INTRODUCTION

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A PRIMARY ROLE OF THE SYMPATHETIC nervous system in humans is the defense of blood pressure after an orthostatic challenge (33). The distance of the heart from the head and feet in the upright position makes regulatory systems that maintain cardiac output and blood pressure essential in a normal gravitational environment. These mechanisms underlie orthostatic tolerance. Activation of the baroreceptor reflex arc by afferent signals from the carotid, aortic, and cardiopulmonary baroreceptors is integrated in the central nervous system, resulting in efferent output to the heart and vasculature. This is manifest as a change in heart rate, myocardial contractility, venous capacitance, and arteriolar resistance.

The hazards to humans in space have recently been highlighted, even in the nonscientific literature (6, 23). One of the primary effects of microgravity are changes induced in the cardiovascular system. In the absence of a gravitational field, there is no arterial or venous pressure gradient between the heart, head, and feet (43). This results in significant physiological adaptive responses, including central displacement of blood volume and equalization of pressures within the arterial circulation. This in turn leads to adaptive neurohumoral responses, a decrease in circulating blood volume (3), and an increase in venous compliance of the splanchnic circulation. Although these responses are adaptive in a microgravity environment, they are maladaptive on return to a normal or near-normal gravitational field. The mechanisms underlying orthostatic intolerance are multifactorial and include a decrease in circulating blood volume, an increase in venous compliance, a decrease in the ability of the active muscle to pump venous blood to the heart (9), alterations in baroreflex responses (8), and vascular contractile hyporesponsiveness.

Recent research has focused on systemic vascular changes that occur during cardiovascular deconditioning (10, 11, 13, 14, 30, 34). It is now well established that end-organ hyporesponsiveness to -adrenergic agonist stimulation results in impaired blood pressure and cardiac output responses to baroreceptor activation. This hyporesponsiveness has been demonstrated in both the arterial and venous circulations, including large conduit arterial vessels [aorta, femoral, and carotid (11, 13, 30)], resistance arterioles in skeletal muscle (10), mesenteric microvessels (1, 24), and veins (14, 35). The potential underlying mechanisms remain undetermined, but they could include vascular atrophy (12), decreased expression of ₁-adrenergic receptors (2), decreased expression of ryanodine receptors important in modulating intracellular Ca²⁺ responses to agonist stimulation (28), or an increased production of vasodilators, for example, nitric oxide (NO) (34).

Although the systemic circulation has been the major focus of efforts to understand underlying vasoregulatory mechanisms, the pulmonary circulation could also be critically important in orthostatic intolerance. The pulmonary circulation plays a pivotal role in determining the loading conditions of the heart and thus cardiac output and blood pressure. Shoukas et al. (36, 37) clearly demonstrated that the baroreceptor reflex can exert significant control of pulmonary resistance and capacitance. Thus changes in the responsiveness of the pulmonary vasculature may significantly alter cardiac output and predispose to orthostatic intolerance. We hypothesized that 1) pulmonary vascular contractile responses are impaired in the hindlimb-unweighted (HLU) rat and 2) these impaired responses are secondary to upregulation of endogenous endothelial NO synthase (eNOS). To test these hypotheses, we 1) measured vasoconstrictor and vasodilator responses in pulmonary arteries from HLU and control (C) rats and 2) determined eNOS, inducible NO synthase (iNOS), and soluble guanlyl cyclase (sGC) expression in lung, and eNOS expression in pulmonary vascular tissue from C and HLU rats. Our results indicate that vasoconstrictor responses are impaired in the pulmonary artery of HLU rats, endothelial-dependent vasodilator responses and NO-mediated endothelial-independent responses are enhanced in vessels from HLU rats, and the attenuated pulmonary vasoconstrictor responses are due in part to increased eNOS activity.

	METHODS

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Animal model. Sprague-Dawley rats (Charles River), ~6-9 mo of age and weighing 400-500 g, were housed separately at 22°C. The animals were fed regular rat chow and water ad libitum. Animals were HLU for 21 days. HLU was accomplished by using the following technique: animals were briefly anesthetized with ketamine-xylazine (10 and 1 mg/kg, respectively) for the placement of a customized body jacket. The jacket was sewn snugly around the animal and to a piece of flexible tygon tubing that runs along the dorsum of the animal to provide firm but flexible support. The tail was then taped to the tygon tubing. The animals were allowed to recover from the anesthetic while being suspended by a tether that maintained them in a 35-45° head-down tilt. The animals are free to move around the circumference of the cage on their front limbs. Because of the length of the tether, the change in the angle at the perimeter of the cage vs. the center is negligible. The animal is not tail suspended because the force of suspension is distributed along the dorsum.

Animal protocols. The study was approved by the Institutional Animal Care and Use Committee and complies with the American Physiological Society guidelines.

In vitro isometric tension measurements. Vessel rings were suspended for isometric tension recording in 5-ml organ chambers. The rings were mounted horizontally between two stainless steel stirrups. One of the stirrups was anchored to a micromanipulator and the other to a force transducer (FT-03) in a myograph (150-µm stirrups) (Mangus, Iwashiya Kishimoto Medical Instruments, Kyoto, Japan). All cumulative concentration-effect curves were performed on vessel rings beginning at their optimum resting tone. This is accomplished by stretching arterial rings at 10-min intervals in increments of 200 mg to reach optimal tone. Vessel rings were equilibrated in oxygenated Krebs-Ringer physiological salt solution (95% O₂-5% CO₂). Optimal resting tone was the minimum level of stretch allowed for the largest contractile response to KCl (60 mM). This was demonstrated to be ~600 mg in the pulmonary artery segments studied. After pulmonary artery rings had been stretched to their optimal resting tone, contractile response to 60 mM KCl was determined. After removal of KCl from the organ chambers and the return of isometric tension to prestimulation value, dose-response curves were generated to phenylephrine (PE; 10⁹ to 10⁴ M) in one-half log order concentrations. Dose-response curves were also generated using a nonadrenergic vasoconstrictor, U-46619 (10¹⁰ to 10⁶ M). Vasorelaxant responses to the endothelial-dependent vasodilator ACh (10⁹ to 10⁴ M) and endothelial-independent vasodilator sodium nitroprusside (SNP; 10⁹ to 10⁴ M) were tested. Dose-response curves to the above agonists were repeated in the presence of N^G-nitro-L-arginine methyl ester (L-NAME; 10⁵ M). All of the vascular responses were performed on each of four rings per animal, and the responses were averaged for each animal. The protocol was as follows: contractile responses were tested to the agonists KCl, PE, and U-46619. Dilator responses were then tested to ACh and SNP. Constrictor responses were further tested in the presence of the NOS inhibitor L-NAME (10⁵ M). Data were collected on-line using a MacLab system and analyzed using Dose Response software (AD Instruments, Mountain View, CA).

Western blot analysis for eNOS, iNOS, and sGC protein. The details of Western blot analysis have been published previously (22). Briefly, the right lobe from each rat lung was homogenized in homogenization buffer and centrifuged, and the protein content was analyzed by the method of Bradford. Then, 100 µg of protein from the lung or pulmonary artery tissue were electrophoresed in a 7.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The blots were incubated with a monoclonal anti-eNOS or anti-iNOS (1:1,000 dilution for each antibody; Transduction Laboratories, Lexington, KY) or polyclonal sGC primary antibody (1:500 dilution, Cayman Chemicals, Ann Arbor, MI) and then by a goat anti-mouse or anti-rabbit horseradish peroxidase-labeled secondary antibody (Bio-Rad). eNOS, iNOS, and sGC protein signals were detected using enhanced chemiluminescence detection (Amersham, Buckinghamshire, UK) reagent. The relative amounts of eNOS, iNOS, and sGC ₁-subunit were quantitated with a densitometer using Imagequant software (Molecular Dynamics, Sunnyvale, CA). All Western blots were repeated three times for each protein detection.

Data analysis. In all experiments, four vessel rings from each animal were tested. Pulmonary artery vasoconstrictor responses were expressed as tension in milligrams. Vasodilator responses were expressed as percent relaxation of a preconstricted tension. EC₅₀ and maximal response (E_max) were calculated by using nonlinear logistic regression analysis with the software PRIZM (Graphpad, Mountain View, CA). All data are reported as means ± SE. Statistical differences were determined by unpaired Student's t-test.

	RESULTS

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There was no significant difference in the body mass of C and HLU rats at the time of experimentation (441 ± 19 vs. 391 ± 56, respectively; n = 5; not significant).

Determination of optimal passive tension. In a preliminary study of C animals (n = 3), optimal passive tension was determined in the pulmonary arteries. Responses to stimulation with 60 mM KCl revealed an increase in active developed tension with each stepwise increment. The maximum response was achieved at a passive tension of ~600 mg. This passive tension was therefore chosen for all subsequent experiments.

Contractile responses to agonists. The contractile response to KCl was significantly decreased in vessels from HLU compared with C rats (344 ± 54 vs. 498 ± 62 mg, respectively; n = 5; P = 0.03; Fig. 1). PE caused a dose-dependent increase in tension in pulmonary artery rings from C and HLU rats. However, the response to PE was significantly attenuated in vessels from HLU compared with C rats. (Fig. 2A, Table 1). U-46619, a thromboxane analog and a potent nonadrenergic agonist, also induced a dose-dependent contractile response in pulmonary artery rings from C and HLU animals. The responses to U-46619 were also significantly attenuated in the vessels from HLU rats (Fig. 2B, Table 1). This indicates that the attenuated responses observed in vessels from HLU animals are not likely due to changes in a specific receptor. These observations could be due to changes distally in contractile mechanisms or result from the modulating influence of altered vasodilator mechanisms.

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Vasoconstrictor response to 60 mM KCl in pulmonary artery segments from control (C) and hindlimb-unweighted (HLU) rats. Values are means ± SE; n = 5 rats. *P = 0.03.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Vasoconstrictor responses to phenylephrine (PE; A) and U-46619 (B) in pulmonary artery segments from C and HLU rats in absence and presence of NG-nitro-L-arginine methyl ester (L-NAME). Values are means ± SE. Vasoconstrictor responses were attenuated in pulmonary arteries from HLU rats (*P < 0.05). Differential vasoconstrictor responses were not observed after inhibition (L-NAME, 105 M).

Effect of L-NAME on responses to PE and U-46619. To determine the effect of NOS inhibition on the contractile response, pulmonary vascular ring vessels were preincubated with L-NAME (10⁵ M) for 20 min before agonist stimulation. Addition of L-NAME did not change baseline tension. As expected, preincubation with L-NAME caused a significant increase in the contractile responses to PE in vessels from both HLU and C animals (Fig. 2A, Table 2). Preincubation with L-NAME also markedly enhanced the contractile response to U-46619 in vessels from HLU and C animals (Fig. 2B, Table 3). However, the magnitude of the enhanced responses to PE and U-46619 were significantly greater in vessels from HLU compared with C animals. The differential responses to vasoconstrictor stimuli observed before L-NAME administration was completely abolished after NOS inhibition (Fig. 2, A and B, Table 2), indicating that changes in the NOS pathway in HLU animals modulated the vasoconstrictor responses to PE and U-46619.

Vasorelaxant responses. To determine whether endothelial-dependent and -independent vasodilator function was enhanced after HLU, pulmonary arteries were precontracted with PE (EC₅₀, ~10⁶ M), and dose-response curves to ACh and SNP were generated. The vasorelaxant response to ACh was significantly enhanced in vessels from HLU rats compared with those from C rats (Fig. 3A, Table 3). Moreover, the vasorelaxant response to SNP was also significantly increased in vessels from HLU animals (Fig. 3B, Table 3). Thus both endothelial-dependent and -independent vasorelaxant responses are accentuated in the pulmonary vasculature of HLU animals.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Endothelial-dependent ACh (A) and -independent [sodium nitroprusside (SNP); B] nitric oxide-mediated vasodilation in pulmonary artery segments from HLU and C rats. Vessels were preconstricted with PE to approximately EC50, and dose responses were performed. Values are means ± SE. Both endothelial-dependent (*P = 0.02) and -independent ( P = 0.003) vasodilator responses were enhanced in pulmonary artery segments from HLU rats.

eNOS, iNOS, and sGC expression. Responses to vasoconstrictor stimuli are modulated by vasodilator pathways, including the endothelial-dependent NO pathway. These modulating effects reflect basal or unstimulated vasodilator pathway activity in contrast to stimulated vasodilator pathway activity, as seen after ACh administration. To determine whether a change in expression of eNOS is responsible for altered endothelial-dependent NO-dependent vasorelaxation observed in the HLU vessels, immunoblotting was performed in pulmonary artery (same tissue as used in bioassays) and lung tissue from HLU and C rats. eNOS expression was significantly enhanced in lung tissue (Fig. 4A) and pulmonary artery (Fig. 4B) from HLU compared with C rats, whereas the expression of iNOS in lung tissue was not different (Fig. 4C).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Western blot analysis of endothelial nitric oxide synthase (eNOS) in lung (A) and pulmonary artery (B), inducible nitric oxide synthase (iNOS) in lung (C), and soluble guanlyl cyclase (sGC) 1-subunit (D) from lung of C and HLU rats. Values are means ± SE. There is increased abundance of eNOS in pulmonary artery and lung tissue and sGC 1-subunit in lung tissue, whereas expression of iNOS remains unchanged. C1-C3, C rats 1-3, respectively; HLU1-HLU3, HLU rats 1-3, respectively.

	DISCUSSION

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We have demonstrated for the first time that HLU results in impaired vascular responsiveness to contractile agonists in the pulmonary circulation. This impaired responsiveness appears to be primarily the result of enhanced activity of eNOS. This is supported by the findings that 1) L-NAME markedly enhances contractile responses in pulmonary artery rings and completely abolishes the difference in contractile responses observed between HLU and C rats with both agonists tested, 2) endothelial-dependent vasodilation is significantly enhanced in the pulmonary artery rings from HLU rats, and 3) eNOS expression is increased in the lung as well as in the pulmonary artery of HLU rats. An increase in the expression of sGC in the lung may be responsible for an endothelial-independent, NO-dependent component of the enhanced vasorelaxant responses observed.

Several investigators have demonstrated impaired vascular responsiveness to agonist stimulation in HLU rats (11, 14, 30). This vascular hyporesponsiveness has been demonstrated in a variety of vessels, including rat aorta, femoral arteries, carotid artery, and mesenteric veins. The mechanisms responsible for vascular hyporesponsiveness are protean and are likely dependent on the vascular bed studied. Recently, Sangha et al. (34) demonstrated that removal of the endothelium from carotid arteries, but not aorta or femoral vessels, normalized the contractile response to norepinephrine. Thus there appears to be both an endothelial-dependent and -independent mechanism underlying vascular hyporesponsiveness. Our data in the pulmonary vascular bed suggest that enhanced endothelial NO production is responsible for the attenuated contractile responses to PE and U-46619 in the pulmonary vessels from HLU rats, because NOS inhibition restores vascular responsiveness and completely obliterates the differential responses observed between HLU and C animals.

It is well established that flow-induced changes in shear stress result in a significant increase in eNOS activity (4) and expression (5). Specifically, shear stress and cyclic stretch have been shown to increase the expression of endothelial NOS at a transcriptional and posttranscriptional level. This is now recognized as an important mechanism in the local regulation of blood flow. The redistribution of blood flow associated with HLU (26) could significantly alter the shear forces to which vessels are exposed and may result in the upregulation of eNOS. This could provide a basis for the enhanced endothelial-dependent vasorelaxant responses to ACh in the pulmonary artery.

HLU results in a cephalad redistribution of blood, with potential flow increases to the dependent regions of the body, including lungs, and flow decreases in nondependent vascular beds. This is consistent with the findings of Colleran et al. (7), who demonstrated an increase in flow to the dependent skull and forelimb bones, with decreased flow to the elevated nonactive hindlimb bones. This is also consistent with the findings of Jasperse et al. (19) and Delp et al. (12), who demonstrated a decrease in the eNOS expression (19) and endothelial-dependent dilation (12, 19) in the soleus feed arteries of HLU rats, a vascular bed that would likely have decreased blood flow and vascular shear stress in these conditions. This has been substantiated by the work of Delp et al. (12), who estimated shear stress by using flow and diameter parameters and demonstrated significant decreases in shear stress in soleus muscle feed arteries after 10 min of HLU (12).

Our data clearly support a role for increased NO production in pulmonary vascular hyporesponsiveness. The source of increased basal and stimulated NO appears to be eNOS. The role of NO in vascular hyporesponsiveness in the systemic circulation has been studied (34, 40). In contrast to our findings, it has been demonstrated, by using both a vessel ring bioassay and expression data, that iNOS is increased in the aorta of HLU rats (34, 40). Whereas iNOS expression is primarily induced by cytokines in vascular and nonvascular tissue, low levels of constitutively expressed iNOS have been demonstrated in several tissues, including cardiovascular and kidney tissues. In addition, dysregulation of iNOS expression has been demonstrated in animal models of hypertension (41, 42). Thus low-level expression of iNOS may have a physiological regulatory role distinct from that of cytokine-mediated induction and massive NO release. Hence, the upregulation of iNOS may not be the consequence of "sick rats" in which cytokines have been stimulated and iNOS induced. Our results, which indicate that iNOS is not upregulated, in contrast with findings of others who did demonstrate upregulation (34, 40), may reflect differences between the pulmonary and systemic circulations.

The enhanced vasodilator responsiveness to ACh suggests that NO may represent a significant component of the vascular hyporesponsiveness. ACh stimulation results in the release of at least two vasodilator molecules, NO and endothelium-derived hyperpolarizing factor. NO is the primary vasodilator molecule in larger vessels, whereas endothelium-derived hyperpolarizing factor appears to be the predominant vasodilator regulating flow in the microvasculature (39). In the proximal pulmonary artery preparation, enhanced basal and stimulated NO production appear to be the primary cause of vascular contractile hyporesponsiveness, because NOS inhibition with L-NAME abolishes the differential contractile responses observed between C and HLU rats.

The enhanced response to ACh in HLU rats may be the result of enhanced NO activity. Increased stimulated NO activity may result from an increase in NOS expression or an increase in the bioavailability of the released NO. Bioavailability of NO has emerged as a central mechanism of vasodilator and/or endothelial function and dysfunction in diseases, such as atherosclerosis, hypertension, and hypercholesterolemia. Vascular production of reactive oxygen species (ROS) and the subsequent reaction and scavenging of NO is thought to be an important mechanism of vascular dysfunction in atherosclerosis (27) and hypertension (38). This is manifest as a decrease in arteriolar dilation to ACh that can be reversed with superoxide dismutase. ROS have not only been implicated in the scavenging of NO, thereby causing endothelial dysfunction, but they are also critical in the normal regulation of tone (pressure-flow autoregulation) in the vasculature (18). Moreover, angiotensin II-induced ROS production by the NADP/NADPH oxidase system has emerged as a novel pathway for vascular signaling (17, 31). Microgravity is associated with downregulation of the renin angiotensin system (16) and with a decrease in wall stretch. Thus, it is entirely possible that decreased vascular ROS production may contribute to enhanced endothelial-dependent, NO-dependent signaling.

sGC is the primary target enzyme for NO. The factors controlling transcriptional regulation of sGC are not well defined. In models of hypoxia, sGC is upregulated and may parallel the upregulation of both eNOS and iNOS (22). This suggests that NOS activity, NO, and cGMP in the vascular smooth muscle may contribute to regulation of sGC. More specifically, Fillippov et al. (15) demonstrated that NO decreases sGC subunit mRNA stability via a transcription- and translation-dependent mechanism (15). In our HLU model, sGC ₁-subunit expression is increased, as is the expression of eNOS. This could explain the enhanced NO-dependent vasodilator responses. Others have observed differences in endothelial-independent vasodilator responses to HLU. McCurdy et al. (25) examined the vasodilator responses in soleus and gastrocnemius muscle bed and demonstrated a decreased vasodilator response to SNP and adenosine. Although the responses observed are opposite, they are not inconsistent. In the HLU rat, the muscle beds are elevated and inactive, whereas the lungs are dependent and for this reason are likely to be exposed to altered pressure and flow patterns. This idea is supported by the work of Colleran et al. (7), who demonstrated an increased flow to the skull and forelimb bones but a decreased flow to the hindlimb bones in HLU rats (7).

Because differences in endothelial-dependent vasoregulatory mechanisms have been demonstrated between right and left porcine pulmonary vessels (20), we have consistently used the same side (right) for physiological and biochemical study in our rodent model. Thus the changes observed are not due to differences in right vs. left vasoregulation.

What is the potential pathophysiological consequence of impaired pulmonary artery vasoreactivity? The primary pathophysiological abnormality associated with microgravity-induced orthostatic intolerance is controversial. An impaired stroke volume response to an orthostatic challenge can contribute significantly to this pathophysiological process (3, 21). The importance of the sympathetic nervous system in regulating the systemic vasculature (venous capacitance and systemic resistance) is also well established. Although changes in venous capacitance properties are critical in regulation of right ventricular stroke volume, changes in capacitive properties of the pulmonary vascular bed can also contribute significantly to the left ventricular stroke volume response. In a canine model using cardiopulmonary bypass, Shoukas et al. (36) demonstrated that changes in carotid sinus pressure from 50 to 200 mmHg increased systemic reservoir volume by ~7.5 ml/kg and pulmonary reservoir volume by ~1 ml/kg. This represents a ~15% change in pulmonary blood volume (assuming an ~80 ml/kg total circulating volume in a dog with ~8% of the circulating volume in the total pulmonary system) (32). Although the degree of the stimulus (50-200 mmHg) can only be produced artificially, and sympathetic stimulation with an orthostatic challenge is unlikely to be of this magnitude, the data above emphasize the point that changes in pulmonary compliance may regulate left ventricular filling and thereby stoke volume. Thus, after orthostatic stress, impaired responsiveness of the pulmonary vasculature and its influence as blood volume redistribution may be significant, particularly in the setting of attenuated systemic vascular responses.

The physiological experiments were performed in the rat pulmonary artery, whereas immunoblotting experiments were performed on lung tissue and in the case of eNOS, the pulmonary artery. The consistency of the findings between eNOS expression in the lung and pulmonary artery gives us some confidence that the changes in the lung may be representative of the pulmonary artery. It is acknowledged, however, that protein expression of NOS isoforms and sGC may not represent only vascular tissue. Although nonvascular tissue does express various isoforms of NOS and sGC, the pulmonary endothelial cells are the primary source of eNOS, whereas iNOS can be induced in pneumocytes in various inflammatory disease states (29). The finding that iNOS expression was unchanged represents a good internal control. In summary, we have demonstrated for the first time that contractile vasoregulated responses in the pulmonary arteries of the rat are attenuated after HLU. This attenuated vascular responsiveness is largely due to an increase in NOS activity secondary to increased expression of eNOS. In addition, stimulated endothelial-dependent vasorelaxation is enhanced. Impaired contractile responses may have important implications for blood volume distribution and stroke volume responses to an orthostatic challenge after exposure to a period of microgravity.