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(Circulation. 1996;93:1020-1025.)
© 1996 American Heart Association, Inc.

Articles

Expression of Opioid Receptors During Heart Ontogeny in Normotensive and Hypertensive Rats

Reuven Zimlichman, MD; Dov Gefel, MD; Heskel Eliahou, MD; Zipora Matas, PhD; Boaz Rosen, MD; Svetlana Gass, BSc; Catherine Ela, PhD; Yael Eilam, PhD; Zvi Vogel, PhD; Jacob Barg, MD, PhD

From the Department of Internal Medicine (R.Z., D.G., B.R.), Hypertension Clinic (H.E.), Clinical Chemistry Laboratory (Z.M.), and Cardiovascular and Hypertension Research Laboratory (S.G., J.B.), Wolfson Medical Center, Tel Aviv University Sackler School of Medicine, Holon, Israel; Department of Bacteriology (C.E., Y.E.), Hebrew University-Hadassah Medical School, Jerusalem, Israel; Department of Neurobiology (Z.V., J.B.), Weizmann Institute of Science, Rehovot, Israel; and Therapeutic Community (J.B.), Ramot Yehuda, Zoharim, Israel.

Correspondence to Jacob Barg, MD, Cardiovascular and Hypertension Research Laboratory, Wolfson Medical Center, Box 5, 58100 Holon, Israel. E-mail bnbarg@weizmann.weizmann.ac.il.

	Abstract

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Background The opioidergic systems are involved in modulating nociceptive stimuli. In addition, recent results suggest that endogenous and exogenous opioids could play a role in the modulation of blood pressure and cardiac functions. However, little is known regarding the expression and role of opioid-binding sites in the heart. The decreased sensitivity to noxious stimuli in hypertensive rats raises the possibility of different developmental pattern expression of opioid-binding sites in normotensive versus hypertensive rats.

Methods and Results Opioid receptor expression in hearts from hypertensive and normotensive rats was studied during heart development by binding assays. From P1 until P90, the development of the heart in the two rat strains was accompanied by a gradual increase in the density of -opioid receptors. Hearts from hypertensive rats expressed significantly higher levels of receptors compared with those of normotensive rats. At ages older than P7, µ-opioid receptors could not be detected in hearts of both strains, whereas -opioid–binding sites gradually increased until reaching adult levels. Seven-day-old cardiomyocyte cultures of both rat strains expressed similar densities of or receptors to those observed in hearts from 7-day-old neonates. The µ-binding sites were not detected in cardiomyocyte cultures. Similar to the in vivo state, cultured myocytes from hypertensive rats had significantly higher levels of -binding sites (1.5-fold) compared with those of normotensive rats. The sites are pertussis toxin sensitive, and the state of coupling of the receptor to G protein is similar for the two rat strains.

Conclusions The role of opioid-binding sites in the heart is not completely clear. Hypertensive rats are known to be less sensitive to noxious stimuli compared with normotensive rats. It is controversial whether the site of application of noxious stimuli plays an important role in the sensitivity to pain in hypertensive rats. We suggest that the opioidergic system could play a role in the modulation of blood pressure in addition to its known effect on nociception.

Key Words: receptors, opioid • cardiomyocytes, cultured • hypertension

	Introduction

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Opioid drugs are widely used as analgesics in patients with pain.¹ For example, opioids are often used to ease the pain of the mother during birth.² Morphine or its derivatives are used for relief of chronic pain in patients with malignant diseases,³ as well as during acute myocardial infarction.⁴ Not only is the nervous system affected by opioids, but there is considerable evidence to support a role for endogenous opioid agonists and their receptors in the regulation of other systems, including cardiovascular functions.^{5 6 7} However, although numerous studies have followed the expression of opioid receptors and peptides in brain,^{8 9 10 11} little is known about the developmental profile of opioid-binding sites in heart.

In several studies, it has been proposed that endogenous and exogenous opioids are involved in the modulation of hypertension and other cardiovascular conditions.^{12 13} For example, naloxone, the opioid antagonist, reverses hypotension¹⁴ and improves recovery from shock.¹⁵ It was also reported that naloxone blocks the arrhythmic effects induced by ouabain,¹⁶ hypoxia,¹⁷ or coronary artery ligation.¹⁸ Increasing amounts of anatomic, physiological, and pharmacological evidence suggest that pain-inhibitory circuitry is linked with the cardiovascular system.^{19 20 21} For example, induction of hypertension in rats by various methods is associated with increased resistance to noxious stimulations.²² SHR are less sensitive to nociceptive stimuli than are normotensive WKY rats.²² These observations support the notion of a relation among the cardiovascular system, hypertension, and the opioidergic system. Moreover, these reports raise the possibility of variations in the opioidergic system of the heart in WKY rats and SHR and emphasize the need for studying the developmental expression of opioid receptors during heart ontogeny.

Recently, it was shown that - and -opioid receptors are present in rat cardiac sarcolemma.^{23 24} Moreover, several studies reported the identification of mRNA for preproenkephalin A in rat ventricles, cultured rat ventricular myocytes,²⁵ and dog heart.²⁶ Activation of receptor with the opioid agonist U50488 in cultured rat ventricular cardiomyocytes was shown to increase the formation of IP₃ and IP₄.²⁷ These effects were opioid receptor mediated since they were blocked by opioid antagonists. These observations establish a role of the opioidergic system in heart function. Opioid receptors were detected in hearts from adult rats, but their developmental expression is unknown. Moreover, no information is available regarding the expression of opioid receptors in normotensive versus hypertensive rats. In this investigation, we studied the developmental profile of opioid receptors in the hearts of WKY rats and SHR and related the profile of opioid receptor expression with hypertension. We found a different pattern of opioid receptor expression during the ontogeny of the heart in the two rat strains and show that the genetically hypertension-prone rats express higher B_max values of -opioid receptors compared with normotensive rats.

	Methods

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Animals
Hearts from WKY rats and SHR of different ages were excised and stored frozen at –70°C until use in opioid receptor–binding assays. The animals were handled according to the "Guide for the Care and Use of Laboratory Animals," as adopted and promulgated by the National Institutes of Health.

Cardiomyocyte Cultures
Cultures of cardiomyocytes were prepared as previously described²⁸ with several modifications. Briefly, myocardial cells were isolated from ventricular fragments of hearts from 1-day-old WKY rats or SHR by serial trypsinization and suspended in Ham's F-10 medium containing 20% heat-inactivated FCS and penicillin/streptomycin antibiotic solution. The cell suspension was enriched with myocytes by preplating on tissue culture dishes for 1 hour. The myocyte-enriched suspension was collected and diluted with the same medium to 5x10⁵ cells/mL. The cultures were maintained in humidified 5% CO₂/95% air atmosphere at 37°C for 7 days before the experiments were performed. At the time of the experiment, the cells were in confluent monolayers consisting of 85% to 90% myocytes that exhibited spontaneous contractions.

Crude Membrane Preparation and Binding Assay
Hearts were washed twice with PBS, pH 7.4, homogenized in 20 vol of 50 mmol/L Tris-HCl buffer, pH 7.4, with a polytron (Kinematica). The nuclear fraction was removed by centrifugation at 1000g, and the crude membrane-containing particulate fraction was collected by a 20-minute centrifugation at 40 000g.¹⁰ The pellet was than resuspended in 50 mmol/L Tris-HCl buffer and used for binding assay. Opioid receptors were monitored by determining the specific binding with 1 or 6 nmol/L of the nonselective opioid ligand [³H]diprenorphine (Amersham). The nonspecific binding (determined in the presence of 5 mmol/L etorphine) was subtracted. The opioid receptor types were determined using 100 nmol/L specific µ, , and agonists as displacers. These included DAMGE, DPDPE, and U50488, respectively. Estimation of B_max values was obtained from the specific binding at 6 nmol/L of [³H]diprenorphine as previously described.^{8 9 10 11} In several cases, the receptor was determined using homologous competition-binding assay of the agonist [³H]U69593 (Amersham). The amount of protein was determined as previously described²⁹ with bovine serum albumin as a standard. Binding data are presented as mean±SEM unless otherwise indicated. B_max, K_d, and IC₅₀ values were calculated from three or four independent experiments with INPLOT 4 computer program (GraphPad Software). The total B_max value was calculated as the sum of the high- and low-affinity sites. The figures and calculation of the binding parameters were drawn with SIGMAPLOT 4.11 computer program (Jandel Scientific) as previously described.³⁰

	Results

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Opioid receptors are found in hearts of WKY rats and SHR and were detected as early as P1 (Figs 1 and 2). As shown in Figs 2 and 3, the development of the rat's heart was accompanied by a gradual increase in the B_max values of the -opioid receptors. At all ages studied, two binding sites were detected in each of the two rat strains. The K_d values for the high-affinity binding sites varied from 0.9±0.4 to 3.1±1.7 nmol/L and from 0.4±0.2 to 0.8±0.3 nmol/L for WKY rats and SHR, respectively. The K_d values for the low-affinity binding sites varied from 29±8 to 220±39 nmol/L and from 11±4 to 200±28 nmol/L for WKY rats and SHR, respectively. There were no significant differences in K_d values of the two binding sites between the two rat strains at all the ages studied. On the other hand, hearts of SHR expressed significantly higher B_max values of receptors compared with those of WKY throughout the developmental period (from P1 until P90) (Fig 2). The amounts of -binding sites during development ranged from 13.5±1.3 to 42.5±4.1 fmol/mg protein for WKY rats and from 19.4±1.7 to 53.1±3.8 fmol/mg protein for SHR. As an additional control, we studied the binding parameters of -opioid receptor in another rat strain (Sprague-Dawley) at P7 and P60. Two binding sites were found: high-affinity binding sites of 0.7±0.4 and 1.6±0.6 nmol/L for P7 and P60, respectively, and low-affinity binding sites of 64±11 and 78±21 nmol/L, respectively. These values were not different from the values obtained for the corresponding ages of WKY rats or SHR. In contrast, the B_max values (15.4±2.1 and 28.2±2.9 fmol/mg protein for P7 or P60, respectively) of receptors in Sprague-Dawley were significantly lower from those of SHR at P7 and P60 (P<.05) but did not differ from those of WKY rats.

View larger version (30K): [in this window] [in a new window]   Figure 1. Homologous competition curves of [3H]U69593 binding at various developmental stages of WKY and SHR hearts. In all curves, two binding sites fitted best as detected with the INPLOT 4 program. The Kd values ranged from 0.4 to 3.1 nmol/L for high-affinity binding sites and from 11 to 220 nmol/L for low-affinity binding sites for both rat strains. Data are the mean±SEM of three experiments performed in duplicate.

View larger version (16K): [in this window] [in a new window]   Figure 2. Age-dependency of the development of -opioid receptors in rat hearts. Two binding sites were resolved for receptors in WKY rats or SHR. A, Bmax values obtained for the sum of high- and low-affinity sites; B shows the high-affinity values; and C shows the low-affinity values. Data are the mean±SEM of three experiments performed in duplicate. *P<.05 or **P<.01, significantly different from the value at P1. The Bmax values of the sum of the two binding sites of SHR at all ages tested are significantly different from those of WKY rats (P<.05). #P<.05, significantly different from the corresponding age of WKY rats.

View larger version (28K): [in this window] [in a new window]   Figure 3. Opioid receptor types in hearts of WKY rats and SHR expressed as percentage of total opioid-binding sites with the specific binding determined with 1 nonselective opioid ligand [3H]diprenorphine. The nonspecific binding (determined in the presence of 5 mmol/L etorphine) was subtracted. The opioid receptor types were determined with 100 nmol/L specific µ (DAMGE), (DPDPE), and (U50488) agonists as displacers. The relative percentages of µ (A and D), (B and E), and (C and F) receptors in hearts from P1, P7, and P14 of WKY rats (A, B, and C) or SHR (D, E, and F). Data are the mean±SEM of three experiments performed in duplicate. *P<.05, significantly different from P1.

At an early developmental period (eg, P1 or P7), all three receptor types, µ, , and , were present in the heart (Fig 3A and 3D). However, after day 7 (eg, P14 and P30), µ receptors were not detected and -binding sites increased gradually until reaching adult levels at P14 (Fig 3B and 3E). Interestingly, during this developmental period, only minor changes in the relative proportions of -opioid receptor were observed (Fig 3C and 3F).

Cardiomyocyte cultures could serve as a convenient system to study the factors that may affect the state of binding to the receptor. We found that 7-day-old cardiomyocyte cultures express similar B_max values for or receptors as observed in hearts from 7-day-old neonates in both rat strains (Fig 4). Comparable with the in vivo situation, no µ-binding sites were detected in 7-day-old cardiomyocyte cultures. As described above, a higher B_max value of receptors (28.9±2.8 fmol/mg protein) was observed in P7 SHR neonates compared with P7 WKY neonates (19.8±2.1 fmol/mg protein). A similar observation was also made with cardiomyocyte cultures. As shown in Fig 4, 7-day-old SHR cultures express higher levels of receptors compared with WKY cultured cardiomyocytes (24.1±2.3 versus 16.2±1.4 fmol/mg protein).

View larger version (23K): [in this window] [in a new window]   Figure 4. Expression of µ-, -, and -opioid receptors in crude membranes of P7 hearts (A) or of 7-day-old cardiomyocyte cultured cells (B) obtained from WKY rats and SHR. The percentage of each opioid receptor type was determined by binding of 1 nmol/L [3H]diprenorphine in the presence of 100 nmol/L of the specific competitors DAMGE, DPDPE, and U69593. The Bmax values of the opioid receptors were obtained by binding of 6 nmol/L of [3H]diprenorphine as described in "Methods." Data are the mean±SEM of three or four experiments performed in duplicate. *P<.05, significantly different from WKY.

These differences in B_max values of receptors in the two strains could possibly be attributed to the state of coupling of the receptor to G proteins, as was suggested in previous studies for neural opioid receptors.¹⁰ However, as shown in Fig 5, Gpp(NH)p in various concentrations similarly inhibited the binding of [³H]U69593 to cardiomyocyte crude membranes from either WKY or SHR cultures. This finding suggests a similar state of coupling of the -opioid receptor to G proteins in both WKY and SHR cultured cardiomyocytes.

View larger version (20K): [in this window] [in a new window]   Figure 5. Concentration dependence of Gpp(NH)p inhibition of [3H]U69593 specific binding to crude membranes of 7-day-old cultured cardiomyocytes. The crude membranes were incubated with 1 nmol/L [3H]U69593 in the presence or absence of indicated concentrations of Gpp(NH)p. Data are the mean±SEM of three experiments performed in duplicate. *P<.01, significantly different from binding without Gpp(NH)p.

Several studies using various neuronal systems reported that the receptor agonist U50488 inhibits the activity of adenylate cyclase, leading to a reduction in the production of cAMP.^{31 32} This inhibition of adenylate cyclase activity is mediated via PTX-sensitive G proteins.³² These results raise the possibility that -agonist binding to the receptor in heart could also be affected by pretreatment with PTX. As shown in Fig 6, PTX dose-dependently inhibited the binding of [³H]U69593 in cultured cardiomyocytes from both rat strains. The PTX treatment did not affect the binding of the opioid antagonist [³H]diprenorphine. These results agree with previous results observed with neural cells and tissues.³³

View larger version (24K): [in this window] [in a new window]   Figure 6. Dose-dependency of the effect of PTX preincubation on the specific binding of [3H]U69593 and [3H]diprenorphine to crude membranes of 7-day-old cultured cardiomyocytes. The cultured cells were treated with the indicated concentrations of PTX 4 hours before the binding assay. Specific binding was determined with 1 nmol/L of [3H]U69593 or [3H]diprenorphine. Note that the binding of [3H]diprenorphine was not affected by preincubation with PTX. Data are the mean±SEM of three experiments performed in duplicate. *P<.01, significantly different from PTX-untreated cultures.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we focused on the postnatal development of opioid receptors in hearts of WKY rats and SHR. At early stages of heart ontogeny (until P7) all three receptors are present, with the -binding sites possessing more than 80% of the total opioid receptors. The -binding sites increase gradually with postnatal age while receptors are only moderately increasing. A comparable developmental pattern for -binding sites was reported in other organs,^{8 9 10 11} whereas and µ profiles differ. For example, an increase in -binding sites was observed during the ontogeny of guinea pig cerebellum.⁹ Similarly, the receptors in rat spinal cord increase in density during postnatal development.³⁴ It was previously shown that most of the opioid binding sites are located on the plasma membrane; however, opioid receptors were also observed at several other cell compartments. Opiate receptor densities may change differentially with development at the various compartments. To avoid this issue, we studied the expression of the receptors in the cell using crude membranes, thus measuring the total amount of opioid binding sites in the cell.

We found a strain-specific developmental pattern of opioid receptors in hearts of WKY rats and SHR. In both rat strains, there is a gradual increase in the B_max value of receptors with two binding affinities. However, SHR express higher B_max values of receptors compared with WKY during the late postnatal period.

An interesting finding is that µ receptors are expressed only at early periods of heart ontogeny. No µ-binding sites were detected in P14 or later stages. Similarly, no µ receptors were found in 7-day-old cardiomyocytes. This observation raises the possibility that under culture conditions a faster rate of cardiomyocyte differentiation may occur. In addition, this loss of µ receptors in the adult heart raises the question of the target for morphine use during acute myocardial infarction and requires a careful study of opioid receptor differentiation in human heart.

The cellular mechanisms regulating the expression of various neurotransmitter receptors in heart are mostly enigmatic. Clearly, numerous signals, including signaling by growth factors, are involved in these processes. Changes in receptor profile may result from a decline or an increase in the number of binding sites^{8 9} or alterations in receptor affinity to the ligands tested.¹⁰ We found that in both rat strains there is a period in which the density of µ (but not of and ) receptors decreases dramatically. It is conceivable that during heart ontogeny, there are changes in receptor repertoire of the existing heart cells or, alternatively, that the new developing cells do not express the receptors or do lose them during maturation. At the same period, it appears that heart cells bearing receptors do develop with age. From P1 until P14, a rapid increase in B_max value of the high-affinity -binding sites is evident, whereas the low-affinity binding sites show only minor fluctuations (see Fig 2). This developmental pattern is similar for both rat strains and was shown in other organs as well.^{8 9 10 11} Because an increase in B_max value was evident in cardiomyocyte cultures as well as in hearts before the period of development of high blood pressure (in the SHR), it is unlikely that the increase in receptor density could be the result of left ventricular hypertrophy.

The age-associated changes in opioid receptors, together with the alterations reported for met-enkephalin, leu-enkephalin, and for the mRNA encoding for the precursor peptide pro-enkephalin,²⁵ suggest that the cardiac opioidergic system undergoes major alterations during heart ontogeny. These changes seem to be strain specific, as the development of the receptor sites in WKY rats is different from that in SHR (as is evident from the higher B_max values). Since the patterns of Gpp(NH)p inhibition of the binding of U69593 to cardiomyocyte crude membranes are similar for the two rat strains, we excluded the possibility that this strain-dependent variation in receptor apparent density is due to the state of coupling of the receptor to G proteins. We, therefore, suggest that the differences in B_max values reflect authentic changes in receptor density rather then alterations in the state of receptor coupling. Our findings about PTX inhibition of U69593 binding agree with previous reports about -agonist inhibition of adenylate cyclase and of voltage-dependent Ca²⁺ channels in neuronal cells.^{32 35} It is therefore likely that the receptor in heart is coupled to PTX-sensitive G proteins.

The relationship of hypertension and pain perception has become of interest^{36 37} ; several studies reported that hypertensive humans as well as hypertensive rats are hypoanalgesic to noxious stimuli.^{22 38} Specific brain regions were implicated in the modulation of both pain and regulation of blood pressure,¹⁹ eg, hypothalamus, nucleus tractus solitarius, locus coruleus, nucleus reticularis gigantocellularis, and raphe nucleus. Pharmacological studies suggest an interaction between the regulatory systems of pain and blood pressure. Morphine induces a decrease in blood pressure, whereas hypotensive drugs are known to modify noxious stimulation.^{37 38}

In the present study, we have shown that hypertensive rats, which usually have increased blood pressure at approximately 60 days postpartum,³⁹ have higher B_max values for the -opioid receptor compared with normotensive rats. The presence of opioid receptors in heart is likely an important factor in the regulation of heart function and supports the notion for an interaction of the opioidergic system with the cardiovascular system. Considering both rat strains, it is tempting to associate our findings with those indicating that genetically hypertension-prone rats are less sensitive to nociceptive stimuli compared with normotensive rats. Possibly, the differences in the level of pain perception and of control of blood pressure in the two rat strains reflects the difference in -opioid receptor expression.

	Selected Abbreviations and Acronyms

 

DAMGE = Tyr-D-Ala-Gly-NMe-Phe-Gly-ol DPDPE = Tyr-D-Pen-Gly-Phe-D-Pen IP = inositol phosphate P = postnatal day PTX = pertussis toxin SHR = spontaneously hypertensive rats U50488 = 3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzenacetamide methanesulfonate hydrate WKY = Wistar-Kyoto

	Acknowledgments

 
This work was supported in part by grants from the USA-Israel Binational Science Foundation and the Israel Ministry of Health.

Received June 28, 1995; revision received October 5, 1995; accepted October 11, 1995.

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