intrasinus pressure of 125 mm Hg. The pulmonary vascular compliance during the control runs increased from 0.303 to 0.329 ml/mm Hg per kg when ISP was increased from 50 to 200 mm Hg. After epinephrine infusion, the pulmonary compliance was 0.273 ml/mm Hg per kg and showed no changes with intrasinus pressure. Similar results were obtained for the systemic vascular compliance which was 2.07 ml/mm Hg per kg at an ISP of 50 mm Hg and increased to 2.39 ml/mm Hg per kg to an ISP of 200 mm Hg during control runs. After epinephrine infusion, the systemic compliance was 1.89 ml/mm Hg per kg and again showed no changes with ISP. These data indicate that the baroreceptor reflex can exert control of pulmonary and systemic vascular resistance and capacitance and epinephrine can greatly attenuate the reflex control of both vascular beds.

To quantitatively understand how the carotid sinus baroreceptor reflex modifies the Starling curve (i.e., the aortic flow (AF)-mean right atrial pressure (MRAP) relationship), experiments were performed in closed-chest, naturally breathing, and anesthetized dogs before and after vagotomy. Mean aortic pressure (MAP) was fixed at approximately 100 mm Hg and the pressure in the isolated carotid sinus (ISP) was varied from 75 to 150 mm Hg in steps of 25 mm Hg. At each ISP, MRAP was slowly increased and decreased while measuring AF by a previously implanted electromagnetic flow probe. A family of AF-MRAP relation curves specified at the different ISP's were thus obtained. Third-order polynomials in MRAP and ISP adequately fit these curves. Whether the vagi were intact or cut, there was no significant difference between the AF-MRAP relation curves obtained at an ISP of 75 or 100 mm Hg. However, decreasing ISP from 125 to 100 mm Hg caused a 24% increase in AF, and increasing ISP to 150 mm Hg caused a 15% decrease in AF in the dogs with intact vagus nerves. For the vagotomized dogs, the same decrease or increase in ISP caused a 17% increase or a 21% decrease in AF, respectively. When MAP was allowed to change by the reflex, only insignificant changes in AF occurred. We conclude that the carotid sinus reflex significantly alters the flow-generating ability of the heart-lung compartment by as much as 40% but this becomes clearly observable only if the reflex change in aortic pressure is prevented.

A thesis recently developed from a series of experiments on the isolated canine left ventricle is described. It is claimed that the ventricular presure/volume ratio at end-systole is relatively insensitive to cardiac loading and varies greatly in response to changes in ventricular contractility. The clinical viability of this basic finding rests on the substitution of diameter for volume in this formulation. Diameter can be measured using a noninvasive ultrasonic technique in the clinic. Accordingly, end-systolic pressure/diameter ratio was studied in the isolated preparation and found to be similarly insensitive to loading conditions and sensitive to inotropic interventions. A further analysis of the pressure/diameter ratio in the ventricle of the conscious dog is in progress. In parallel with these studies, use of the pressure/diameter ratio to evaluate contractility in cardiac patients is being tested. The preliminary findings from conscious dogs and clinic patients are briefly discussed.

Acute shifts in the pressure-volume relationship of the left ventricle occur under several conditions. One potential mechanism for this phenomenon is ventricular interdependence, that is, the influence of right ventricular filling on the left ventricle. This mechanism was studied in an isolated cross-circulated ejecting canine heart preparation with balloons sewn into both right and left ventricles and connected to volumetric chambers allowing continuous and accurate simultaneous measurement of right and left ventricular pressures and volume. Sets of left ventricular pressure-volume loops were obtained under a variety of right ventricular volumes. Experiments were repeated with and without the pericardium. Both diastole and systole were studied. We show that in the pericardium-free heart working over a normal range of volumes, the right ventricle exerts little influence on left ventricular pressure. With the pericardium present, there is a small but significant effect of right ventricular volume on left ventricular pressure during systole and diastole. Over a wider range of volumes imposed in the arrested heart, the right ventricle does influence left ventricular pressure even without the pericardium. Thus we have demonstrated that ventricular interdependence is not likely to lead to large acute pressure-volume shifts, either during diastole or systole, except in the presence of considerably above normal right ventricular volumes.

The ability of the aortic arch baroreceptors to change vascular capacity was measured and, in the same animal, compared with carotid sinus reflex changes in capacity. Seven dogs were anesthetized with pentobarbital sodium and perfused with constant flow. Changes in external reservoir volume reflected reciprocal changes in total systemic vascular capacity and changes in arterial pressure parallel changes in total peripheral resistance. The aortic arch and carotid sinus baroreceptor areas were isolated, and the pressures were controlled separately. With carotid sinus pressure held constant at 125 mmHg, aortic arch pressure was increased and decreased between 225 and 50 mmHg, and the changes in reservoir volume and systemic arterial pressure were measured. Results from increasing and decreasing aortic arch or carotid sinus pressure were not significantly different and were averaged. The mean change in reservoir volume was 1.9 +/- 0.2 ml/kg and the change in mean arterial pressure was 18.7 +/- 3.7 mmHg. The changes in reservoir volume and arterial pressure caused by the aortic arch reflex were not influenced by the level of carotid sinus pressure. Carotid sinus pressure changes between 200 and 50 mmHg at a constant aortic arch pressure caused reservoir volume and arterial pressure to change by 7.2 +/- 0.9 ml/kg and 45.1 +/- 4.1 mmHg, respectively. The level of aortic arch pressure did not modify these responses.

As a means of assessing ventricular performance, we analyzed the time-varying ratio of instantaneous pressure, P(t), to instantaneous volume, V(t), in the canine left ventricle. Intraventricular volume was measured by plethysmography, while the right heart was totally bypassed. The cardiac nerves were sectioned, and an epinephrine infusion was used to alter the contractile state. The instantaneous pressure-volume ratio was defined as E(t) = P(t)/[V(t) - V d ], where V d is an experimentally determined correction factor. We found that (1) all the E(t) curves thus defined were similar in their basic shape and attained their peak near the end of the ejection phase regardless of the mechanical load, the contractile state, or the heart rate, (2) under a constant heart rate and contractile state extensive changes in preload, afterload, or both did not alter the peak value of E(t), Emax, or the time to Emax from the onset of systole, Tmax, and (3) these parameters of E(t) markedly changed with epinephrine infusion or increases in heart rate. At an epinephrine infusion rate of 2 µg/kg min -1 , Emax increased to 12.2 ± 4.5 ( SD ) mm Hg/ml (N = 9) from its control value of 6.6 ± 1.2 mm Hg/ml before the infusion. Simultaneously, Tmax shortened from 191 ± 29 msec to 157 ± 26 msec. Increases in the paced heart rate proportionally shortened Tmax (45% per 100-beats/min change in heart rate) without any effect on Emax. We concluded that E(t), represented by Emax and Tmax, explicitly reflects the ventricular contractility.

To understand more fully the role of the arterial baroreceptor reflex on systemic and pulmonary vascular resistance, we studied the influence of the carotid sinus baroreceptor reflex control system on the entire systemic and pulmonary arterial pressure-flow relationships. Ten pentobarbital-anesthetized dogs, whose carotid sinuses were isolated, were used in a preparation in which the right and left hearts were bypassed to control systemic and pulmonary blood flows. At intrasinus pressures of 50, 125, and 200 mm Hg, systemic and pulmonary arterial pressures were measured in response to step changes in systemic and pulmonary blood flows from 0 up to 200 ml/min per kg. The systemic arterial pressure-flow relationship exhibited a marked nonlinearity, especially at either high or low flows. A third-order polynomial fit was found to represent the steady state systemic arterial pressure-flow relationship best. Blood flow autoregulation was reflected as a secondary change in systemic arterial pressure at constant flow approximately 15 seconds after the initial response. Blood flow autoregulation was seen in the entire systemic vascular bed over the entire range of flows studied. The degree of autoregulatory significantly contributed to the shape of the systemic arterial pressure-flow relationship. The steady state arterial pressure-flow relationship shifted upward and toward the pressure axis, increasing the calculated incremental resistance and total peripheral resistance as intrasinus pressure was decreased. The systemic zero-flow arterial pressure was found to increase with decreases in intrasinus pressure. The pulmonary arterial pressure-flow relationship was found to be linear in the range of flows studied from 25 up to 200 ml/min per kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of carotid sinus and aortic arch reflex control of total peripheral resistance was studied in eight dogs anesthetized with sodium pentobarbital and placed on constant flow cardiac bypass. Carotid sinus and aortic arch baroreceptor areas were isolated and separately perfused at controlled pressures. Combinations of carotid sinus and aortic arch pressures were delivered at random in steps of 25 mm Hg over the 50-225 mm Hg pressure range, and systemic arterial pressure was measured. Changes in arterial pressure reflected changes in total peripheral resistance. A multiple linear regression showed that both carotid sinus and aortic arch pressures exhibited a sigmoidal relationship with arterial pressure. Independent of carotid and aortic baroreceptor pressures, arterial pressure was found to be a periodic function of time (period = 2 hours) in all dogs. The average carotid sinus reflex open loop gain was found to be 0.231 +/- 0.092, while average aortic arch open loop gain was 0.141 +/- 0.088. The gain of either the carotid sinus or aortic arch reflex was not influenced by the absolute pressure level of the other receptor area. In a separate series of experiments performed in the same dogs, we tested the hypothesis that a nonlinear temporal summation of the reflex control of total peripheral resistance might exist when the inputs to carotid and aortic baroreceptors are changed simultaneously. With both inputs held at the region of maximum gain, 25 mm Hg step changes were imposed first on carotid sinus pressure, then on aortic arch pressure, and then on both simultaneously. A temporal inhibition of the two reflexes showed that simultaneous excitation of both receptors resulted in a smaller reflex response than the sum of individual responses.

The direct effects of vasopressin on the resistance and capacitance properties of the pulmonary and systemic vasculature were studied in nine aneural dogs on systemic and pulmonary bypass. The systemic and pulmonary pressure-flow, the systemic and pulmonary arterial pressure-volume, and the systemic and pulmonary venous pressure-volume relationships were determined for five levels of infused vasopressin. Vasopressin levels of approximately 10, 30, 150, 300, and 500 pg/ml were achieved by intravenous infusions. Samples of venous blood were drawn before and after each set of pressure-flow and pressure-volume relationships for the determination of vasopressin level by radioimmunoassay. A linear relationship was found between vasopressin level and systemic vascular resistance. Systemic vascular resistance increased 0.072 +/- 0.011 mmHg.kg.min.ml-1 for a change in vasopressin level of 100 pg/ml. Vasopressin did not affect pulmonary vascular resistance or any vascular compliance. High doses of infused arginine vasopressin were necessary to elicit substantial vasoconstriction.

We have studied the effect of the carotid sinus baroreceptor reflex on respiration in 10 vagotomized, spontaneously breathing, pentobarbital anesthetized dogs. The carotid body chemoreceptor reflex response was eliminated by surgically excluding the carotid bodies from the carotid sinus baroreceptor area. Steady state frequency, tidal volume, and minute ventilation were measured after 25 mm Hg step changes in intrasinus pressure between 50 and 200 mm Hg. Over this range, the step decreases in intrasinus pressure caused concomitant increases in mean arterial pressure from 86 to 182 mm Hg. All of the respiratory response curves were sigmoidal in shape. Decreasing intrasinus pressure from 200 to 50 mm Hg caused respiratory frequency to increase from 4.8 to 9.7/min, and tidal volume to decrease from 704 to 515 ml. The calculated total ventilation, however, increased from 3180 to 4530 ml/min. The time of inspiration decreased from 3.7 to 2.4 seconds, and the time of expiration decreased from 9.8 to 4.1 seconds. These ventilatory responses are shown to be baroreceptor reflex mediated, and not secondary to changes in arterial pressure. These findings indicate that not only does the carotid sinus baroreceptor reflex control arterial pressure, but it also simultaneously influences ventilation, through changes in both respiratory frequency and tidal volume.

SUMMARY The instantaneous isovolumic and ejecting pressure-volume relationship of the right ventricle was studied in 11 cross-circulated, isolated canine hearts to characterize the right ventricular contractile state. Accurate measurement of volume was achieved by the use of a water-filled, thin latex balloon in the right ventricle connected to a special volume loading and transducing chamber. Pressure was measured with a miniature pressure transducer mounted within the balloon. Wide variations in loading conditions were achieved by changing the volume of air above the volumetric chamber. The pressure and volume data were collected from multiple beats under a constant contractile state in the same mode of contraction while the left ventricle was vented to air. Linear regression analysis applied to each of the isochronal pressure-volume data sets at 20-msec intervals from the onset of contraction showed a highly linear correlation between the pressure and the volume. Both the slope and the volume intercept of the regression lines changed with time throughout the cardiac cycle. The maximal slope of the regression line (E,,,,) averaged 2.50 ± 0.49 mm Hg/ml (mean ± SD) for ejecting beats and 2.68 ± 0.55 mm Hg/ml for isovolumic beats. Epinephrine infusions of 12.5 fig/min and 25.0 /ig/min increased E^u by 31% and 82%, respectively (P < 0.005). We conclude that: (1) The instantaneous pressure-volume relationships of the right ventricle in the isovolumic and ejecting modes can be regarded as linear, at least within the physiological range; however, these two modes of contraction did not yield an identical relationship. (2) The slope of these pressure-volume relationship curves changes with a change in the contractile state. Ore Res 44: 309-315, 1979 IT IS WELL KNOWN that there are major anatomical and physiological differences between the right and left ventricles. Compared with the left ventricle, the right ventricle has a greater regional variation in wall thickness and a more complex geometric shape. The developed pressure in systole is much smaller, and intraventricular pressure falls considerably while ejection proceeds. Much recent research has been focussed on left ventricular function, yet there is little quantitative information concerning the right ventricle, particularly with respect to the question of how to characterize contractile state and pumping ability. Those indices of contractile state used in characterizing left ventricular function have not been quantified in and shown to be valid for the right ventricle. Since there are major differences between left and right ventricles, we investigated whether right ventricular contractile state could be described in a similar fashion as left ventricular contractile state. In this study we determined the time-varying ratio of instantaneous pressure to volume, which has been shown to be sensitive to changes in left ventricular contractile state and nearly independent of

Two-dimensional echocardiography can provide serial cross-sectional images of the left ventricular cavity. We examined whether such serial images from steady-state ejecting hearts would allow three-dimensional reconstruction and accurate volume estimation without major geometric assumptions. Cross-circulated, paced dog hearts were suspended in a blood-filled tank. Serial cross-sectional images were taken at 3-mm intervals along the vertical axis. Left ventricular cavity and muscle areas of each image were planimetered with a light-pen system and summated for volume: total volume = sigma (areas x 3 mm). Direct left ventricular volume was measured through the cardiac cycle with a volumetric chamber connected to a balloon in the ejecting left ventricle. In six hearts, 67 separate direct volume measurements (range 9.5--54.7 ml) from various points in the cardiac cycle were compared with the simultaneous echo volume measurements. By least squares linear regression, echo volume = 1.01 (direct volume) - 0.44 ml; r = 0.972, SEE = 2.93 ml. Provided accurate cross-sectional localization is available, these studies suggest that extremely accurate steady-state left ventricular volume can be determined noninvasively in the ejecting heart from multiple cross-sectional images.

To quantify the relative contribution of blood flow redistribution and active changes in vascular capacity in the regulation of cardiac output, blood flow and volumes in two parallel vascular beds were measured in response to varying carotid sinus pressures. In nine dogs, carotid sinuses were isolated and intrasinus pressure was controlled. Two external reservoirs were placed between the caval veins and the right heart to measure changes in vascular capacity in splanchnic and extrasplanchnic vascular beds. At intrasinus pressures of 50 and 200 mm Hg, we have simultaneously measured arterial resistances, compliances, changes in flows, and "unstressed vascular volume," and time constants of venous drainage in the splanchnic and extrasplanchnic vascular beds. Compliances and time constants of venous drainage were found to be nearly equal in the two beds. A decrease in intrasinus pressure from 200 to 50 mm Hg resulted in a small redistribution of blood flow (about 5% of cardiac output) from the extrasplanchnic compartment to the splanchnic vascular bed. Changes in reservoir volumes were found to be around 7.0 ml/kg. The splanchnic vascular bed was responsible for a greater change in reservoir volume for a given change in intrasinus pressure. With any change in intrasinus pressure, the change in arterial resistance in the extrasplanchnic vascular bed was greater than that of the splanchnic vascular bed. Blood flow redistribution was not found to be a significant factor contributing to changes in reservoir volume. The changes in reservoir volume seen, must have been due to active changes in vascular capacity in the two channels chosen.

Thirteen pentobarbital-anesthetized dogs whose carotid sinuses were isolated and perfused at a constant pressure were placed on total cardiac bypass. With systemic venous pressure held at 0 mmHg (condition 1), arterial inflow was stopped for 20 s at intrasinus pressures of 50, 125, and 200 mmHg. Zero-flow arterial pressures under condition 1 were 16.2 +/- 1.3 (SE), 13.8 +/- 1.1, and 12.5 +/- 0.8 mmHg, respectively. In condition 2, the venous outflow tube was clamped at the instant of stopping the inflow, causing venous pressure to rise. The zero-flow arterial pressures were 19.7 +/- 1.3, 18.5 +/- 1.4, and 16.4 +/- 1.2 mmHg for intrasinus pressures of 50, 125, and 200 mmHg, respectively. At all levels of intrasinus pressure, the zero-flow arterial pressure in condition 2 was higher (P less than 0.005) than in condition 1. In seven dogs, at an intrasinus pressure of 125 mmHg, epinephrine increased the zero-flow arterial pressure by 3.0 mmHg, whereas hexamethonium and papaverine decreased the zero-flow arterial pressure by 2 mmHg. Reductions in the hematocrit from 52 to 11% resulted in statistically significant changes (P less than 0.01) in zero-flow arterial pressures. Thus zero-flow arterial pressure was found to be affected by changes in venous pressure, hematocrit, and vasomotor tone. The evidence does not support the literally interpreted concept of the vascular waterfall as the model for the finite arteriovenous pressure difference at zero flow.

Carotid sinus reflex interactions were studied in 10 dogs anesthetized with pentobarbital sodium. The right and left carotid sinus regions were isolated and perfused at controlled pressures. Pressure in the right and left carotid sinuses were independently varied, and the resulting steady-state reflex changes in arterial pressure, heart rate, respiratory frequency, tidal volume, and total ventilation were measured. Reflex changes when carotid sinus pressure was changed on one side were strongly influenced by pressure in the contralateral carotid sinus (P less than 0.05). Right carotid sinus gain was found to be 0.628 +/- 0.058 at a left carotid sinus pressure of 50 mmHg and 0.148 +/- 0.027 when left carotid sinus pressure was 200 mmHg. Similar results were found for left carotid sinus gain. Suppression was also found for heart rate, respiratory rate, tidal volume, and total ventilation. The hypothesis that rapid resetting of one carotid sinus baroreflex might influence responses from the other side was also tested. Although ipsilateral resetting was consistently observed, no contralateral component of the resetting was detected. An additional inhibitory summation between the right and left carotid sinuses was found such that simultaneous excitation of both receptors resulted in a smaller reflex response than did the sum of individual responses. Sympathetic denervation of the carotid sinus region had no effect.

To attain a quantitative understanding of carotid sinus baroreceptor reflex control of cardiac output, we studied the reflex control of total systemic vascular capacity in vagotomized dogs. In experiments measuring blood volume shifts caused by the carotid sinus reflex (series 1), venous return was diverted into a reservoir while cardiac output and central venous pressure were maintained at constant levels. The pressure in the isolated carotid sinuses (ISP) was lowered or raised in 25-mm Hg steps between 75 and 200 mm Hg. This procedure mobilized blood into or out of the reservoir, indicating a decrease or an increase in total vascular capacity, respectively. The mean maximum volume shift, 3.6 ml/kg body weight, occurred in the same ISP region, 135 ± 12.5 mm Hg, where reflex control of total peripheral resistance was strongest. The total volume shift was approximately 7.5 ml/kg for ISP changes from 75 to 200 mm Hg. When mean arterial blood pressure was maintained constant during the ISP step changes, the volume shift almost doubled. In experiments measuring the reflex effect on total systemic vascular compliance (series 2) and in experiments determining the reflex control of arterial compliance (series 3), total systemic vascular and lumped arterial compliances were measured in the same dogs that were used in series 1 experiments. The total systemic vascular and arterial compliances were approximately 2.0 ml/mm Hg kg -1 and 0.0677 ml/mm Hg kg -1 , respectively. The reflex did not affect these compliances. We concluded that the reflex controls the total systemic venous capacity to a degree that changes cardiac output potentially by 30-10% per 25-mm Hg change in ISP.