Endothelial Nitric Oxide Synthase (eNOS) and the Cardiovascular System: in Physiology and in Disease States.

Endothelial nitric oxide synthase (eNOS) plays a critical role in regulating and maintaining a healthy cardiovascular system. The importance of eNOS can be emphasized from the genetic polymorphisms of the eNOS gene, uncoupling of eNOS dimerization, and its numerous signaling regulations. The activity of eNOS on the cardiac myocytes, vasculature, and the central nervous system are discussed. The effects of eNOS on the sympathetic autonomic nervous system (SANS) and the parasympathetic autonomic nervous system (PANS), both of which profoundly influence the cardiovascular system, will be elaborated. The relationship between the eNOS protein with cardiovascular autonomic reflexes such as the baroreflex and the Exercise Pressor Reflex will be discussed. For example, the effects of endogenous nitric oxide (NO) are shown to be mediated by the eNOS protein and that eNOS-derived endothelial NO is most effective in regulating blood pressure oscillations via modulating the baroreflex mechanisms. The protective action of eNOS on the CVS is emphasized here because dysfunction of the eNOS enzyme is intricately correlated with the pathogenesis of several cardiovascular diseases such as hypertension, arteriosclerosis, myocardial infarction, and stroke. Overall, our current understanding of the eNOS protein with a focus on its role in the modulation, regulation, and control of the cardiovascular system in a normal physiological state and in cardiovascular diseases are discussed.

Changing single side-chains can greatly enhance the resistance of a membrane protein to irreversible inactivation.

The thermal inactivation rates of a set of 20 cysteine-substituted variants of the integral membrane protein diacylglycerol kinase were measured. Two of the mutations, I53C and I70C, were found to significantly prolong the half-life of the enzyme in detergent solution. By combining the single mutants to create a double mutant, I53C/I70C, the half-life of the enzyme was improved from less than a minute at 70 degrees C to 51 minutes. These results demonstrate that individual side-chain substitutions can significantly improve the properties of membrane proteins in detergent solution.

Inactivation mechanism of the membrane protein diacylglycerol kinase in detergent solution.

We have examined the irreversible inactivation mechanism of the membrane protein diacylglycerol kinase in the detergents n-octyl-beta-D-glucopyranoside (OG) at 55 degrees C and n-decyl-maltopyranoside (DM) at 80 degrees C. Under no inactivation conditions did we find any direct evidence for the chemical modifications that are commonly found in soluble proteins. Moreover, protein inactivated at 55 degrees C in OG could be reactivated by an unfolding and refolding protocol, suggesting that the protein is inactivated by a stable conformational change, not a covalent modification. We also found that the inactivation rate decreased with both increasing protein concentration and increasing thermodynamic stability, consistent with an inactivation pathway involving transient dissociation and/or unfolding of the protein. Our results suggest that the primary cause of diacylglycerol kinase inactivation is not low solubility, but poor intrinsic stability in the detergent environment.

Robustness of protein folding kinetics to surface hydrophobic substitutions.

We use both combinatorial and site-directed mutagenesis to explore the consequences of surface hydrophobic substitutions for the folding of two small single domain proteins, the src SH3 domain, and the IgG binding domain of Peptostreptococcal protein L. We find that in almost every case, destabilizing surface hydrophobic substitutions have much larger effects on the rate of unfolding than on the rate of folding, suggesting that nonnative hydrophobic interactions do not significantly interfere with the rate of core assembly.

Effects of opioid receptor activation on cardiovascular responses and extracellular monoamines within the rostral ventrolateral medulla during static contraction of skeletal muscle.

During static muscle contraction, activation of opioid receptors alters the extracellular glutamate concentrations within the rostral ventrolateral medulla (RVLM). In addition, microdialysis of glutamate in the ventrolateral medulla (VLM) increases the release of norepinephrine (NE), dopamine (DA), and serotonin (5-HT). Therefore, we hypothesized that extracellular concentrations of these monoamines as well as cardiovascular responses during static skeletal muscle contraction would be modulated following administration of [D-Ala(2)]methionine enkephalinamide (DAME), an opioid receptor agonist, into the RVLM. Microdialysis of 100 microM DAME into the RVLM of 10 rats significantly (P<0.01) decreased extracellular levels (in pg/10 microl) of NE (from 3.3+/-0.3 to 1.9+/-0.3), DA (from 5.5+/-0.2 to 3.7+/-0.3), and 5-HT (from 6.1+/-0.8 to 3.6+/-0.2) during static exercise. After microdialysis of DAME, the exercise pressor reflex also significantly (P<0.01) decreased mean arterial pressure (MAP) by 13+/-3 mmHg and heart rate (HR) by 16+/-6 bpm, compared with control (MAP=22+/-4 mmHg and HR=31+/-7 bpm). Subsequently, after 30 min microdialysis of naloxone, an opioid receptor antagonist, muscle contraction increased the extracellular monoamine levels (in pg/10 microl, 3.8+/-0.3 NE; 5.2+/-0.3 DA; and 5.5+/-0.4 5-HT) similar to the control groups and evoked a reversal of cardiovascular responses. Similarly, 30 min of microdialyzing naloxone, added to the perfusing medium containing DAME, reversed the attenuating effects of DAME on monoamines, MAP, and HR during a muscle contraction. Furthermore, microdialysis of 100 microM naloxone alone for 30 min potentiated cardiovascular responses and monoamine levels during a muscle contraction. In summary, the present data demonstrates that microdialysis of DAME into RVLM attenuates the exercise pressor reflex mediated increases in MAP, HR and extracellular levels of biogenic monoamines. A subsequent microdialysis of naloxone reversed the effects suggesting that an opioidergic mechanism within RVLM modulates the exercise pressor reflex. Overall, the present study provides further insights into the opioidergic modulation of the exercise pressor reflex.

Effects of nitric oxide and GABA interaction within ventrolateral medulla on cardiovascular responses during static muscle contraction.

We hypothesized that nitric oxide (NO) has opposing roles in regulating cardiovascular responses within the rostral (RVLM) and caudal (CVLM) ventrolateral medulla by modulating release of gamma-aminobutyric acid (GABA). We have measured GABA concentrations within the RVLM and CVLM during increases in mean arterial pressure (MAP) and heart rate (HR) following a 2-min tibial nerve stimulation-evoked static muscle contraction before and after microdialysis of the NO precursor, L-arginine (1.0 microM), for 30 min, and after the NO inhibitor, L-NMMA (1.0 microM), for 30 min. In eight anesthetized rats, muscle contraction significantly increased MAP, HR and GABA levels within the RVLM area (from 0.53+/-0.09 to 1.22+/-0.10 ng/10 microl). Following microdialysis of L-arginine, muscle contraction augmented GABA levels (from 0.45+/-0.07 to 2.18+/-0.09 ng/10 microl) and attenuated changes in MAP and HR. Subsequent application of L-NMMA significantly decreased GABA levels (from 0.47+/-0.08 to 0.22+/-0.07 ng/10 microl) but potentiated MAP and HR responses to a muscle contraction. In contrast, muscle contraction significantly increased MAP and HR but decreased GABA concentrations within the CVLM (from 1.20+/-0.20 to 0.78+/-0.17 ng/10 microl). Following microdialysis of L-arginine, muscle contraction significantly attenuated GABA levels (from 1.34+/-0.19 to 0.33+/-0.10 ng/10 microl) and augmented changes in MAP and HR in response to muscle contraction. A subsequent microdialysis of L-NMMA into the CVLM reversed the effects of L-arginine. These results demonstrate that NO within the RVLM and CVLM differentially modulates cardiovascular responses during static muscle contraction and that NO influences exercise-induced cardiovascular responses by modulating GABA release within the ventrolateral medulla.

Simultaneous glutamate and gamma-aminobutyric acid release within ventrolateral medulla during skeletal muscle contraction in intact and barodenervated rats.

The purpose of this study was to determine if baroreflex modulates cardiovascular responses and neurotransmitter release within rostral (RVLM) and caudal (CVLM) ventrolateral medulla during static contraction of skeletal muscle using anesthetized rats. We evoked cardiovascular responses by a static muscle contraction and measured simultaneous release of glutamate and gamma-aminobutyric acid (GABA) in both the RVLM and CVLM using microdialysis probes, two inserted bilaterally into the RVLM and two into the CVLM. In intact anesthetized rats, a muscle contraction increased release of glutamate concomitantly in both the RVLM and CVLM along with significant increases in heart rate and arterial blood pressure. In contrast, concentrations of GABA increased within the RVLM, but decreased significantly within the CVLM during the pressor response. These changes were due to contraction-evoked activation of muscle afferents since tibial nerve stimulation following muscle paralysis failed to evoke glutamate, GABA, or any cardiovascular changes. On the other hand, static muscle contractions in baroreceptor denervated rats augmented the increases in heart rate and blood pressure. Furthermore, muscle contraction significantly enhanced the release of glutamate in the RVLM but attenuated its release in the CVLM. In addition, concentrations of GABA within the RVLM were attenuated following a muscle contraction in denervated rats without any changes in GABA within the CVLM. These results demonstrate that the baroreceptors influence cardiovascular responses to static muscle contraction associated with dynamic changes in glutamate and GABA release within the RVLM and CVLM.

Computer-based redesign of a protein folding pathway.

A fundamental test of our current understanding of protein folding is to rationally redesign protein folding pathways. We use a computer-based design strategy to switch the folding pathway of protein G, which normally involves formation of the second, but not the first, beta-turn at the rate limiting step in folding. Backbone conformations and amino acid sequences that maximize the interaction density in the first beta-hairpin were identified, and two variants containing 11 amino acid replacements were found to be approximately 4 kcal mol-1 more stable than wild type protein G. Kinetic studies show that the redesigned proteins fold approximately 100 x faster than wild type protein and that the first beta-turn is formed and the second disrupted at the rate limiting step in folding.

Maturation depresses cGMP-mediated decreases in [Ca2+]i and Ca2+ sensitivity in ovine cranial arteries.

Because cerebrovascular cGMP levels vary significantly during maturation, we examined the hypothesis that the ability of cGMP to relax cerebral arteries also changes during maturation. In concentration-response experiments, potassium-induced tone in basilar arteries was significantly more sensitive to a nonmetabolizable cell-permeant cGMP analogue 8-(p-chlorophenylthio)-cGMP (8-pCPT-cGMP) in term fetal [-log one-half maximal concentration (EC(50)) = 4.4 +/- 0.1 M] than in adult (-log EC(50) = 4.0 +/- 0.1 M) ovine basilar arteries. Serotonin-induced tone also revealed significantly greater sensitivity to the cGMP analogue in fetal (-log EC(50) = 4.9 +/- 0.1 M) than in adult (-log EC(50) = 4.7 +/- 0.1 M) basilars. In fura 2-loaded preparations, 8-pCPT-cGMP had no significant effect on cytosolic calcium concentrations in potassium-contracted arteries but at 6 microM significantly reduced calcium only in fetal basilars (Delta = 33 +/- 8%). Higher 8-pCPT-cGMP concentrations reduced cytosolic calcium in both fetal and adult basilars. Similarly, in both potassium- and 5-hydroxytryptamine (5-HT)-contracted preparations, low concentrations of 8-pCPT-cGMP reduced myofilament calcium sensitivity only in fetal basilars (Delta = 29 +/- 6 and Delta = 42 +/- 10%, respectively), whereas higher concentrations reduced calcium sensitivity in both fetal and adult arteries. In beta-escin-permeabilized arteries, equivalent reductions in basal and agonist-enhanced myofilament calcium sensitivity were produced by much lower 8-pCPT-cGMP concentrations in fetal (172 and 61 microM, respectively) than in adult (410 and 231 microM, respectively) basilars. The mechanisms mediating cGMP-induced vasorelaxation appear similar in fetal and adult arteries, with the exception that they are much more sensitive to cGMP in fetal than adult arteries. These age-related differences in the sensitivity of cytosolic calcium concentration, basal, and agonist-enhanced myofilament calcium sensitivity to cGMP can easily explain why both potassium- and 5-HT-induced tone are more sensitive to cGMP in fetal than adult cerebral arteries.

Effects of maturation on mechanisms of cGMP-induced cerebral vasodilatation.

In light of observations that cerebrovascular levels of cGMP vary during maturation, the present study examines the possibility that the mechanisms mediating cGMP-induced cerebral vasodilatation also change during maturation. Specifically, these experiments explore age-related changes in the ability of cGMP to both: (1) depress cytosolic calcium concentration, and (2) attenuate contractile protein calcium sensitivity in alpha-toxin and beta-escin permeabilized preparations as well as fura-2 loaded arteries. The present data demonstrate that: (1) cGMP attenuates cytosolic calcium concentration at lower concentrations than required to reduce myofilament calcium sensitivity; (2) both potassium-induced and 5HT-induced contractions were more sensitive to cGMP in fetal than adult arteries; (3) all potassium-induced increases in cytosolic calcium were resistant to the effects of cGMP, but those produced by 5HT were sensitive to attenuation by cGMP, and more so in fetal than in adult basilar arteries, and (4) cGMP attenuated both basal and agonist-enhanced myofilament calcium sensitivity. Overall, these data demonstrate that the mechanisms mediating the multiple vasoactive effects of cGMP are more potent in immature than in mature cerebral arteries and are heavily influenced by both the artery type and the method of contraction.

Maturation attenuates the effects of cGMP on contraction, [Ca2+]i and Ca2+ sensitivity in ovine basilar arteries.

The present study explores the hypothesis that age-related variations in cerebrovascular responses to vasodilators reflect corresponding age-dependent differences in the mechanisms coupling changes in cytosolic cGMP to vasorelaxation. The experiments focused on cGMP's ability to decrease either [Ca2+]i or myofilament Ca2+ sensitivity, because both effects can contribute to cGMP-induced vasodilation. Use of the cGMP analog 8-pCPT-cGMP minimized problems associated with limited cell permeation or cGMP hydrolysis. In fetal basilars contracted with 10 microM serotonin, the EC30 for 8-pCPT-cGMP-induced relaxation was 6 microM. In fura-2 loaded fetal basilars, pretreatment with 6 microM 8-pCPT-cGMP significantly depressed the sensitivity of [Ca2+]i to 5HT, and also myofilament sensitivity to calcium, but only in fetal arteries. In fetal basilar arteries contracted with 120 mM potassium, the EC30 for 8-pCPT-cGMP-induced relaxation was 25 microM. In fura-2 loaded ovine arteries, pretreatment with 25 microM 8-pCPT-cGMP had no effect on the ability of graded concentrations of potassium to elevate [Ca2+]i but reduced potassium's ability to induce contraction and attenuated myofilament calcium sensitivity; these latter effects were significant only in fetal arteries. In alpha-toxin permeabilized preparations, 25 microM 8-pCPT-cGMP significantly depressed both basal- and agonist-stimulated myofilament calcium sensitivity, only in fetal but not in adult basilars. Together, these results demonstrate that: (1) sensitivity to cGMP is greater in fetal than adult sheep arteries independent of method of contraction; (2) cGMP can reduce [Ca2+]i but only in agonist-contracted and not in potassium-contracted arteries; (3) and cGMP attenuates myofilament calcium sensitivity regardless of method of contraction. Overall, the data demonstrate that variations in the ability of cGMP to produce vasodilatation reflect age-, artery-, and agonist-dependent differences in the combination of mechanisms mediating responses to cGMP.

Structural transitions in the protein L denatured state ensemble.

We use a broad array of biophysical methods to probe the extent of structure and time scale of structural transitions in the protein L denatured state ensemble. Measurement of amide proton exchange protection during the first several milliseconds following initiation of refolding in 0.4 M sodium sulfate revealed weak protection in the first beta-hairpin and helix. A tryptophan residue was introduced into the first beta-hairpin to probe the extent of structure formation in this part of the protein; the intrinsic fluorescence of this tryptophan was found to deviate from that expected given its local sequence context in 2-3 M guanidine, suggesting some partial ordering of this region in the unfolded state ensemble. To further probe this partial ordering, dansyl groups were introduced via cysteine residues at three sites in the protein. It was found that fluorescence energy transfer from the introduced tryptophan to the dansyl groups decreased dramatically upon unfolding. Stopped-flow fluorescence studies showed that the recovery of dansyl fluorescence upon refolding occurred on a submillisecond time scale. To probe the interactions responsible for the residual structure observed in the denatured state ensemble, the conformation of a peptide corresponding to the first beta-hairpin and helix of protein L was studied using circular dichroism spectroscopy and compared to that of full-length protein L and previously characterized peptides corresponding to the isolated helix and second beta-hairpin.

Developmental changes in ryanodine- and IP(3)-sensitive Ca(2+) pools in ovine basilar artery.

To explore the hypothesis that cerebrovascular maturation alters ryanodine- and inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) pool sizes, we measured total intracellular Ca(2+) with (45)Ca and the fractions of intracellular Ca(2+) released by IP(3) and/or caffeine in furaptra-loaded permeabilized basilar arteries from nonpregnant adult and term fetal (139-141 days) sheep. Ca(2+) mass (nmol/mg dry weight) was similar in adult (1.60 +/- 0.18) and fetal (1.71 +/- 0.16) arteries in the pool sensitive to IP(3) alone but was significantly lower for adult (0.11 +/- 0.01) than for fetal (1.22 +/- 0.11) arteries in the pool sensitive to ryanodine alone. The pool sensitive to both ryanodine and IP(3) was also smaller in adult (0.14 +/- 0.01) than in fetal (0.85 +/- 0.08) arteries. Because the Ca(2+) fraction in the ryanodine-IP(3) pool was small in both adult (5 +/- 1%) and fetal (7 +/- 4%) arteries, the IP(3) and ryanodine pools appear to be separate in these arteries. However, the pool sensitive to neither IP(3) nor ryanodine was 10-fold smaller in adult (0.87 +/- 0.10) than in fetal (8.78 +/- 0.81) arteries, where it accounted for 72% of total intracellular membrane-bound Ca(2+). Thus, during basilar artery maturation, intracellular Ca(2+) mass plummets in noncontractile pools, decreases modestly in ryanodine-sensitive pools, and remains constant in IP(3)-sensitive pools. In addition, age-related increases in IP(3) efficacy must involve factors other than IP(3) pool size alone.

Modulation of pressor response to muscle contraction via monoamines following AMPA-receptor blockade in the ventrolateral medulla.

We hypothesized that cardiovascular responses to static muscle contraction are mediated via changes in extracellular concentrations of monoamines (norepinephrine, dopamine and serotonin) following the administration of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an AMPA-receptor antagonist) into the rostral (RVLM) or caudal (CVLM) ventrolateral medulla. For the RVLM experiments (n= 8), a 2-min static muscle contraction increased the mean arterial pressure (MAP) and heart rate (HR) by 23 +/- 2 mmHg and 28 +/- 8 bpm, respectively. During this contraction, the concentrations of norepinephrine, dopamine, and serotonin within the RVLM increased by 278 +/- 52%, 213 +/- 23%, and 232 +/- 24%, respectively. Microdialysis of CNQX (1.0 microM) for 30 min into the RVLM attenuated the increases in MAP and HR ( 11 +/- 2 mmHg and 14 +/- 5 bpm) without a change in developed muscle tension. The levels of norepinephrine, dopamine, and serotonin within the RVLM were also attenuated. In contrast, microdialysis of CNQX into the CVLM (n= 8) potentiated the contraction-evoked responses in MAP ( 21 +/- 2 vs 33 +/- 5 mmHg) and HR ( 25 +/- 5 vs 46 +/- 8 bpm) without any effect on the monoamine levels within the CVLM region. These results suggest that AMPA-receptor blockade within the RVLM and CVLM has opposing effects on cardiovascular responses during static muscle contraction. In addition, such receptor blockade modulates extracellular concentrations of monoamines within the RVLM but not in the CVLM. These results provide evidence that AMPA receptors within the ventrolateral medulla play a role in exercise pressor reflex.