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Am J Physiol Heart Circ Physiol 279: H502-H510, 2000;
0363-6135/00 $5.00
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Vol. 279, Issue 2, H502-H510, August 2000

Acute hypoxia modulates 5-HT receptor density and agonist affinity in fetal and adult ovine carotid arteries

Danilyn M. Angeles, James Williams, Lubo Zhang, and William J. Pearce

Center for Perinatal Biology, Departments of Physiology and Pharmacology, Loma Linda University School of Medicine, Loma Linda, California 92350


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In light of recent observations that receptor-ligand binding and coupling are physiologically regulated, the present study examined the hypothesis that the direct effects of hypoxia on vascular contractility involve modulation of pharmacomechanical coupling via changes in agonist affinity and/or receptor density. Because the direct effects of hypoxia on vascular smooth muscle contractility can vary with age, we carried out these experiments using both fetal and adult arteries. In common carotid arteries from near-term fetal and adult sheep, hypoxia (PO2 = 9-12 Torr for 30 min) reduced the maximum responses to potassium by 17.8 ± 3.5% (fetus) and 20.5 ± 2.2% (adult), significantly reduced the pD2 for 5-HT in the fetus (7.01 ± 0.1 to 6.3 ± 0.2) but not the adult (6.1 ± 0.1 to 6.0 ± 0.1), and significantly reduced 5-HT-induced maximum contractions (as % maximum response to 120 mM K+) not in the fetus (from 114 ± 7 to 70 ± 10%, not significant) but only in the adult (from 83 ± 15 to 25 ± 7%, P < 0.05) arteries. Hypoxia significantly attenuated 5-HT binding affinity (pKA, determined by partial irreversible blockade with phenoxybenzamine) in both fetal (from 6.5 ± 0.2 to 6.0 ± 0.2) and adult arteries (from 6.2 ± 0.2 to 5.7 ± 0.1) and also decreased receptor density (fmol/mg protein, determined by competitive binding with ketanserin and mesulergine) in adult (from 18.3 ± 1.1 to 10.9 ± 1.0) but not in fetal (21.0 ± 1.0 to 23.2 ± 1.4) arteries. These results suggest that acute hypoxia modulates receptor-ligand binding via age-dependent modulation of agonist affinity and receptor density. These effects may contribute to hypoxic vasodilatation and help explain why the effects of hypoxia on vascular contractility differ between fetuses and adults.

ketanserin; competition binding; maturation; mesulergine; ontogeny


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

OWING TO THE CRITICAL REQUIREMENT for oxygen in cellular homeostasis, studies of cellular responses to reduced oxygen availability have comprised an active area of research for many years. In most vascular beds, these responses include extravascular responses, endothelial responses, and direct vascular effects. Extravascular responses to hypoxia typically involve the release of potent vasodilator metabolites such as adenosine (27), hydrogen and potassium ions (16), and prostaglandins (38). Endothelial responses to hypoxia are heterogeneous, tissue and artery specific, and can involve the release of vasodilators such as nitric oxide (42), hyperpolarizing factors, and prostacyclin (44). In addition, in some preparations vascular endothelium has been shown to release contracting factors in response to hypoxia, including endothelium-derived contracting factor (44), endothelin (36), and thromboxane A2 (19).

Direct vascular effects of hypoxia are also varied and complex. Hypoxia can activate ATP-sensitive K+ channels (KATP) channels, possibly through effects on intracellular ATP concentration (4). Adenosine, a vasodilator metabolite commonly released in response to hypoxia, can also modulate the open state probability of KATP channels and thereby further enhance hypoxic vasodilatation (24, 28). Hypoxia can also inhibit Ca2+ influx through oxygen-sensitive, L-type Ca2+ channels and open Ca2+-sensitive K+ channels (8, 13). A net effect of these multiple actions of hypoxia on membrane ion channels is that hypoxia also often decreases the cytosolic calcium concentrations needed to support contraction (31) via membrane hyperpolarization (26) and/or deficits in intracellular calcium mobilization (11). The overall effect of these mechanisms is relaxation of the vascular smooth muscle.

Surprisingly, none of the many published studies of vascular responses to hypoxia has yet examined the effects of acute hypoxia on receptor-ligand interactions. Recent evidence suggests that receptor turnover is often highly dynamic and that many membrane receptors, including the 5-HT2 family, can be quickly downregulated (37). Acute hypoxia has also been reported to decrease agonist binding affinity for kainate and glutamate in brain preparations from both fetal guinea pigs (22) and newborn piglets (9). Such regulation of G protein-coupled receptors typically involves regions of conserved aromatic and charged residues essential for ligand binding, G protein coupling, and internalization (37). Mediators of this regulation include the G protein-related kinases (GRKs) that phosphorylate ligand-bound receptors and thereby impair receptor signaling and agonist sensitivity (33). Hypoxia can also modulate the transcription and expression of multiple proteins involved in vascular pharmacomechanical coupling pathways (5). Taken together, these findings suggest that receptor-ligand binding may be subject to modulation by hypoxia.

The following series of experiments was designed to address the general hypothesis that acute hypoxia directly modulates receptor-ligand binding and coupling to contraction in vascular smooth muscle. In general, the coupling of receptor-ligand binding to contraction is governed by three very basic mechanisms: 1) the affinity of the ligand for the receptor; 2) the number of receptors present; and 3) the coupling efficiency of each receptor to the contractile apparatus (10, 39). Correspondingly, we conducted experiments to measure the effects of hypoxia on agonist affinity, receptor density, and coupling efficiency. To avoid problems related to the simultaneous release of parenchymal vasodilator metabolites in response to hypoxia, we studied the in vitro effects of hypoxia on the common carotid artery, a large conduit artery not typically controlled by tissue metabolites. To eliminate problems related to the presence of multiple receptor subtypes for a single agonist (46), we selected 5-HT as the agonist for these studies because the ovine carotid contains a single serotonergic receptor, the 5-HT2A (40). Because the direct effects of hypoxia on vascular smooth muscle contractility vary significantly with age in common carotid arteries (14, 32, 47), we carried out these experiments using both fetal and adult common carotid arteries. This approach enabled an assessment of our corollary hypothesis that age-related differences in the direct effects of hypoxia on common carotid contractility involve corresponding differences in the effects of hypoxia on ligand-receptor interactions.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General methods. Adult carotid arteries were obtained from healthy adult nonpregnant sheep (18-24 mo old) of either sex euthanized with 100 mg/kg iv pentobarbital sodium. Fetal carotid arteries were obtained from near-term fetuses (139-141 days gestation) of either sex weighing from 2.5 to 4.0 kg, delivered by cesarean section and then euthanized with 100 mg/kg iv pentobarbital sodium. All procedures were reviewed and approved by the Institutional Animal Use and Care Committee of Loma Linda University. After dissection was completed, all arteries were cleaned of extraneous connective and adipose tissue and cut into multiple segments, each 1 mm in length for adult sheep and 3 mm in length for the fetal lamb. Two sets of eight matched segments were obtained from each animal and studied in parallel. To avoid possible endothelium-mediated effects, the endothelium was removed from all segments by passing a roughened hypodermic needle through the lumen of the vessel several times and gently flushing it with cold isotonic Krebs solution. Physically denuded segments were thereafter incubated in the continuous presence of the nitric oxide synthase inhibitors NG-nitro-L-arginine methyl ester (L-NAME, 100 µM) and NG-nitro-L-arginine (L-NNA, 100 µM). The functional integrity of the endothelium was evaluated in all segments by testing the response to 0.1 µM bradykinin in arteries precontracted with 10 µM 5-HT. Segments that relaxed more than 10% in response to bradykinin were discarded.

All segments were equilibrated at optimum resting tensions of ~1 g on paired handmade tungsten wires placed between a low-compliance force transducer (0.6 g/µm displacement, Kulite BG-10) and a post attached to a micrometer used to vary resting tension. The artery segments were equilibrated at 38.5°C (normal ovine core temperature) for 30 min in a bicarbonate-Krebs solution containing (in mM) 122 NaCl, 25.6 NaHCO3, 5.56 dextrose, 5.17 KCl, 2.49 MgSO4, 1.60 CaCl2, 0.114 ascorbic acid, 100 µM L-NNA, 100 µM L-NAME, and 0.027 disodium-EDTA, continuously bubbled with 95% O2-5% CO2. Contractility measurements were recorded, digitized, and normalized via an online computer, as previously described in detail (29).

One set of matched artery segments served as the control group, and the other was equilibrated for 30 min under hypoxic conditions. Hypoxia was produced by bubbling with 95% N2-5% CO2, and the bath oxygen tensions attained (9-12 Torr) were determined using miniature polarographic Clark style electrodes monitored by a high-impedance picoammeter (Diamond General 1231). All electrodes were resintered and calibrated immediately before each use.

Determination of 5-HT dissociation constant. We determined agonist binding affinities using the method of partial irreversible blockade, as previously described (10). Arteries were first contracted by exposure to an isotonic potassium-Krebs solution and then allowed to return to resting levels of tension. We then induced a second contraction using 10 µM serotonin. Once the contractile response had stabilized, endothelial integrity was determined functionally by exposure to 10-7 M bradykinin. After exposure to bradykinin, the segments were washed with normal sodium-Krebs and allowed to return to baseline levels of tension, after which the segments were again contracted with potassium-Krebs to verify reproducibility of contractile responses. After peak tensions were attained, the segments were returned to sodium-Krebs and incubated for 20 min in the presence (four segments) or absence (four segments) of phenoxybenzamine. The phenoxybenzamine concentrations used were 10-150 nM in hypoxic segments and 50-300 nM in the normoxic segments. After 20 min of phenoxybenzamine treatment, the segments were washed four times with an isotonic bicarbonate-Krebs solution, incubated for 30 min in bicarbonate-Krebs containing 10-7 M prazosin (to inhibit alpha -adrenergic receptors) and 2 × 10-7 M cocaine (to inhibit neuronal uptake of 5-HT), and then bubbled with either 95% O2-5% CO2 (normoxic) or 95% N2-5% CO2 (hypoxic) gas. Finally, a concentration-response determination was performed using cumulative increasing concentrations of 5-HT in half-log increments.

The data were analyzed to determine agonist dissociation constants as previously described by our laboratory (6). Briefly, the concentration-response relations in both treated and untreated segments were fit to the logistic equation using nonlinear regression. The coefficients obtained from these fits were used to calculate multiple-matched equieffective concentrations of 5-HT in the control and treated segments. From these dose pairs, the pKA values (negative log of dissociation constant) were determined by fitting to the modified Furchgott equation
log (A)<IT>=</IT>log [(A<A><AC>i</AC><AC>´</AC></A>K<SUB>A</SUB><IT>q</IT>)]<IT>&cjs0823;  </IT>[(K<SUB>A</SUB><IT>+</IT>(<IT>1−q</IT>)A<A><AC>i</AC><AC>´</AC></A>)]
where q is the fraction of active receptors remaining after phenoxybenzamine treatment, A is the 5-HT concentration of agonist before phenoxybenzamine, and Aí is the equal effective concentration of 5-HT after the phenoxybenzamine exposure.

Determination of receptor density and antagonist dissociation constant. From groups of animals different from those used for agonist affinity determinations, two sets of eight common carotid segments were prepared and equilibrated for 20 min in a bicarbonate-Krebs solution, as described previously in METHODS: Determination of 5-HT dissociation constant, and then equilibrated for 30 min with either 95% O2-5% CO2 (normoxic segments) or 95% N2-5% CO2 (hypoxic segments). After this equilibration, all segments were quickly frozen by immersion in liquid nitrogen and stored at -80°C until the time of assay.

Because previous studies of the serotonergic receptor subtype present in ovine common carotid arteries strongly indicated the almost exclusive presence of the 5-HT2A subtype (40), we used tritiated ketanserin as our primary ligand for quantifying 5-HT receptors in our preparation. However, because saturation binding curves were often highly variable and occasionally exhibited nonlinearities in the nonspecific binding curves at high ketanserin concentrations, we chose to measure 5-HT2A receptor densities using a competition model. Among a wide variety of serotonergic ligands evaluated, the best results were obtained using mesulergine as our competitive ligand. To enable highly reliable measures of receptor density using ketanserin and mesulergine, we designed a "double-competition" model, wherein two assays were run in parallel using the same artery homogenate. In the first assay, 0-100 nM mesulergine were used to displace [3H]ketanserin. In the second assay, 0-10 nM ketanserin was used to displace [3H]mesulergine. By solving these two binding curves simultaneously for a single set of coefficients, we obtained highly reliable values for receptor density (Bmax) and the negative log values of the dissociation constants (pKb) for ketanserin dissociation constant (Kd) and mesulergine Kd. We performed this analysis using a custom-made Excel template that fitted quench-corrected counts per minute to antagonist concentrations using the SOLVER routine to minimize least-squares error. Extensive validation experiments indicated optimum conditions of 30-s grinding time in ice-cold 50 mM Tris solution with 500 µM EGTA and 1 mM phenylmethylsulfonyl fluoride (PMSF), binding at 37°C for 15 min, and separation on GF/C filters soaked in 0.3% polyethylenimine in distilled water at neutral pH.

Matched sets of normoxic and hypoxic arteries from each animal were analyzed in parallel within the same assay. After homogenization, the preparations were centrifuged at 400 g for 10 min to remove high-density particulate matter, after which the low-speed supernates were centrifuged at 50,000 g for 30 min. The high-speed pellets were rehomogenized using a Dounce homogenizer in 12 ml of ice-cold 50 mM Tris solution with 500 µM EGTA and 1 mM PMSF. Protein concentrations in both samples were quantified using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Results from this protein analysis were utilized to dilute the homogenates to exactly 1.2 mg/ml. Four hundred microliters of homogenate were then mixed with varying concentrations of cold ligand (50 µl) and 5 nM of hot ligand (50 µl). Quadruplicate samples were then incubated and separated as described above, after which the filters were extracted overnight in scintillation cocktail and counted the following day. The resulting counts per minute data were analyzed to obtain estimates of Bmax, ketanserin pKb, and mesulergine pKb, using the custom-written SOLVER routine, as described above.

Calculation of binding-response relations. To determine the relations between the numbers of receptors bound and the contractile responses, we first used the agonist affinity values obtained in the first protocol to convert the agonist concentrations used into values of fractional receptor occupancy using the equation
Fractional occupancy<IT>=</IT>A&cjs0823;  (A<IT>+K</IT><SUB>A</SUB>)
where A is the agonist concentration and KA is the agonist dissociation constant. These values were then multiplied by the corresponding values of receptor density to determine the femtomoles of receptors bound at each agonist concentration used. These values of femtomoles bound were then plotted against their corresponding values of contractile force. Contractile force values were normalized relative to the maximum response observed in each artery segment in response to 120 mM isotonic potassium-Krebs under normoxic conditions at the beginning of each experiment and under hypoxic conditions after equilibration for 30 min at an oxygen tension of 9-12 Torr.

Statistics. All values were calculated as means ± SE. In cases where multiple segments were studied with the same protocol, results were averaged by animal; n always refers to the number of animals used in a given experimental group. Before statistical analysis, the distributions of all data sets were analyzed for normalcy and were log transformed where necessary. All values were compared using analysis of variance followed by Duncan's multiple range test to assess intergroup differences. Power analyses were performed where no significant differences were observed, and where necessary, additional experiments were completed to attain power values >0.95 for all nonsignificant comparisons.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General results. From 40 adult sheep and 43 fetal lambs, 664 (eight segments per animal) common carotid segments were obtained for this study. The maximum normoxic contractile tensions produced by these arteries in response to 120 mM KCl averaged 2.01 ± 0.19 and 5.77 ± 0.57 g in the fetal and adult arteries, respectively. When precontracted with 10 µM 5-HT, 10-7 M bradykinin relaxed the arteries by 2.9 ± 0.6 and 4.3 ± 0.6%, respectively, indicating that endothelium removal was effective. None of the physically denuded segments prepared for this study relaxed more than 10% in response to 10-7 M bradykinin.

Effects of acute hypoxia on 5-HT concentration-response relations and agonist affinity. In both the fetus and the adult, acute hypoxia significantly reduced the maximum responses to potassium, and these percentage decreases averaged 20.5 ± 2.2% in the adult and 17.8 ± 3.5% in the fetus (Fig. 1). As shown in Fig. 2, acute hypoxia also significantly reduced the pD2 (-log of the EC50) for 5-HT more in fetal (from 7.01 ± 0.1 to 6.3 ± 0.2, P < 0.05) than in adult (6.1 ± 0.1 to 6.0 ± 0.1, not significant) arteries. When fetal and adult artery responses were combined and analyzed together, ANOVA revealed that hypoxia significantly attenuated Emax (percent maximum response as defined by complete depolarization with 120 mM potassium) values. However, when analyzed individually by a post hoc Duncan's analysis, hypoxia significantly reduced Emax in adult (from 83 ± 15 to 25 ± 7%, P < 0.05) but not in fetal (from 114 ± 7 to 70 ± 10%, P > 0.05, not significant) arteries. Most importantly, hypoxia significantly attenuated agonist affinity when both adult and fetal arteries were combined, and also when they were analyzed separately. Hypoxia decreased pKA in the fetal arteries from 6.5 ± 0.2 to 6.0 ± 0.2 (P < 0.05) and in the adult arteries from 6.2 ± 0.2 to 5.7 ± 0.1 (P < 0.05).


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  		Fig. 1.   Cumulative concentration-response relations obtained for 5-HT, expressed relative to the maximum normoxic responses to 120 mM isotonic potassium. Hypoxia significantly reduced maximum response in both fetuses and adults and also produced a rightward shift in the concentration-response curves. All values are expressed as means ± SE for the numbers of animals indicated in Fig. 2. For statistical comparisons, please see Fig. 2.



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  		Fig. 2.   As indicated by these means ± SE, hypoxia reduced the pD2 for 5-HT in fetal (from 7.01 ± 0.1 to 6.3 ± 0.2) but not adult (6.1 ± 0.1 to 6.0 ± 0.1) arteries. Conversely, hypoxia significantly reduced %maximum response (Emax) more in adult (from 83 ± 15 to 25 ± 7%) than in fetal (from 114 ± 7 to 79 ± 10%) arteries. Most importantly, hypoxia significantly attenuated agonist affinity in both fetal (from 6.5 ± 0.2 to 6.0 ± 0.2) and adult (from 6.2 ± 0.2 to 5.7 ± 0.1) arteries. The numbers of animals used for all measurements are indicated in the boxes along the abscissa. *Significant differences as revealed by a Duncan's multiple range analysis.

Effects of acute hypoxia on Bmax and antagonist affinities. As shown in Fig. 3, acute hypoxia significantly reduced Bmax (in fmol/mg protein) in adult common carotid arteries (normoxic: 18.3 ± 1.1, hypoxic: 10.9 ± 1.0) but had no significant effect on fetal common carotid arteries (normoxic: 21.0 ± 1.0, hypoxic: 23.2 ± 1.4). The Bmax value observed in hypoxic adult arteries was also significantly less than that observed in either normoxic or hypoxic fetal arteries (see Fig. 3).


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  		Fig. 3.   As indicated by these competition displacement curves, hypoxia enhanced initial binding (values on ordinate) of both [3H]mesulergine (top) and [3H]ketanserin (middle) in the fetus but depressed initial binding for both ligands in adult artery preparations. For the receptor density values obtained from these curves, acute hypoxia had no significant effect in fetal common carotid arteries (receptor density, Bmax, changed from 21.0 to 23.2 fmol/mg protein) but significantly reduced receptor density in adult common carotid arteries (Bmax decreased from 18.3 to 10.9 fmol/mg protein). The numbers of observations for each measurement are indicated at the base of each bar. *Significant difference as revealed by a Duncan's multiple range analysis of the Bmax values.

As shown in Fig. 4, acute hypoxia and maturation had no significant effects on the binding affinities of mesulergine and ketanserin. In fetal arteries, normoxic and hypoxic pKb values for mesulergine averaged 8.8 ± 0.2 and 8.9 ± 0.2, respectively. Corresponding adult values averaged 9.1 ± 0.1 and 9.0 ± 0.2. For ketanserin, normoxic and hypoxic pKb values averaged 9.1 ± 0.3 and 9.6 ± 0.1 in the fetus and 9.4 ± 0.1 and 9.5 ± 0.1 in the adult. None of these values were significantly different from one another, as indicated using two-way ANOVA.


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  		Fig. 4.   Acute hypoxia and maturation had no significant effect on binding affinities for mesulergine and ketanserin, as indicated by a two-way analysis of variance. Values indicate means ± SE for the numbers of observations indicated at the base of each bar.

Effects of acute hypoxia on binding-response relations. As shown in Fig. 5, top left, the relation between femtomole receptor bound and contractile response was virtually linear in normoxic adult arteries. Acute hypoxia altered the shape of this relation but had relatively little effect on the amounts of tension produced for each receptor bound. For example, in normoxic adult arteries 0.3 µM 5-HT produced 5.8 fmol/mg of bound receptors, which produced 20.4% of the maximum normoxic response to potassium. In hypoxic adult arteries, 3 µM 5-HT produced 6.5 fmol/mg of bound receptors, and these produced 20.1% of the maximum normoxic response to potassium. Thus at these 5-HT concentrations the ratio of contractile tension to femtomole per milligram bound was quite similar in normoxic (approx 3.5% per fmol/mg bound) and hypoxic (approx 3.1% per fmol/mg bound) arteries. Because hypoxia also depressed the maximum contractile responses to potassium, we normalized the contractile responses to 5-HT relative to the maximum responses to potassium under hypoxic conditions to eliminate nonspecific effects of hypoxia on contractility (Fig. 5, bottom panels). After this normalization, the contractile responses observed were still similar in normoxic (approx 3.5% initial potassium-induced tension per fmol/mg bound) and hypoxic (approx 3.9% initial potassium-induced tension per fmol/mg bound) arteries, indicating that in adult arteries the majority of the response to acute hypoxia occurs at the level of ligand-receptor binding and not downstream from this event.


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  		Fig. 5.   Relations between the numbers of receptors bound and the resulting contractile response were dramatically different in adult (left) and fetal (right) arteries. These relations were affected little by correction for the general effect of hypoxia on potassium-induced tone (bottom). The linear binding-response relations in the normoxic adult arteries suggest the absence of receptor reserve, whereas the hyperbolic shapes for the fetal arteries suggest a significant receptor reserve. Hypoxia had relatively little effect on the contractile force produced by a given number of receptors in adult arteries but significantly depressed the force produced in fetal arteries, signifying that it is only in the fetus that a significant effect of hypoxia occurs downstream of receptor-ligand binding. Values indicate means ± SE for the numbers of observations indicated for Fig. 2.

In the fetus, the effects of hypoxia on the 5-HT receptor binding-response relations were markedly different from those observed in the adult arteries. First, the basic relations between femtomoles bound and contractile responses were all nonlinear rectangular hyperbolas, indicating the probable presence of receptor reserve in these arteries. Second, hypoxia significantly depressed the amount of tension produced at corresponding numbers of receptor bound. For example, 0.1 µM 5-HT yielded ~5.4 fmol/mg receptor bound, which produced 52.8 ± 5.9% of the maximum normoxic response to potassium. Correspondingly, under hypoxic conditions, 0.3 µM 5-HT also yielded approximately the same number of bound receptors (5.7 fmol/mg protein), but these produced only 29.9 ± 6.2% of the maximum normoxic response to potassium. Even when fetal contractile responses were normalized relative to the maximum hypoxic response to potassium, the contractile responses produced at same femtomoles bound remained markedly different in normoxic and hypoxic arteries. Together, these results suggest that a significant portion of the response to hypoxia in fetal carotid arteries occurs downstream from ligand-receptor binding.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although a broad variety of both direct (4, 8, 13, 44) and indirect (30) vascular mechanisms have been shown to contribute to hypoxic vasodilatation, virtually nothing is known of the direct effects of hypoxia on vascular receptor-ligand interactions. Given that recent findings strongly suggest that ligand-receptor interactions are labile (37), subject to multiple mechanisms of physiological regulation (33, 37), and may be influenced by hypoxia (5), the present studies were designed to address the hypothesis that acute hypoxia directly modulates receptor-ligand binding and coupling to contraction in vascular smooth muscle. To this end, the experimental design examined each of the three basic components that together govern the relation between agonist concentration and contractile response. These include: 1) the affinity of the ligand for the receptor; 2) the number of receptors present; and 3) the coupling efficiency of each receptor to the contractile apparatus (10, 39).

Age-dependent vascular effects of acute hypoxia. One interesting aspect of responses to hypoxia is that the direct effects of hypoxia on vascular contractility, in vitro, can vary with age. For example, rates of relaxation to acute hypoxia are typically much slower, but magnitudes of relaxation are much greater, in fetal and newborn than in adult ovine carotid arteries (14, 32). In these studies, fetal and newborn carotid responses to acute hypoxia were similar in both rate and magnitude. Thus in the interest of minimizing animal use and facilitating interpretation of our data in relation to, and in comparison with, the large body of previously published results from near-term fetal lambs, only fetal carotids were used in these studies. Because previous studies have also indicated that the vascular endothelium participates in carotid responses to hypoxia in an age-dependent manner (47), we examined only endothelium-denuded preparations to eliminate any potential effects of endothelial vasoactive factors on carotid ligand-receptor interactions. With this approach, we evaluated the hypothesis that age-related differences in the direct effects of hypoxia on common carotid contractility in vitro involve corresponding differences in the effects of hypoxia on ligand-receptor interactions.

Effects of acute hypoxia on receptor affinity for 5-HT. The ability of hypoxia to relax agonist-induced artery tone has been documented in many previous studies (23, 30), the majority of which typically attribute the response to the vascular and nonvascular mechanisms already cited. More recently, however, several studies in nonvascular tissues have begun to examine the effects of acute hypoxia on ligand-receptor interactions. In brain preparations from both fetal guinea pigs (22) and newborn piglets (9), approximately 1 h of acute hypoxia decreased agonist binding affinity for kainate and glutamate, respectively. In contrast, in the rat brain stem 5-15 min of hypoxia had no effect on agonist affinity for substance P (21). Although the receptor types and tissues examined in these studies differed considerably, together the data suggest that acute hypoxia of more than 15 min duration can influence agonist binding affinity in at least some nonvascular preparations. In vascular preparations of uterine artery, chronic hypoxia of several weeks duration can decrease agonist affinity for 5-HT (15), but the minimum duration of exposure necessary to attain this effect remains uncertain. The present results suggest that as little as 30 min of exposure to severe hypoxia can significantly decrease agonist affinity, at least for 5-HT in ovine common carotid arteries. Interestingly, this effect was equivalent in both fetal and adult arteries, despite significant age-related differences in other components of the pathway coupling 5-HT to contraction.

Contemporary views of G protein-coupled receptors suggest they have two distinct affinity states with high and low agonist binding affinity, respectively (12, 18). Transitions between these two affinity states play a key role in receptor desensitization and appear to be mediated, at least in part, via the actions of GRKs (33). If hypoxia could in some way stimulate GRK activity, it is possible that via this mechanism it could also modulate agonist binding affinity. Consistent with this possibility, exposure of neonatal rats to as little as 2 min of hypoxia can increase GRK activity up to 2.5-fold in neonatal rat liver cells (12). Whereas the present data provide no indication as to the possible involvement of altered GRK activity in the observed effects of hypoxia on agonist binding affinity, the data are consistent with such an effect and suggest that further examinations of this mechanism may be fruitful.

Effects of acute hypoxia on receptor density. In addition to its reported effects on agonist affinity (15), chronic exposure to hypoxia over many days or weeks has also been shown to decrease receptor density in several tissues, including beta -adrenergic receptors in the heart (45) and alpha -adrenergic receptors in cerebral arteries (41). Shorter durations of exposure to hypoxia, in the range of 2 h or less, can also decrease the densities of beta -adrenergic receptors in the heart (20) and receptors for adenosine (17) and substance P (21) in brain tissues, but the effects of these shorter exposure times on vascular receptor densities have not yet been reported. The present study extends this observation to ovine carotid arteries and demonstrates that exposure to severe hypoxia (PO2approx 10 Torr) for as little as 30 min can significantly decrease serotonergic receptor density by up to 40%, at least in the adult ovine common carotid artery. Somewhat unexpectedly, the ability of acute hypoxia to decrease receptor density was observed in adult, but not in fetal, preparations.

Hypoxic reductions of 5-HT receptor density in the present studies probably cannot be attributed to modulation of radioligand binding affinities, because these values were unaffected by hypoxia or age for both antagonists used (Fig. 4). In addition, the double competition method developed to measure receptor densities was extremely robust and highly reproducible, suggesting that artifactual errors probably contributed little to our measurements of receptor density. These considerations suggest that short-term severe hypoxia did indeed decrease receptor density, although the mechanisms involved remain uncertain. As suggested by Roth and his colleagues (37), 5-HT receptors can be rapidly downregulated via receptor-mediated endocytosis, a process that appears to internalize phosphorylated plasma membrane receptors into intracellular endosomes (7). For beta -adrenergic receptors in fetal rat liver, these receptor endosomes are typically of very low density and thus should be excluded from the high-density pellet we used for membrane receptor measurements (12). Hypoxic acceleration of receptor phosphorylation and internalization, perhaps via enhanced GRK activity as already mentioned, could efficiently explain the decrease in receptor density observed in the present studies.

In some preparations, internalized receptors can be recycled in as little as 20 min, as suggested by posthypoxic recovery of isoproterenol-stimulated adenyl cyclase activity in the fetal rat liver (12). In other preparations, such as the chick heart, receptor recovery following hypoxia can require up to 2 h of reoxygenation (20). Although the reasons for this variability in recovery time remain uncertain, decreased intracellular availability of GTP is apparently not involved (20). In our ovine carotid arteries, receptor density was clearly depressed after 30 min of exposure to hypoxia, suggesting that at this time receptor recovery was depressed relative to the rate of internalization. This observation, in turn, raises the possibility that hypoxia may act not only by accelerating the rate of internalization and/or receptor degradation but also by retarding the rate of receptor recovery. Further experiments will be required to differentiate among these possibilities, but the absence of hypoxic depression of receptor density in fetal arteries predicts that the mechanisms responsible are either absent or undeveloped in immature arteries.

Effects of acute hypoxia on binding-response relations. In addition to its possible effects on receptor affinity and density, hypoxia can also potently influence multiple downstream mechanisms coupling receptor activation to contraction. For example, hypoxia can modulate the agonist-induced entry of extracellular calcium (31), phospholipase C activity (34), inositol trisphosphate production (35), and ultimately the number of activated myosin cross bridges (23). From these many effects, we sought to estimate their relative magnitudes independent of hypoxic effects on ligand-receptor interactions. To achieve this, we calculated the number of 5-HT receptors bound at each agonist concentration we examined. As indicated in METHODS: Calculation of binding-response relations, this calculation corrected for vessel-to-vessel differences in receptor density and affinity under hypoxic conditions and provided an estimate of the contractile force produced for each femtomole of receptors bound. As indicated in Fig. 5, the relation between the numbers of receptors bound and the contractile force produced was little affected by hypoxia in the adult arteries. This observation persisted even when contractile responses were corrected for age-related differences in the effects of hypoxia on potassium-induced tone (Fig. 5, bottom). Although hypoxia increased the curvilinear character of the curves relating femtomoles of receptors bound to contractile responses, in absolute terms the size of the contractile response produced for each femtomole bound was quite similar under hypoxic and normoxic conditions in adult carotid arteries. Together, these observations suggest that the effects of hypoxia on agonist-receptor interactions constitute a major component of the overall actions of acute hypoxia, at least for 5-HT in adult ovine carotid arteries. In this tissue, hypoxia appears to reduce cell surface receptor density and agonist affinity but has relatively little effect on their intrinsic efficacy.

In contrast to the adult, the basic relations between the number of receptors bound and contractile responses were all nonlinear rectangular hyperbolas in the fetus, indicating the probable presence of receptor reserve (39). The presence of spare receptors in the fetus might help explain why hypoxia had little effect on receptor density, particularly if these receptors were uncoupled or inaccessible to the mechanisms mediating receptor downregulation in the adult. More importantly, the relations between the numbers of receptors bound and contractile force were dramatically influenced by acute hypoxia in the fetus (Fig. 5). Independent of the method used to normalize the contractile responses observed during hypoxia, any given number of receptors bound always produced less contractile tone under hypoxic than under normoxic conditions. This observation suggests that mechanisms downstream from agonist-receptor binding contribute significantly to hypoxic vasodilatation in the fetus. Given that contractile tone is more dependent on calcium sensitization (1, 3) and the entry of extracellular calcium (2) in fetal than adult arteries, it seems probable that hypoxia may decrease contractile tone in fetal arteries at least in part by altering calcium handling. Consistent with this possibility, hypoxia has been shown to modulate agonist-induced entry of extracellular calcium (31) as well as mobilization of intracellular calcium (11).

As an overview, taken together, the present results emphasize that carotid artery responses to acute hypoxia involve significant direct vascular effects. Most importantly, these data demonstrate that hypoxia can modulate agonist-receptor interactions and that the impact of these effects is age specific. In mature arteries, reductions in agonist affinity and density appear to be the predominant effects of acute hypoxia leading to vasodilatation; effects on mechanisms downstream from agonist-receptor binding appear to contribute relatively little to the overall response to acute hypoxia. In immature arteries, acute hypoxia also depresses agonist affinity but has little effect on receptor density. Instead, hypoxia appears to depress the ability of the bound receptors to elicit a contractile response. Why the effects of hypoxia vary between arteries of differing age remains unclear but could reflect differences in the ambient normal arterial oxygen tensions typical of fetuses (approx 25 Torr) and adults (approx 100 Torr). In addition, the present observations are subject to some limitations. All experiments were carried out in endothelium-denuded preparations so that the participation of the endothelium in these responses remains unknown. The experiments were also conducted in large conduit arteries, and thus the applicability of these results to smaller resistance arteries is uncertain. Finally, these studies used 5-HT exclusively and thus their relevance to other agonists is unknown. Aside from these limitations, the present results suggest a novel mechanism of action for acute hypoxia that helps explain why fetal and adult arteries respond so differently to acute hypoxia. Interestingly, both age groups are highly capable of vasodilating in response to acute hypoxia, although via distinctly different mechanisms. The molecular bases for these mechanistic differences and their potential for pharmacological manipulation remain promising topics for future investigation.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants HL-54120 and HD-31266 and the Loma Linda University School of Medicine.


    FOOTNOTES

The work reported here was completed as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physiology for D. M. Angeles.

Address for reprint requests and other correspondence: W. J. Pearce, Center for Perinatal Biology, Loma Linda Univ. School of Medicine, Loma Linda, CA 92350 (E-mail: wpearce{at}som.llu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 22 September 1999; accepted in final form 1 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 279(2):H502-H510
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