AJP - Heart AJP: Advances in Physiology Education
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 284: H1104-H1109, 2003. First published December 5, 2002; doi:10.1152/ajpheart.00441.2002
0363-6135/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
284/4/H1104    most recent
00441.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (20)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Turnbull, L.
Right arrow Articles by Baker, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Turnbull, L.
Right arrow Articles by Baker, A. J.
Vol. 284, Issue 4, H1104-H1109, April 2003

alpha 1-Adrenergic receptor responses in alpha 1AB-AR knockout mouse hearts suggest the presence of alpha 1D-AR

Lynne Turnbull, Diana T. McCloskey, Timothy D. O'Connell, Paul C. Simpson, and Anthony J. Baker

Departments of Medicine, Radiology and Cardiovascular Research Institute, University of California, San Francisco 94143; and the Veterans Affairs Medical Center, San Francisco, California 94121


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Two functional alpha 1-adrenergic receptor (AR) subtypes (alpha 1A and alpha 1B) have been identified in the mouse heart. However, it is unclear whether the third known subtype, alpha 1D-AR, is also present. To investigate this, we determined whether there were alpha 1-AR responses in hearts from a novel mouse model lacking alpha 1A- and alpha 1B-ARs (double knockout) (ABKO). In Langendorff-perfused hearts, alpha 1-ARs were stimulated with phenylephrine. For ABKO hearts, phenylephrine reduced left ventricular pressure and coronary flow (to 87 ± 2% and 86 ± 4% of initial, respectively, n = 11, P < 0.01). These effects were blocked by prazosin and 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspirol[4,5]decane-7,9-dione} dihydrochloride, suggesting that alpha 1D-AR-mediated responses were present. In contrast, right ventricular trabeculae from ABKO hearts did not respond to phenylephrine, suggesting that in ABKO perfused hearts, the effects of phenylephrine were not mediated by direct actions on cardiomyocytes. A novel finding was that alpha 1-AR stimulation caused positive inotropy in the wild-type mouse heart, in contrast to negative inotropy observed in mouse cardiac muscle strips. We conclude that mouse hearts lacking alpha 1A- and alpha 1B-ARs retain functional alpha 1-AR responses involving decreases of coronary flow and ventricular pressure that reflect alpha 1D-AR-mediated vasoconstriction. Furthermore, alpha 1-AR inotropic responses depend critically on the experimental conditions.

Langendorff-perfused heart; phenylephrine; myocardial contractility; coronary arteries


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MOUSE HEART contains mRNA for three subtypes of the alpha 1-adrenergic receptor (AR): alpha 1A, alpha 1B, and alpha 1D (1). However, protein has been detected by binding or function for only two alpha 1-AR subtypes, alpha 1A and alpha 1B (22). Therefore, it is unknown whether the alpha 1D-AR protein is present in the heart and what its function might be.

Previous studies found that alpha 1D-ARs were localized to extracardiac blood vessels. In the rat, alpha 1D-ARs mediate contraction of blood vessels, including the aorta, mesenteric, and femoral arteries (3, 5, 6, 18), and are involved in regulating the pressor response to phenylephrine (24). In the mouse, alpha 1D-ARs also mediate contraction in the thoracic aorta, abdominal aorta, and mesenteric arteries (20, 21) and have recently been shown to be involved in regulating systemic blood pressure (17).

The presence of mRNA for the alpha 1D-AR in whole mouse heart homogenate and the presence of alpha 1D-ARs in the systemic vasculature raise the possibility that alpha 1D-ARs are expressed in the mouse heart in the coronary vessels.

Therefore, the goal of this study was to determine whether there are functional alpha 1D-ARs in the mouse heart. Our approach used hearts from a novel mouse model lacking alpha 1A-ARs and alpha 1B-ARs, or alpha 1A/alpha 1B-AR double knockout (ABKO) (T. D. O'Connell, S. Ishizaka, A. Nakamura, P. M. Swigart, M. C. Rodrigo, S. Cotechia, D. G. Rokosh, W. Grossman, E. Foster, P. C. Simpson, unpublished observations). Langendorff-perfused hearts were stimulated with alpha 1-AR agonists and antagonists to determine whether there was a residual alpha 1-AR response in ABKO hearts that might reflect alpha 1D-AR function. We found that alpha 1-AR stimulation of the ABKO mouse heart resulted in decreased coronary flow and systolic pressure. These effects were blocked by the alpha 1-AR antagonist prazosin and the alpha 1D-AR subtype-selective antagonist 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspirol[4,5]decane-7,9-dione} dihydrochloride (BMY-7378). Our results demonstrate that the ABKO mouse heart has functional alpha 1D-ARs and further suggest that alpha 1D-ARs are involved in coronary vasoconstriction.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal methods. All procedures were approved by the Animal Studies Subcommittee of the San Francisco Veterans Affairs Medical Center. KO mice were generated as recently described (T. D. O'Connell, S. Ishizaka, A. Nakamura, P. M. Swigart, M. C. Rodrigo, S. Cotechia, D. G. Rokosh, W. Grossman, E. Foster, P. C. Simpson, unpublished observations). alpha 1B-AR KO mice (2) were mated with alpha 1A-AR KO mice (13) to produce F1 generation mice heterozygous for both KOs. F1 heterozygous mice were mated to produce F2 wild-type (WT) and ABKO mice, and breeding pairs from these lines produced offspring that were used for these experiments. WT and ABKO (mixed C57BL/6, FVBN, and 129SvJ) adult mice of both sexes were used (n = 47, average age 16 ± 1 wk, average wt 29.8 ± 0.7 g). Mice were anesthetized with pentobarbital sodium (1 mg/g, Abbott Laboratories; Chicago, IL), heparinized (2 U/g, Elkins-Sinn; Cherry Hill, NJ), and the hearts were rapidly excised for the studies using perfused hearts or trabeculae.

Isolated heart preparation. Excised hearts were placed in ice-cold arrest solution composed of (in mM) 120 NaCl, 30 KCl, and 0.1 CaCl2, and the aortic arch was dissected. The aorta was cannulated (20 gauge) and perfused with a modified Krebs-Henseleit solution composed of (in mM) 118 NaCl, 4.7 KCl, 1.66 MgSO4, 1.18 KH2PO4, 0.5 sodium EDTA, 25 NaHCO3, 5.55 glucose, 5 sodium pyruvate, and 2.5 CaCl2, using the Langendorff technique. Perfusion was at constant pressure (70 mmHg). The perfusate was oxygenated and maintained at a pH of 7.4 by vigorous bubbling with 95% O2-5% CO2. The right atrium was trimmed and the sinoatrial node was crushed. Hearts were electrically stimulated at the atrioventricular junction with a coaxial stimulation electrode (Harvard Apparatus; Holliston, MA) connected to a square pulse stimulator (model SD9, Grass-Telefactor; West Warwick, RI). Hearts were paced at 6 Hz with square pulses (width 4 ms) and at supramaximal voltage.

Heart temperature was constantly monitored via a thermistor probe placed in the right ventricle and was maintained at 37°C. To ensure adequate oxygenation of the preparation, the apparatus (Constant Pressure Non-Circulating System, Radnoti Glass Technology; Monrovia, CA) was modified to include a recirculating shunt from the top of the aortic cannula to the bubbling reservoir. Transit time for the perfusate from the reservoir to the cannula was reduced to <10 s, regardless of coronary flow, thus limiting changes in perfusate gas composition during transit to the heart. The perfusate was passed through an in-line filter (5.0 µm) to remove particulate matter.

Coronary flow was measured by collecting the coronary effluent for 1 min. In five hearts, we experimentally reduced coronary flow by clamping the aortic inflow without altering perfusion pressure.

To monitor left ventricular pressure development a small fluid-filled balloon made of plastic wrap was attached to a short length of polyethylene-50 tubing and attached to a pressure transducer (model TRN050; Kent Scientific; Litchfield, CT). The balloon was inserted into the left ventricle via an opening in the left atrium. The balloon was filled with degassed water in 5-µl increments up to a final volume of 30-40 µl and adjusted to maintain the left ventricular end-diastolic pressure at 8-10 mmHg. The contribution of the balloon alone to measured pressure was negligible.

Left ventricular pressure signals were digitized (0.2-1 kHz sampling) and stored on a laboratory computer. Data were analyzed to obtain pressure development and timing parameters.

Inotropic responses. Hearts were preincubated with the beta -antagonist timolol (10 µM) and then stimulated with the non-subtype-selective alpha 1-agonist phenylephrine (PE; 10 µM). PE was delivered via an infusion pump at a rate of 1% of coronary flow. In some experiments, hearts were also preincubated with the non-subtype-selective alpha 1-antagonist, prazosin (5 µM) or the subtype-selective alpha 1D-antagonist BMY-7378 (500 nM). Stock solutions of PE, timolol, and BMY-7378 were prepared in the perfusate. Prazosin was dissolved in 100% ethanol and then diluted in perfusate to make a stock solution. The final concentration of ethanol delivered was 0.4%. Control experiments showed that infusion of the perfusate or the ethanol-perfusate vehicle did not affect perfused heart function. L-Phenylephrine hydrochloride, timolol (maleate salt), and BMY-7378 dihydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). Prazosin-HCl was purchased from Research Biochemicals International (Natick, MA). All chemicals were of analytic grade.

Right ventricular trabeculae. Excised hearts were perfused through the aorta with a modified Krebs-Henseleit solution composed of (in mM) 112 NaCl, 15 KCl, 1.2 MgCl2, 2.0 NaH2PO4, 24 NaHCO3, 1.2 NaSO4, 10 glucose, 30 2,3-butanedione monoxime (BDM), and 1 CaCl2. The perfusate was oxygenated with 95% O2-5% CO2 to maintain a pH of 7.4 at 22°C. The right ventricle was opened, and a trabecula that was free-running between the right ventricular wall and the tricuspid valve was dissected out.

Trabeculae were placed in a muscle chamber and attached to the apparatus by mounting the ends on stainless steel pins (100 µm diameter) attached to a micromanipulator at one end to vary length and a force transducer (model AE-80, SensoNor) at the other end. Trabeculae were superfused at 22°C with Krebs-Henseleit solution (as above without BDM, with KCl 5 mM, and with CaCl2 2 mM). Sarcomere length was assessed by the diffraction of light by muscle sarcomeres and diastolic sarcomere length was set to 2.1 µm. Trabeculae were field stimulated using platinum wire electrodes at a frequency of 0.5 Hz and supramaximal voltage. Trabeculae were preincubated with timolol (10 µM), and alpha 1ARs were stimulated with PE (10 µM) added to the superfusate.

Statistics. Results are presented as means ± SE. Student's t-tests, one-way ANOVA, and Student-Newman-Keuls test were used to determine differences between means. Values of P < 0.05 were considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Residual response to alpha 1-AR stimulation in ABKO perfused heart. Examples of the effects of PE stimulation on contraction of WT and ABKO perfused hearts are shown in Fig. 1. Figure 1A shows a representative slow time-base recording of left ventricular pressure of a WT perfused heart with addition of PE indicated by the arrow. PE caused a complex contractile response involving an early transient negative inotropic phase, followed by a sustained positive inotropic phase. This finding was unexpected because previous studies from this laboratory and others (8-11, 14, 16) have demonstrated that muscle strips from mouse hearts display a sustained negative inotropic response to PE.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 1.   Residual response to alpha 1-adrenergic reagent (AR) stimulation in alpha 1A- and alpha 1B-double knockout (ABKO) hearts. Representative slow time-based tracings of left ventricular (LV) pressure (LVP) responses to phenylephrine (PE) recorded from wild-type (WT) (A) and ABKO (B) hearts. Hearts were preincubated with the beta -AR antagonist timolol. PE was added, as indicated by arrow. A: in WT hearts, PE caused a transient fall of pressure, followed by a sustained positive inotropic phase. B: knockout of the alpha 1A- and alpha 1B-AR subtypes abolished the positive response to PE; however, ABKO hearts had a residual sustained negative inotropic response.

In contrast to the effects of PE in WT hearts, in ABKO hearts (Fig. 1B), PE induced a small, sustained negative inotropic response that developed gradually and lacked the complex time course that characterized the response of WT hearts to PE. PE did not significantly affect left ventricular end-diastolic pressure in WT or ABKO hearts.

For all hearts studied, the effect of PE stimulation on contraction of WT and ABKO perfused hearts is summarized in Fig. 2A. Data are presented as a percentage of the left ventricular developed pressure measured before addition of PE (% control). Figure 2A shows that for WT hearts, PE caused a transient negative inotropic phase, followed by a sustained positive inotropic phase. For all WT hearts, PE increased left ventricular developed pressure to a final maximum of 118 ± 5% of control. For all ABKO hearts studied, PE caused a gradual reduction of developed pressure to below control levels, to a final minimum of 87 ± 2% of control. PE did not affect contraction kinetics in WT or ABKO hearts (data not shown).


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2.   Contrasting responses to PE in ABKO and WT perfused hearts. A: pooled data summarizing the effects of PE on LV developed pressure (LVDP) in WT (n = 8) and ABKO (n = 11) hearts. Data were normalized to developed pressure before PE addition (arrow) and shown as means ± SE. *P < 0.05 vs. time 0. B: representative slow time-based recording of developed force in an intact trabecula isolated from an ABKO heart. PE had no effect on developed force when added (arrow). Both perfused heart and trabeculae preparations were preincubated with timolol.

No response to alpha 1-AR stimulation in ABKO trabeculae. To investigate whether the negative inotropic response to PE observed in ABKO hearts was mediated by alpha 1-ARs localized to cardiomyocytes, we determined the effect of PE on contractions of isolated muscle strips. Figure 2B shows a slow time-base recording of peak twitch force of a right ventricular trabecula from an ABKO heart. PE had no effect on trabecula contraction force, suggesting that the cardiomyocytes in trabeculae from ABKO hearts do not contain alpha 1-ARs. For all trabeculae studied, developed force was 99.4 ± 1.8% of control 10 min after PE stimulation and 106 ± 2.4% of control after 30 min of PE stimulation (n = 9). These values were not statistically different from those before PE addition. In contrast, right ventricular trabeculae from WT littermates demonstrated a marked negative inotropic response to PE (unpublished data) consistent with previous studies of mouse myocardium by us and by others (8-10, 14, 16).

Inotropic effects of PE in WT and ABKO hearts are mediated by alpha 1-ARs. To determine whether the negative inotropic response to PE in ABKO hearts was mediated by alpha 1-AR stimulation, we used the non-subtype-selective alpha 1-AR antagonist prazosin. Figure 3 shows that in the presence of prazosin, PE stimulation did not elicit a contractile response in either WT or ABKO hearts. Figure 4 summarizes the effects of PE stimulation on all WT and ABKO hearts studied in the absence and presence of prazosin. Data were normalized to the left ventricular developed pressure before PE addition. Figure 4 shows that the increase in developed pressure, caused by PE stimulation of WT hearts, was abolished by prazosin. Likewise, the fall of developed pressure caused by PE stimulation of ABKO hearts was also abolished by prazosin. These findings indicate that the effects of PE on the function of both WT and ABKO hearts were mediated by alpha 1-ARs.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3.   Responses to PE in ABKO and WT hearts were abolished by prazosin. Representative slow time-based tracings of LVP responses to PE recorded from (A) WT and (B) ABKO hearts. Hearts were preincubated with the alpha 1-AR antagonist prazosin (PRZ) in the presence of timolol. PE was added, as indicated by arrow.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 4.   ABKO and WT pressure responses were mediated by alpha 1-ARs. Pooled data showing the effects of the alpha 1-AR antagonist PRZ and the alpha 1D-AR antagonist 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspirol[4,5]decane-7,9-dione} dihydrochloride [BMY-7378 (BMY)] on LVDP responses to PE stimulation in WT and ABKO hearts. PRZ abolished changes in LVDP in response to PE stimulation in both WT and ABKO hearts. BMY-7378 did not reduce the response of WT hearts to PE stimulation; however, BMY-7378 abolished the pressure response to PE stimulation in ABKO hearts. Data were normalized to developed pressure before PE addition and shown as means ± SE; n, numbers in parentheses. All experiments performed in the presence of timolol. dagger P < 0.01 vs. control and vs. PE + PRZ.

Negative inotropic effect of PE in ABKO heart is mediated by alpha 1D-ARs. Because ABKO myocardium does not contain alpha 1A-ARs or alpha 1B-ARs, the residual alpha 1-AR response likely reflects alpha 1D-ARs. To more directly test for alpha 1D-AR function, we used the subtype-selective alpha 1D-AR antagonist BMY-7378. Figure 4 shows that specific antagonism of alpha 1D-ARs with BMY-7378 did not reduce the positive inotropic response of WT hearts to PE; indeed the response of WT to PE tended to be greater in the presence of BMY-7378 (although the difference did not reach statistical significance, P > 0.05). This suggests that for WT hearts, the positive inotropic response to PE was mediated by alpha 1A-ARs and/or alpha 1B-ARs, but not by alpha 1D-ARs.

In contrast, Fig. 4 also shows that the negative inotropic response of ABKO hearts to PE was completely abolished by BMY-7378. This suggests that for ABKO hearts the negative inotropic response to PE was mediated by alpha 1D-ARs.

PE reduced coronary flow. alpha 1-AR stimulation had no effect on ABKO trabeculae suggesting an absence of alpha 1-ARs on cardiomyocytes. Therefore the negative inotropy observed with PE stimulation in ABKO perfused hearts may involve alpha 1-ARs that are not located on cardiomyocytes. To investigate the possibility of alpha 1-ARs localized to the coronary vasculature, coronary flow was monitored during alpha 1-AR stimulation with PE in ABKO hearts. Figure 5A shows the time course of effects on coronary flow due to PE addition and washout for ABKO hearts. Data are shown as a percentage of the coronary flow before PE addition. PE caused a decline in coronary flow to 78 ± 3% of control that was sustained over the period of PE stimulation. The coronary flow recovered to control levels after PE removal (30-min washout). To test whether PE effects on coronary flow were mediated by alpha 1-ARs, we used prazosin. For all experiments, the effects of PE on coronary flow and the effects of prazosin are summarized in Fig. 5B. Under basal conditions, coronary flow was not different between WT and ABKO hearts (22.1 ± 1.2 vs. 23.3 ± 1.5 ml · min-1 · g-1, respectively). In WT hearts, PE caused a reduction of coronary flow that was statistically significant (85 ± 3% control, P < 0.01); this decrease in coronary flow was abolished with prazosin (99 ± 2% control). In ABKO hearts, PE decreased coronary flow to 86 ± 4% control (P < 0.01), a decrease similar to that observed in WT hearts. Furthermore, decreased coronary flow in ABKO hearts was also abolished by treatment with prazosin (98 ± 6% control). This demonstrates that the reductions of coronary flow in both WT and ABKO hearts were mediated by alpha 1-AR stimulation.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5.   alpha 1-AR stimulation reduced coronary flow (CF). A: time course of effect of PE-induced reduction of CF in ABKO hearts (n = 3). CF recovered on PE washout. B: PE caused reduced CF in both WT and ABKO hearts. The reduction observed in CF was abolished by preincubation with PRZ or BMY. Data were normalized to CF before PE addition and shown as means ± SE; n, numbers in parentheses. All experiments were performed in the presence of timolol. dagger P < 0.01 vs. control or vs. PE + PRZ.

To investigate whether the reductions in coronary flow with alpha 1-AR stimulation were mediated by the alpha 1D-AR subtype, we used BMY-7378. For both WT and ABKO hearts, BMY-7378 abolished alpha 1-AR-mediated decreases of coronary flow (100 ± 4% control vs. 99 ± 2% control, respectively). This demonstrates that alpha 1D-ARs mediate decreased coronary flow with PE stimulation.

Reduced coronary flow and reduced developed pressure. To investigate whether decreased coronary flow in ABKO hearts during PE stimulation could be responsible for the observed decrease in left ventricular developed pressure, in control experiments we reduced coronary flow experimentally. Figure 6 shows that in WT hearts there was a close to linear relationship between coronary flow and left ventricular developed pressure (fitted regression was statistically significant: R = 0.871, n = 5, P < 0.0001). Furthermore, Fig. 6 shows that this regression relation overlapped the data for coronary flow and developed pressure obtained with PE stimulation of ABKO hearts (open circle). This overlap is consistent with PE stimulation of ABKO hearts causing reduced coronary flow, which then leads to decreased developed pressure.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 6.   Decreased CF and reduced pressure development. LVDP is shown in response to experimentally reduced CF (solid symbols, n = 5 hearts). Line shows a linear regression fit to the data. Open symbol shows means ± SE for PE stimulation of ABKO hearts.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There were three major findings in this study. First, ABKO mouse hearts contain functional alpha 1-ARs that mediate reductions in coronary flow and pressure development. Second, these alpha 1-AR responses in ABKO hearts could be abolished by the subtype-specific antagonist BMY-7378, suggesting they were caused by alpha 1D-ARs. Third, in contrast to previous reports of negative inotropy with alpha 1-AR stimulation of mouse muscle strips, we found a marked positive inotropy with alpha 1-AR stimulation of the Langendorff-perfused WT mouse heart.

alpha 1D-AR in heart. In this study, we found that hearts lacking the alpha 1A- and alpha 1B-AR subtypes still responded to PE and that this response was abolished by prazosin and BMY-7278. This demonstrates that there are functional alpha 1-ARs in ABKO hearts and that these alpha 1-ARs are most likely alpha 1D-ARs. In contrast, trabeculae from ABKO hearts did not respond to PE. This demonstrates that for trabeculae from ABKO hearts there are no alpha 1-ARs on cardiomyocytes that influence contractility. Together these findings suggest that the decreased pressure of ABKO hearts with PE stimulation does not result from effects mediated by alpha 1-ARs on cardiac myocytes. Instead, the findings suggest that the response of ABKO hearts to PE was mediated by alpha 1D-ARs localized to cells within the vasculature. Consistent with this, PE caused a decreased coronary flow in ABKO hearts, suggesting that alpha 1D-ARs in ABKO hearts are localized to the coronary vasculature. These findings suggest that the decreased pressure with PE stimulation of ABKO hearts arises secondarily due to the reduced coronary flow. This suggestion is supported by two findings. First, for ABKO hearts, decreases of left ventricular pressure and coronary flow in response to PE were both blocked in parallel by BMY-7378. Second, we found that reducing coronary flow experimentally reproduced the decline in pressure observed with PE. These findings are consistent with but not proof of a causal relation between alpha 1D-AR-mediated decreases of both coronary flow and left ventricular pressure in ABKO hearts.

The expression of the alpha 1D-AR in the mouse heart has been uncertain. Receptor-binding studies of mouse heart homogenate detect no alpha 1D-AR protein (19), although the mRNA for this alpha 1-AR subtype is present (1). The present study provides functional evidence for alpha 1D-ARs in the heart. Because ABKO hearts lack alpha 1A-ARs and alpha 1B-ARs, we suggest that the alpha 1-AR subtype responsible for the residual alpha 1-AR response is the alpha 1D-AR. Furthermore, we suggest that alpha 1D-ARs are located in the coronary vasculature and cause vasoconstriction. These suggestions are consistent with the role of alpha 1D-ARs in regulating blood pressure (17) and aortic contraction (20, 21) in the mouse.

Similar to the mouse heart, previous receptor binding and functional studies of rat heart had reported very little or no alpha 1D-AR protein present (3, 23). However, more recent studies (15) using immunoreactive blotting show that alpha 1D-AR protein is present in the rat heart. We attempted to use the same commercially available alpha 1D-AR antibody (based on the human alpha 1D-AR) used by Shen et al. (15) (catalog no. SC-10721; lot no. A23, Santa Cruz Biotechnology) to immunohistochemically detect alpha 1D-ARs in Western blots and cryosections prepared from ABKO mouse hearts. However, the results proved inconsistent both within and between experiments suggesting insufficient specificity of antibody binding to mouse alpha 1D-ARs. Therefore, until it is possible to specifically detect the mouse alpha 1D-AR protein, the results presented here represent a pharmacological and physiological approach and provide evidence for a vascular localization for the alpha 1D-AR in the mouse heart.

alpha 1-AR stimulation in WT heart. The effect of alpha 1-AR stimulation on myocardial contractility has been controversial. Previous studies demonstrate that alpha 1-AR inotropic effects show considerable variation between different species and preparations. For example, in whole hearts and muscle strips from the rat, rabbit, guinea pig, hamster, and dog, alpha 1-AR stimulation causes positive inotropy (for a review, see Ref. 7). In contrast, recent studies of muscle strips and isolated myocytes from mouse heart found alpha 1-AR stimulation caused negative inotropy (8-10, 14, 16).

This is the first report of the effects of alpha 1-AR stimulation on pressure development in the isovolumic Langendorff mouse heart. In contrast to the negative inotropy with alpha 1-AR stimulation in mouse cardiomyocytes and muscle strips, in the whole heart we found that alpha 1-AR stimulation caused a significant positive inotropic response. It is unclear what causes the contrasting responses to alpha 1-AR stimulation for whole mouse heart versus mouse muscle strips. Differences in experimental conditions such as temperature and pacing rate may be important, or there may be elements of the whole heart that are not reflected in the trabeculae.

Consistent with our results, a recent study (4) found PE stimulation of the working mouse heart preparation caused a small but not statistically significant increase in the rate of pressure development.

Similar to the ABKO heart, for the WT mouse heart, alpha 1-AR stimulation reduced coronary flow. Therefore, the positive inotropic response to alpha 1-AR stimulation in the WT perfused heart may have been underestimated due to the concurrent decrease in coronary flow, which could have inhibited pressure development. Consistent with this, abolishing decreases of coronary flow with BMY-7378 tended to increase the inotropic response of WT hearts to PE.

In summary, our findings suggest that there are functional alpha 1-ARs in hearts lacking alpha 1A- and alpha 1B-ARs. We suggest that the alpha 1-AR response in hearts lacking alpha 1A- and alpha 1B-ARs arises from alpha 1D-ARs located in the coronary vasculature. We suggest that these alpha 1D-ARs act as coronary vasoconstrictors. Finally, we suggest that myocardial responses to alpha 1-AR stimulation depend critically on the experimental conditions.


    ACKNOWLEDGEMENTS

We thank Manoj Rodrigo, Marietta Paningbatan, and Gregory Simpson for expert technical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-56257 and P01 HL-68738 project 3 (to A. J. Baker), HL-54890 and HL-31113 (to P. C. Simpson), and postdoctoral fellowship HL-10422 (to D. T. McCloskey), and by a Grant-in-Aid from the American Heart Association, Western States Affiliate (to A. J. Baker). A. J. Baker is an Established Investigator of the American Heart Association.

Address for reprint requests and other correspondence: A. J. Baker, Univ. of California, San Francisco, Veterans Affairs Medical Center, Cardiology Div. 111C, 4150 Clement St., San Francisco, CA 94121 (E-mail: ajbaker{at}itsa.ucsf.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. Section 1734 solely to indicate this fact.

First published December 5, 2002;10.1152/ajpheart.00441.2002

Received 24 May 2002; accepted in final form 25 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alonso-Llamazares, A, Zamanillo D, Casanova E, Ovalle S, Calvo P, and Chinchetru MA. Molecular cloning of alpha 1D-adrenergic receptor and tissue distribution of three alpha 1-adrenergic receptor subtypes in mouse. J Neurochem 65: 2387-2392, 1995[ISI][Medline].

2.   Cavalli, A, Lattion AL, Hummler E, Nenniger M, Pedrazzini T, Aubert JF, Michel MC, Yang M, Lembo G, Vecchione C, Mostardini M, Schmidt A, Beermann F, and Cotecchia S. Decreased blood pressure response in mice deficient of the alpha 1b-adrenergic receptor. Proc Natl Acad Sci USA 94: 11589-11594, 1997[Abstract/Free Full Text].

3.   Deng, X, Chemtob S, and Varma D. Characterization of the alpha 1D-adrenoceptor subtype in rat myocardium, aorta and other tissues. Br J Pharmacol 119: 269-276, 1996[ISI].

4.   Grupp, IL, Lorenz JN, Walsh RA, Boivin GP, and Rindt H. Overexpression of alpha 1B-adrenergic receptor induces left ventricular dysfunction in the absence of hypertrophy. Am J Physiol Heart Circ Physiol 275: H1338-H1350, 1998[Abstract/Free Full Text].

5.   Hrometz, SL, Edelmann SE, McCune DF, Olges JR, Hadley RW, Perez DM, and Piascik MT. Expression of multiple alpha 1-adrenoceptors on vascular smooth muscle: correlation with the regulation of contraction. J Pharmacol Exp Ther 290: 452-463, 1999[Abstract/Free Full Text].

6.   Kenny, B, Chalmers D, Philpott P, and Naylor A. Characterization of an alpha 1D-adrenoceptor mediating the contractile response of rat aorta to noradrenaline. Br J Pharmacol 115: 981-986, 1995[ISI].

7.   Li, K, He H, Li C, Sirois P, and Rouleau JL. Myocardial alpha 1-adrenoceptor: inotropic effect and physiologic and pathologic implications. Life Sci 60: 1305-1318, 1997[ISI][Medline].

8.   McCloskey, DT, Rokosh DG, O'Connell TD, Keung EC, Simpson PC, and Baker AJ. alpha 1-adrenoceptor subtypes mediate negative inotropy in myocardium from alpha 1A/C-knockout and wild type mice. J Mol Cell Cardiol 34: 1007-1017, 2002[ISI][Medline].

9.   Montgomery, DE, Wolska BM, Pyle WG, Roman BB, Dowell JC, Buttrick PM, Koretsky AP, Del Nido P, and Solaro RJ. alpha -Adrenergic response and myofilament activity in mouse hearts lacking PKC phosphorylation sites on cardiac TnI. Am J Physiol Heart Circ Physiol 282: H2397-H2405, 2002[Abstract/Free Full Text].

10.   Nishimaru, K, Kobayashi M, Matsuda T, Tanaka Y, Tanaka H, and Shigenobu K. alpha -Adrenoceptor stimulation-mediated negative inotropism and enhanced Na+/Ca2+ exchange in mouse ventricle. Am J Physiol Heart Circ Physiol 280: H132-H141, 2001[Abstract/Free Full Text].

11.   Nishimaru, K, Sekine T, Tanaka Y, Tanaka H, and Shigenobu K. Temperature sensitive effects of alpha -adrenergic stimulation in mouse ventricular myocardia. Res Commun Mol Pathol Pharmacol 104: 173-180, 1999[ISI][Medline].

13.   Rokosh, DG, and Simpson PC. Knockout of the alpha 1A/C-adrenergic receptor subtype: the alpha 1A/C is expressed in resistance arteries and is required to maintain arterial blood pressure. Proc Natl Acad Sci USA 99: 9474-9479, 2002[Abstract/Free Full Text].

14.   Sakurai, K, Norota I, Tanaka H, Kubota I, Tomoika H, and Endoh M. Negative inotropic effects of angiotensin II, endothelin1 and phenylephrine in indo-1 loaded adult mouse ventricular myocytes. Life Sci 70: 1173-1184, 2002[ISI][Medline].

15.   Shen, H, Peri K, Deng X, Chemtob S, and Varma D. Distribution of alpha 1-adrenoceptor subtype proteins in different tissues of neonatal and adult rats. Can J Physiol Pharmacol 78: 237-243, 2000[ISI][Medline].

16.   Tanaka, H, Manita S, Matsuda T, Adachi M, and Shigenobu K. Sustained negative inotropism mediated by alpha -adrenoceptors in adult mouse myocardia: developmental conversion from positive response in the neonate. Br J Pharmacol 114: 673-677, 1995[ISI][Medline].

17.   Tanoue, A, Nasa Y, Koshimizu T, Shinoura H, Oshikawa S, Kawai T, Sunada S, Takeo S, and Tsujimoto G. The alpha 1D-adrenergic receptor directly regulates arterial blood pressure via vasoconstriction. J Clin Invest 109: 765-775, 2002[ISI][Medline].

18.   Testa, R, Destefani C, Guarneri L, Poggesi E, Simonazzi I, Taddei C, and Leonardi A. The alpha 1d-adrenoceptor subtype is involved in the noradrenaline-induced contractions of rat aorta. Life Sci 57: PL159-PL163, 1995[ISI][Medline].

19.   Wang, BH, Du XJ, Autelitano DJ, Milano CA, and Woodcock EA. Adverse effects of constitutively active alpha 1B-adrenergic receptors after pressure overload in mouse hearts. Am J Physiol Heart Circ Physiol 279: H1079-H1086, 2000[Abstract/Free Full Text].

20.   Yamamoto, Y, and Koike K. alpha 1-Adrenoceptor subtypes in the mouse mesenteric artery and abdominal aorta. Br J Pharmacol 134: 1045-1054, 2001[ISI].

21.   Yamamoto, Y, and Koike K. Characterization of alpha 1-adrenoceptor-mediated contraction in the mouse thoracic aorta. Eur J Pharmacol 424: 131-140, 2001[ISI][Medline].

22.   Yang, M, Reese J, Cotecchia S, and Michel MC. Murine alpha 1-adrenoceptor subtypes. I. Radioligand binding studies. J Pharmacol Exp Ther 286: 841-847, 1998[Abstract/Free Full Text].

23.   Yang, M, Verfurth F, Buscher R, and Michel M. Is alpha 1D-adrenoceptor protein detectable in rat tissues? Naunyn Schied Arch Pharmacol 355: 438-446, 1997[ISI][Medline].

24.   Zhou, L, and Vargas HM. Vascular alpha 1D-adrenoceptors have a role in the pressor response to phenylephrine in the pithed rat. Eur J Pharmacol 305: 173-176, 1996[ISI][Medline].

Am J Physiol Heart Circ Physiol 284(4):H1104-H1109


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. T. McCloskey, S. Turcato, G.-Y. Wang, L. Turnbull, B.-Q. Zhu, T. Bambino, A. P. Nguyen, D. H. Lovett, R. A. Nissenson, J. S. Karliner, et al.
Expression of a Gi-coupled receptor in the heart causes impaired Ca2+ handling, myofilament injury, and dilated cardiomyopathy
Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H205 - H212.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
G.-Y. Wang, D. T. McCloskey, S. Turcato, P. M. Swigart, P. C. Simpson, and A. J. Baker
Contrasting inotropic responses to {alpha}1-adrenergic receptor stimulation in left versus right ventricular myocardium
Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H2013 - H2017.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
L. Turnbull, H.-Z. Zhou, P. M. Swigart, S. Turcato, J. S. Karliner, B. R. Conklin, P. C. Simpson, and A. J. Baker
Sustained preconditioning induced by cardiac transgenesis with the tetracycline transactivator
Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H1103 - H1109.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
D. T. McCloskey, L. Turnbull, P. M. Swigart, A. C. Zambon, S. Turcato, S. Joho, W. Grossman, B. R. Conklin, P. C. Simpson, and A. J. Baker
Cardiac transgenesis with the tetracycline transactivator changes myocardial function and gene expression
Physiol Genomics, June 16, 2005; 22(1): 118 - 126.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
M. A. Uberti, C. Hague, H. Oller, K. P. Minneman, and R. A. Hall
Heterodimerization with {beta}2-Adrenergic Receptors Promotes Surface Expression and Functional Activity of {alpha}1D-Adrenergic Receptors
J. Pharmacol. Exp. Ther., April 1, 2005; 313(1): 16 - 23.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
Z.-S. Jiang, W. Srisakuldee, F. Soulet, G. Bouche, and E. Kardami
Non-angiogenic FGF-2 protects the ischemic heart from injury, in the presence or absence of reperfusion
Cardiovasc Res, April 1, 2004; 62(1): 154 - 166.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
284/4/H1104    most recent
00441.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (20)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Turnbull, L.
Right arrow Articles by Baker, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Turnbull, L.
Right arrow Articles by Baker, A. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online