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BACKGROUND Vasopressin is frequently used to treat catecholamine-resistant vasodilatory shock. It enhances the vasoconstrictor effects of catecholamines at concentrations of vasopressin that have none or only minimal intrinsic pressor effects. However, the vascular mechanisms underlying this combined pharmacological approach have not been fully elucidated. METHODS We used isometric tension measurements in vascular rings to investigate potential cellular mechanisms. Vascular rings (0.2 mm diameter) were harvested from the superior mesenteric artery of Wistar rats (2 to 4 months of age). Dose-response relationships were derived for vasopressin (VP) and norepinephrine (NE), in the absence and presence of a subpressor dose of VP (10(-9) M). The contribution of tyrosine kinase (TK), the TK pathway proteins SRC and PYK2, as well as protein kinase C (PKC) were determined by measuring the modulating influence of specific inhibitors on the pressor response to NE (10(-5) M) alone and the augmented pressor response to VP (10(-9) M). RESULTS VP (10(-9) M) had only minimal pressor effect alone (10% of maximal response), but significantly increased the E(max) response to NE (587.8 ± 40.7 vs 331.2 ± 10.4 mg). TK inhibition completely abolished the pressor response to NE (100% vs 1.0% 0.5%) and the augmented VP response alone (100% vs 2.0% ± 1.01%). Both responses were significantly, but equally, decreased by SRC inhibition (63% ± 4.0% and 69% 1.0%). In contrast, inhibition of the TK molecule PYK2 with salicylate had differential inhibitory effects on the vasoconstrictor responses. Salicylate caused a greater inhibition of VP-induced augmented NE response in comparison with NE alone (62.1% ± 7% and 15% ± 2%). Inhibition of either the µ or γ PKC isoform significantly decreased both responses, but the magnitude of the inhibition was significantly different for each isoform. Inhibition of the γ PKC isoform significantly decreased the vasoconstriction responses to both NE and VP plus NE (82.9 ± 3.9 vs 32.8 ± 3.8). Inhibition of the µ PKC isoform significantly decreased both responses and completely abolished the VP-augmented response to NE. CONCLUSION These data are consistent with a central role for TK in mediating both the NE response and the VP-augmented response. Moreover, PYK2 and the µ and γ isoforms of PKC seem to play a preferential role in mediating the augmented VP response. The apparent divergent roles of these pathways in mediating NE- versus VP-augmented pressor responses could potentially lead to new targeted therapies in catecholamine-resistant shock.

Ionizing radiation has been implicated in the development of significant cardiovascular complications. Since radiation exposure is associated with space exploration, astronauts are potentially at increased risk of accelerated cardiovascular disease. This study investigated the effect of high atomic number, high-energy (HZE) iron-ion radiation on vascular and endothelial function as a model of space radiation. Rats were exposed to a single whole-body dose of iron-ion radiation at doses of 0, 0.5 or 1 Gy. In vivo aortic stiffness and ex vivo aortic tension responses were measured 6 and 8 months after exposure as indicators of chronic vascular injury. Rats exposed to 1 Gy iron ions demonstrated significantly increased aortic stiffness, as measured by pulse wave velocity. Aortic rings from irradiated rats exhibited impaired endothelial-dependent relaxation consistent with endothelial dysfunction. Acute xanthine oxidase (XO) inhibition or reactive oxygen species (ROS) scavenging restored endothelial-dependent ...

Background Emerging evidence suggests that phosphoinositide 3-kinase (PI3K) may modulate cardiac inotropy; however, the underlying mechanism remains elusive. We hypothesized that β(2)-adrenergic receptor (AR)-coupled PI3K constrains increases in cardiac inotropy through cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase (PDE) activation. Methods We tested the effects of PI3K and PDE4 inhibition on myocardial contractility by using isolated murine cardiac myocytes to study physiologic functions (sarcomere shortening [SS] and intracellular Ca(+) transients), as well as cAMP and PDE activity. Results PI3K inhibition with the reversible inhibitor LY294002 (LY) resulted in a significant increase in SS and Ca(2+) handling, indicating enhanced contractility. This response depended on G(iα) protein activity, because incubation with pertussis toxin (an irreversible G(iα) inhibitor) abolished the LY-induced hypercontractility. In addition, PI3K inhibition had no greater effect on SS than both a PDE3,4 inhibitor (milrinone) and LY combined. Furthermore, LY decreased PDE4 activity in a concentration-dependent manner (58.0% of PDE4 activity at LY concentrations of 10 μM). Notably, PI3K(γ) coimmunoprecipitated with PDE4D. The β(2)-AR inverse agonist, ICI 118,551 (ICI), abolished induced increases in contractility. Conclusions PI3K modulates myocardial contractility by a cAMP-dependent mechanism through the regulation of the catalytic activity of PDE4. Furthermore, basal agonist-independent activity of the β(2)-AR and its resultant cAMP production and enhancement of the catalytic activity of PDE4 through PI3K represents an example of integrative cellular signaling, which controls cAMP dynamics and thereby contractility in the cardiac myocyte. These results help to explain the mechanism by which milrinone is able to increase myocardial contractility in the absence of direct β-adrenergic stimulation and why it can further augment contractility in the presence of maximal β-adrenergic stimulation.

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.

There is increasing evidence that upregulation of arginase contributes to impaired endothelial function in aging. In this study, we demonstrate that arginase upregulation leads to endothelial nitric oxide synthase (eNOS) uncoupling and that in vivo chronic inhibition of arginase restores nitroso-redox balance, improves endothelial function, and increases vascular compliance in old rats. Arginase activity in old rats was significantly increased compared with that shown in young rats. Old rats had significantly lower nitric oxide (NO) and higher superoxide (O2−) production than young. Acute inhibition of both NOS, with NG-nitro-l-arginine methyl ester, and arginase, with 2( S)-amino- 6-boronohexanoic acid (ABH), significantly reduced O2− production in old rats but not in young. In addition, the ratio of eNOS dimer to monomer in old rats was significantly decreased compared with that shown in young rats. These results suggest that eNOS was uncoupled in old rats. Although the expression of arginase 1 and eNOS was similar in young and old rats, inducible NOS (iNOS) was significantly upregulated. Furthermore, S-nitrosylation of arginase 1 was significantly elevated in old rats. These findings support our previously published finding that iNOS nitrosylates and activates arginase 1 (Santhanam et al., Circ Res 101: 692–702, 2007). Chronic arginase inhibition in old rats preserved eNOS dimer-to-monomer ratio and significantly reduced O2− production and enhanced endothelial-dependent vasorelaxation to ACh. In addition, ABH significantly reduced vascular stiffness in old rats. These data indicate that iNOS-dependent S-nitrosylation of arginase 1 and the increase in arginase activity lead to eNOS uncoupling, contributing to the nitroso-redox imbalance, endothelial dysfunction, and vascular stiffness observed in vascular aging. We suggest that arginase is a viable target for therapy in age-dependent vascular stiffness.

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.

We have previously shown that microgravity and simulated microgravity induce an increase in human and rat aortic stiffness. We attempted to elucidate the mechanism(s) responsible for this increase in stiffness. We hypothesize that an alteration in vessel wall collagen or elastin content or in extracellular matrix (ECM) cross-linking either individually or in a combination is responsible for the increased vessel stiffness. Rats underwent hindlimb unweighting (HLU) for a period of 7 days to simulate microgravity. The contribution of ECM cross-linking to the vessel wall stiffness was evaluated by measuring aortic pulse wave velocity following inhibition of the cross-linking enzymes lysyl oxidase (LOX) and transglutaminase (tTG) and the nonenzymatic advanced glycation end product cross-linking pathway during HLU. Aortic collagen and elastin content was quantified using established colorimetric assays. Collagen subtype composition was determined via immunofluorescent staining. The increase in aortic pulse wave velocity after HLU was significantly attenuated in the LOX and tTG inhibition groups compared with saline (1.13 ± 0.11 vs. 3.00 ± 0.15 m/s, LOX vs. saline, P < 0.001; 1.16 ± 0.25 vs. 3.00 ± 0.15 m/s, tTG vs. saline, P < 0.001). Hydroxyproline content, a measure of collagen content, was increased in all groups after HLU (2.01 ± 0.62 vs. 3.69 ± 0.68% dry weight, non-HLU vs. HLU, P = 0.009). Collagen subtype composition and aortic elastin content were not altered by HLU. Together, these data indicate that HLU-induced increases in aortic stiffness are due to both increased aortic collagen content and enzyme cross-linking activity.

Protein S-nitrosation is a reversible post-translation modification critical for redox-sensitive cell signaling that is typically studied using the Biotin Switch method. This method and subsequent modifications usually require avidin binding or Western blot analysis to detect biotin labeled proteins. We describe here a modification of the Biotin Switch assay that eliminates the need for Western blot or avidin enrichment protocols and allows direct comparison of the S-nitrosation state proteins from two different samples in the same gel lane or on the same 2D gel. This S-FLOS method offers detection, identification and quantification of S-nitrosated proteins, with the potential for site-specific identification of nitrosation events.

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.

Cardiac myocytes contain two constitutive NO synthase (NOS) isoforms with distinct spatial locations, which allows for isoform-specific regulation. One regulatory mechanism for NOS is substrate ( l -arginine) bioavailability. We tested the hypothesis that arginase (Arg), which metabolizes l -arginine, constrains NOS activity in the cardiac myocyte in an isoform-specific manner. Arg activity was detected in both rat heart homogenates and isolated myocytes. Although both Arg I and II mRNA and protein were present in whole heart, Arg II alone was found in isolated myocytes. Arg inhibition with S -(2-boronoethyl)- l -cysteine (BEC) augmented Ca 2+ -dependent NOS activity and NO production in myocytes, which did not depend on extracellular l -arginine. Arg II coimmunoprecipited with NOS1 but not NOS3. Isolation of myocyte mitochondrial fractions in combination with immuno-electron microscopy demonstrates that Arg II is confined primarily to the mitochondria. Because NOS1 positively modulates myocardial contractility, we determined whether Arg inhibition would increase basal myocardial contractility. Consistent with our hypothesis, Arg inhibition increased basal contractility in isolated myocytes by a NOS-dependent mechanism. Both the Arg inhibitors N -hydroxy- nor - l -arginine and BEC dose-dependently increased basal contractility in rat myocytes, which was inhibited by both nonspecific and NOS1-specific NOS inhibitors N G -nitro- l -arginine methyl ester and S -methyl- l -thiocitrulline, respectively. Also, BEC increased contractility in isolated myocytes from WT and NOS3 but not NOS1 knockout mice. We conclude that mitochondrial Arg II negatively regulates NOS1 activity, most likely by limiting substrate availability in its microdomain. These findings have implications for therapy in pathophysiologic states such as aging and heart failure in which myocardial NO signaling is disrupted.

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.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol. This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

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.

The mechanisms by which nitric oxide (NO) influences myocardial Ca 2+ cycling remain controversial. Because NO synthases (NOS) have specific spatial localization in cardiac myocytes, we hypothesized that neuronal NOS (NOS1) found in cardiac sarcoplasmic reticulum (SR) preferentially regulates SR Ca 2+ release and reuptake resulting in potentiation of the cardiac force-frequency response (FFR). Transesophageal pacing (660 to 840 bpm) in intact C57Bl/6 mice (WT) stimulated both contractility (dP/dt max normalized to end-diastolic volume; dP/dt-EDV) by 51±5% ( P P −/− ) (15±2% increase in dP/dt-EDV; P P −/− (≈2 months of age) also exhibited suppressed frequency-dependent sarcomere shortening and Ca 2+ transients ([Ca 2+ ] i ) compared with WT. SR Ca 2+ stores, a primary determinant of the FFR, increased at higher frequencies in WT (caffeine-induced [Ca 2+ ] i at 4 Hz increased 107±23% above 1 Hz response) but not in NOS1 −/− (13±26%; P −/− ) had preserved FFR in vivo, as well as in isolated myocytes with parallel increases in sarcomere shortening, [Ca 2+ ] i , and SR Ca 2+ stores. NOS1 −/− had increased SR Ca 2+ ATPase and decreased phospholamban protein abundance, suggesting compensatory increases in SR reuptake mechanisms. Together these data demonstrate that NOS1 selectively regulates the cardiac FFR via influences over SR Ca 2+ cycling. Thus, there is NOS isoform-specific regulation of different facets of rate-dependent excitation-contraction coupling; inactivation of NOS1 has the potential to contribute to the pathophysiology of states characterized by diminished frequency-dependent inotropic responses.

We hypothesized that estradiol treatment would improve vascular dysfunction commonly associated with obesity, hyperlipidemia, and insulin resistance. A sham operation or 17β-estradiol pellet implantation was performed in male lean and obese Zucker rats. Maximal vasoconstriction (VC) to phenylephrine (PE) and potassium chloride was exaggerated in control obese rats compared with lean rats, but estradiol significantly attenuated VC in the obese rats. Estradiol reduced the PE EC50in all groups. This effect was cyclooxygenase independent, because preincubation with indomethacin reduced VC response to PE similarly in a subset of control and estrogen-treated lean rats. Endothelium-independent vasodilation (VD) to sodium nitroprusside was similar among groups, but endothelium-dependent VD to ACh was significantly impaired in obese compared with lean rats. Estradiol improved VD in lean and obese rats by decreasing EC50but impaired function by decreasing maximal VD. The shift in EC50corresponded to an upregulation in nitric oxide synthase III protein expression in the aorta of the estrogen-treated obese rats. In summary, estrogen treatment improves vascular function in male insulin-resistant, obese rats, partially via an upregulation of nitric oxide synthase III protein expression. These effects are counteracted by adverse factors, such as hyperlipidemia and, potentially, a release of an endothelium-derived contractile agent.

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.

Advanced glycation end-products (AGEs) initiate cellular inflammation and contribute to cardiovascular disease in the elderly. AGE can be inhibited by Alagebrium (ALT), an AGE cross-link breaker. Moreover, the beneficial effects of exercise on aging are well recognized. Thus, we investigated the effects of ALT and exercise (Ex) on cardiovascular function in a rat aging model. Compared to young (Y) rats, in sedentary old (O) rats, end-systolic elastance (Ees) decreased (0.9±0.2 vs 1.7±0.4 mm Hg/μL, P

Ionizing radiation has been implicated in the development of significant cardiovascular complications. Since radiation exposure is associated with space exploration, astronauts are potentially at increased risk of accelerated cardiovascular disease. This study investigated the effect of high atomic number, high-energy (HZE) iron-ion radiation on vascular and endothelial function as a model of space radiation. Rats were exposed to a single whole-body dose of iron-ion radiation at doses of 0, 0.5 or 1 Gy. In vivo aortic stiffness and ex vivo aortic tension responses were measured 6 and 8 months after exposure as indicators of chronic vascular injury. Rats exposed to 1 Gy iron ions demonstrated significantly increased aortic stiffness, as measured by pulse wave velocity. Aortic rings from irradiated rats exhibited impaired endothelial-dependent relaxation consistent with endothelial dysfunction. Acute xanthine oxidase (XO) inhibition or reactive oxygen species (ROS) scavenging restored endothelial-dependent responses to normal. In addition, XO activity was significantly elevated in rat aorta 4 months after whole-body irradiation. Furthermore, XO inhibition, initiated immediately after radiation exposure and continued until euthanasia, completely inhibited radiation-dependent XO activation. ROS production was elevated after 1 Gy irradiation while production of nitric oxide (NO) was significantly impaired. XO inhibition restored NO and ROS production. Finally, dietary XO inhibition preserved normal endothelial function and vascular stiffness after radiation exposure. These results demonstrate that radiation induced XO-dependent ROS production and nitroso-redox imbalance, leading to chronic vascular dysfunction. As a result, XO is a potential target for radioprotection. Enhancing the understanding of vascular radiation injury could lead to the development of effective methods to ameliorate radiation-induced vascular damage.

Aims Arginase II regulates NOS activity by competing for the substrate l-arginine. Oxidized LDL (OxLDL) is a proatherogenic molecule that activates arginase II. We tested the hypotheses that OxLDL-dependent arginase II activation occurs through a specific receptor, and via a Rho GTPase effector mechanism that is inhibited by statins. Methods and results Arginase II activation by OxLDL was attenuated following preincubation with the LOX-1 receptor-blocking antibody JTX92. This also prevented the dissociation of arginase II from microtubules. LOX-1 −/− mice failed to exhibit the increased arginase II activity seen in WT mice fed a high cholesterol diet. Furthermore, endothelium from LOX-1 −/− mice failed to demonstrate the diet-dependent reduction in NO and increase in ROS that were observed in WT mice. OxLDL induced Rho translocation to the membrane and Rho activation, and these effects were inhibited by pretreatment with JTX92 or statins. Transfection with siRNA for RhoA, or inhibition of ROCK both decreased OxLDL-stimulated arginase II activation. Preincubation with simvastatin or lovastatin blocked OxLDL-induced dissociation of arginase II from microtubules and prevented microtubule depolymerization. Conclusions This study provides a new focus for preventive therapy for atherosclerotic disease by delineating a clearer path from OxLDL through the endothelial cell LOX-1 receptor, RhoA, and ROCK, to the activation of arginase II, downregulation of NO, and vascular dysfunction.

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.

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.

Endothelial dysfunction and increased arterial stiffness contribute to multiple vascular diseases and are hallmarks of cardiovascular aging. To investigate the effects of aging on shear stress-induced endothelial nitric oxide (NO) signaling and aortic stiffness, we studied young (3-4 mo) and old (22-24 mo) rats in vivo and in vitro. Old rat aorta demonstrated impaired vasorelaxation to acetylcholine and sphingosine 1-phosphate, while responses to sodium nitroprusside were similar to those in young aorta. In a customized flow chamber, aortic sections preincubated with the NO-sensitive dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, were subjected to steady-state flow with shear stress increase from 0.4 to 6.4 dyn/cm(2). In young aorta, this shear step amplified 4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence rate by 70.6 +/- 13.9%, while the old aorta response was significantly attenuated (23.6 +/- 11.3%, P0.05). Endothelial NO synthase (eNOS) inhibition, by N(G)-monomethyl-l-arginine, abolished any fluorescence rate increase. Furthermore, impaired NO production was associated with a significant reduction of the phosphorylated-Akt-to-total-Akt ratio in aged aorta (P0.05). Correspondingly, the phosphorylated-to-total-eNOS ratio in aged aortic endothelium was markedly lower than in young endothelium (P0.001). Lastly, pulse wave velocity, an in vivo measure of vascular stiffness, in old rats (5.99 +/- 0.191 m/s) and in N(omega)-nitro-l-arginine methyl ester-treated rats (4.96 +/- 0.118 m/s) was significantly greater than that in young rats (3.64 +/- 0.068 m/s, P0.001). Similarly, eNOS-knockout mice demonstrated higher pulse wave velocity than wild-type mice (P0.001). Thus impaired Akt-dependent NO synthase activation is a potential mechanism for decreased NO bioavailability and endothelial dysfunction, which likely contributes to age-associated vascular stiffness.

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.

In this study, the role of the classroom instructor was redefined from a “lecturer” responsible for delivering the core curriculum to a “facilitator” at the center of an active learning environment. Web-based lectures were used to provide foundation content to students outside of the classroom, which made it possible to improve the quality of student-faculty contact time in the classroom. Students reported that this hybrid format of instruction afforded them a better understanding of the content, a higher probability of retaining the content, and the opportunity to spend more time thinking about the application of the content compared with more traditional lecture-based methods of instruction.

Whether myocardial contractile impairment contributes to orthostatic intolerance (OI) is controversial. Accordingly, we used transient bilateral carotid occlusion (TBCO) to compare the in vivo pressor, chronotropic, and inotropic responses ( parts 1 and 2) to open-loop selective carotid baroreceptor unloading in anesthetized mice. In part 3, in vitro myocyte responses to isoproterenol in mice exposed to hindlimb unweighting (HLU) for ∼2 wk were determined. Heart rate (HR) and mean arterial pressure (MAP) responses to TBCO were measured. In control mice, TBCO increased HR (15 ± 2 beats/min, P < 0.05) and MAP (17 ± 2 mmHg, P < 0.05). These responses were markedly potentiated in denervated control (DC) mice, in which the aortic depressor nerve and sympathetic trunk were sectioned before measurement. Baroreflex responses to TBCO were eliminated by blockade with hexamethonium bromide (10 μg/kg). In HLU (denervated) mice, HR and MAP responses were reduced ∼70% compared with DC mice. In part 2, myocardial contractile responses to TBCO were measured with a left ventricular micromanometer-conductance catheter. TBCO in DC mice increased the slope of the end-systolic pressure-volume relation (end-systolic elastance) by 86 ± 13%. This inotropic response was attenuated (14 ± 10%, P < 0.005) after HLU. In part 3, contractile responses to isoproterenol were impaired in myocytes isolated from HLU mice. In conclusion, selective carotid baroreceptor unloading stimulates HR, blood pressure, and myocardial contractility, and HLU attenuates each response. These findings have important implications for the management of OI in astronauts, the elderly, and individuals subjected to prolonged bed rest.

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.

The online version of the original article can be found underdoi:10.1007/s00421-011-2257-9.M.Khan J.Steppan S.Ryoo L.Santhanam T.Berkowitz D. Nyhan D. E. Berkowitz (&)Department of Anesthesiology and Critical Care Medicine,Johns Hopkins University Medicine, Tower 711, 600 N Wolfe St.,Baltimore, MD 21287, USAe-mail: dberkowi@bme.jhu.eduK. H. SchuleriDepartment of Medicine, Johns Hopkins University Medicine,600 N Wolfe St., Baltimore, MD 21287, USAE. Tuday L. Bugaj A. A. Shoukas D. E. BerkowitzDepartment of Biomedical Engineering,Johns Hopkins University Medicine, 600 N Wolfe St.,Baltimore, MD 21287, USA

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.

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.

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)