A knockout approach indicates a minor vasoconstrictor role for vascular {alpha} 1B-adrenoceptors in mouse

Craig J. Daly, Clare Deighan, Ann McGee, Dawson Mennie, Zeeshan Ali, Melissa McBride and John C. McGrath

Institute of Biomedical and Life Sciences, Division of Neuroscience and Biomedical Systems, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pharmacological analysis alone has failed to clarify the role of the three {alpha}1-adrenoceptor subtypes in modulating vascular tone, due to a lack of sufficiently selective antagonists, particularly for the {alpha} 1B-adrenoceptor, and the complexity when three receptor subtypes are potentially activated by the same agonist. We adopted a combined genetics/ pharmacology strategy based on the {alpha}1B-adrenoceptor knockout (KO) mouse. The potency of three {alpha}1-adrenoceptor antagonists vs. phenylephrine was tested in aorta, carotid, mesenteric, and caudal isolated arteries from KO and wild-type (WT) mice. In the KO mouse the pharmacology became straightforward, showing {alpha}1D in two major conducting arteries (aorta and carotid) and {alpha}1A in two distributing arteries (mesenteric and caudal). By combining antagonist pharmacology and genetics, we provide a simplified analysis of {alpha}1-mediated vasoconstriction, demonstrating that {alpha}1D and {alpha}1A are the major subtypes involved in vasoconstriction, with a minor but definite contribution from {alpha}1B in every vessel.

{alpha}1-adrenoceptors; mouse aorta; arteries; functional genomics


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN THE LAST 5 YEARS GENETICALLY altered mice (over)-expressing or lacking genes of importance within the vascular system have become widely available, with a major focus on the role of the endothelium (7). However, this approach has not yet been used to clarify the role of individual {alpha}1-adrenoceptor subtypes which drive contraction in several diverse vascular beds. Compensatory mechanisms that may come into play as a result of receptor deletion or overexpression have also not been widely studied in native tissues.

The gene sequences for three distinct {alpha}1-adrenoceptor subtypes have been identified in several species; {alpha}1A, {alpha}1B, and {alpha} 1D (8). The mRNA for all three subtypes has been detected together in several blood vessels. However, the presence of mRNA for a particular subtype does not predict a functional role (19). So, which one(s) mediates vasoconstriction? Poor selectivity of antagonists limits pharmacological analysis, particularly since there are three possible receptors. This has blocked consensus on the subtype(s) responsible for vasoconstriction in blood vessels. For example, in the commonly studied rat aorta, pharmacological analysis alone has led different laboratories to conclude that at least three different functional {alpha}1-adrenoceptor subtypes may be involved in mediating vasoconstriction (1, 11, 22). The postjunctional adrenoceptor profile of more distal vessels (e.g., carotid, mesenteric, and caudal) in several species is similarly clouded and is unknown in the mouse.1

The general picture from pharmacological analysis of vasoconstrictor responses in rat is that larger vessels (e.g., aorta and carotid artery) appear to use {alpha}1D (11, 14), whereas many smaller resistance vessels (e.g., mesenteric and caudal artery) appear to use {alpha}1A (12, 21). However, the pharmacology is imprecise, and it is difficult to eliminate the possible presence of {alpha}1B-adrenoceptors in large and small arteries (2, 20; Table 1). The use of transgenic mouse models of gene deletion is a potential adjunct to pharmacological analysis that we have now tested on the {alpha} 1-adrenoceptor family in an attempt to clarify which receptor subtypes have the major and minor functional roles.


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Table 1. Comparison of the potency of 3 antagonists vs. the {alpha}1-adrenoceptor agonist phenylephrine in mouse caudal, mesenteric, and carotid artery

 
The {alpha}1B-adrenoceptor was the first of its type to be cloned (5) and knocked out (4). In the {alpha}1B-adrenoceptor knockout (KO) mouse, arterial pressor responses to phenylephrine were slightly reduced, suggesting a role for the {alpha}1B-adrenoceptor in the regulation of adrenergic responses, yet basal blood pressure was normal (4). In vivo, the influence of physiological reflexes complicates matters and encourages a simpler (in vitro) analysis of vessels isolated from genetically altered models.

We hypothesized that if all three {alpha}1-adrenoceptor subtypes play a (complex interacting) role in vasoconstriction, then the {alpha}1B-KO mouse should enable simplification of the analysis, since 1) comparison of the commonly used selective antagonists should provide a clearer picture in KO vessels, and 2) any compensatory mechanisms that provide for the long-term absence of one particular subtype may be more easily detectable. Investigating the effect that continued absence of one particular receptor subtype may have on the sensitivity of the remaining subtypes (and on antagonist affinities) is the basis of the experiments in this study.

Our approach was to use classic pharmacological methods to classify the postjunctional {alpha}1-adrenoceptor(s) responsible for mediating vasoconstriction in four isolated arteries from WT and KO mice. Aorta, carotid artery, mesenteric artery, and caudal artery were chosen for their different circulatory roles and as examples of predominant {alpha}1A or {alpha}1D involvement in other species (11, 12, 14, 21).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breeding pairs of wild-type (WT) and KO mice were kindly supplied by Professor Susanna Cotecchia (University of Lausanne) and bred in the University of Glasgow. Animals were maintained on a 12:12-h light/dark schedule at 22–25°C and 45–65% humidity and fed ad libitum on a standard rodent diet and tap water. Adult males (28–44 g; 3–4 mo old) were killed by an overdose of anesthetic (pentobarbitone sodium) followed by cervical dislocation.

The generation of {alpha}1B-adrenoceptor KO mice has been described previously (4). In particular, two out of seven chimerical mice, which were mated, gave rise to germ line transmission of the disrupted allele generating heterozygous mice. Heterozygous mice were mated to obtain the homozygous {alpha}1B-adrenoceptor +/+ (WT) and -/- (KO) progeny whose genetic background was therefore a 129/Sv/C57BL/6J mixture. For each genotype, mice from different litters were randomly intercrossed to obtain the WT and KO progeny used in this study. Since 1997, this makes at least 40 intercrosses (not backcrosses). The mice were never intercrossed with other strains or mated with those from the same litters. Since the mice were never backcrossed on a different genetic background, but only intercrossed within the same strain (WT or KO), their genetic background should not be too different from that of the animals described in the original report (4). The WT and KO mice are not strictly congenics since they were not backcrossed on an homogeneous genetic background. However, it can be assumed that a similar degree of genetic mixture has been transmitted throughout both the WT and KO progenies. The WT mice represent a true control since they were derived from the initial intercrosses between the heterozygous mice and bred in parallel with the KO progeny. It is important to highlight that either the WT or KO mice used in the experiments are from different litters. This should exclude the possibility that the phenotypic changes observed in the KO mice result from the fact that other genetic changes, different from the deletion of the {alpha}1B gene, are clustered within one litter. RT-PCR has been used to verify the lack of {alpha}1B-receptors in KO mice.

Vessel isolation.
The full length of aorta from the heart to the diaphragm was carefully removed from the animal and placed in a Petri dish for further dissection. Segments were supported in a 30-ml organ bath between two hooks, one of which was connected by cotton thread to a Grass model FTO3c isometric transducer. Vessel segments were placed under a resting tension of 1 g. This tension was found to be optimum from an earlier length-tension study using KCl as a constrictor agent.

Myograph mounted vessels.
Carotid, mesenteric (1st order branches), and caudal artery (midsection) segments were removed with the aid of a dissection microscope, freed of connective tissue in Krebs and mounted on a wire myograph. Preliminary studies (of mesenteric and caudal) employed the normalization technique of Mulvany and Halpern (1977) (16) to obtain vessel internal diameter and normalized resting tensions. After vessel isolation, all arteries were cleared of connective tissue, cut into 2-mm segments and myograph-mounted in an identical fashion with only the resting tensions being varied for each vessel type (carotid, 0.25 g; mesenteric, 0.17 g; caudal artery, 0.2 g).

All vessels.
All tissue baths contained heated (37°C) and gassed (95% O2-5% CO2) physiological salt solution (PSS) of composition (in mM) 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4· H2O, 1.2 KH2PO4, 24.9 NaHCO3, and 11.1 glucose.

Following a 30- to 40-min equilibration period, an initial concentration of 1 µM phenylephrine was administered to check tissue viability. After a further 30 min, the first of four consecutive concentration response curves (CRCs) was constructed (1 nM to 0.3 mM). After washout, the first of three concentrations of antagonist was added and left to equilibrate for 30 min. A second CRC was then constructed in the presence of the antagonist. The protocol was then repeated for two further increased concentrations of antagonist. Time control experiments in the absence of antagonists were also performed.

Responses are expressed as either "grams force" or "% of max. response." We choose not to express responses as force per cross-sectional area, since it is not possible in our system to accurately distinguish the media from adventitia. Given the possibility that KO vessels may possess altered (reduced) adventitial thickness, we feel this may introduce an unknown variable. All myograph mounted vascular segments are 2 mm in length as determined by the mounting apparatus. However, media thickness is unknown.

The log dose ratios were calculated using the EC50 in the presence and absence of a single concentration of antagonist. Affinity values were then calculated by the method of Arunlakshana and Schild (3) and expressed as pA2 values where the slope of the regression line indicates competitive (overlapping unity) or noncompetitive antagonism. Antagonist concentrations, for pA2 calculations, were within the range 1 nM to 1 µM for each antagonist.

Comparison of WT and KO CRCs were analyzed using two-way ANOVA for repeated measures. EC50 values were checked for normality using Graph Pad Prism (KS values are shown in the RESULTS) and compared using t-tests.

The drugs used were of analytical grade and are listed with their supplier. BMY7378, prazosin HCl, 5-methylurapidil (5MU), and chloroethylclonidine (CEC) were from Research Biochemical International (RBI); phenylephrine HCl and 5-hydroxytryptamine (5-HT) were from Sigma.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thoracic aorta.
KO aorta displayed similar sensitivity to phenylephrine (EC50 7.39, n = 19) compared with WT vessel segments (EC50 7.02, n = 38) (Fig. 1B). Maximum force generated by phenylephrine was also similar (WT 1.07 ± 0.05 g; KO 1.15 ± 0.15 g) (Fig. 1A). Comparison of EC50 values (normally distributed, KS = 0.18 and 0.22 for WT and KO, respectively) showed no significant difference (P > 0.1).



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Fig. 1. Comparison of the phenylephrine concentration response curves (CRCs) in thoracic aorta taken from wild-type (WT; solid symbols) and knockout (KO; open symbols) mice, expressed as either grams force (A) or percent of maximal response (B). Data are means ± SE of tissues from n = 19 and n = 38 animals in KO and WT, respectively.

 
Three {alpha}1-adrenoceptor antagonists (prazosin, 5-methylurapidil, and BMY7378) produced a concentration-dependent rightward shift of the phenylephrine CRC (data summarized in Table 1). The affinities in the control mice were similar to those published for the rat (Table 2). Compared with controls, in the KO higher affinities were found for prazosin (9.8 -> 10.6) and BMY7378 (8.8 -> 9.3) (Table 1). The {alpha}1A-selective antagonist (5MU) displayed no change in affinity.


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Table 2. Comparison of the potency of 4 antagonists vs. the {alpha}1-adrenoceptor agonist phenylephrine in rat and mouse thoracic aorta

 
The irreversible alkylating agent CEC (1–100 µM) caused a decrease in maximum response to phenylephrine in both WT and KO tissues (not shown). However, the results with CEC were highly variable and did not produce concentration-dependent shifts in the CRC. In this respect WT and KO were similar. On this basis we discontinued the use of this compound.

CRCs to 5-HT were not significantly different in either sensitivity or maximum responsiveness between WT (0.61 ± 0.03 g; EC50 6.94 ± 0.12) and KO (0.67 ± 0.04 g; EC50 7.02 ± 0.12) segments of thoracic aorta.

Carotid artery.
KO displayed a greater sensitivity (P < 0.001) to phenylephrine (EC50 6.8, n = 27) compared with WT (EC50 6.3, n = 28) (Fig. 2A). The maximum contraction to phenylephrine was not significantly different (P > 0.05) for KO vs. WT (0.34 ± 0.01 vs. 0.37 ± 0.01 g, respectively).



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Fig. 2. Phenylephrine CRCs in WT (solid symbols) and KO (open symbols) carotid (A), mesenteric (B), and caudal artery (C). Data are means ± SE of n = 28, 6, and 8 animals for carotid, mesenteric, and caudal arteries, respectively. Error bars for carotid artery are smaller than the symbol size. NIF, nifedipine.

 
Antagonist affinities in the KO were similar to those in aorta and are consistent with cloned {alpha}1D, notably high affinity for BMY7378 and low affinity for 5-MU. In the KO, the pA2 of prazosin increased (Table T1). BMY7378 showed no difference between KO and WT in the apparent pA2, but the slope of the Schild plot was extremely shallow (0.4) in the WT, suggesting heterogeneity, whereas in the KO it was 0.9, indicating competitive antagonism.

Mesenteric artery.
Comparison of WT vs. KO phenylephrine response curves revealed no change in maximum response (0.5 ± 0.07 vs. 0.6 ± 0.04 g, respectively) nor in sensitivity (EC50: WT 5.6, KO 5.4; Fig. 2B).

Compared with the aorta and carotid the antagonist potencies in the KO mesenteric artery showed several differences. The affinity of prazosin was lower and therefore more in line with affinity values at cloned {alpha}1-adrenoceptors; this has no consequence for subtype. The affinity of 5MU in KO mesenteric artery (9.7) was an order of magnitude higher than at aorta and carotid, consistent with a change from {alpha} 1D- to {alpha}1A-adrenoceptors. Comparing control with KO showed a rise in 5MU potency (8.9 -> 9.4) consistent with contamination of the control with {alpha}1B-adrenoceptors (Table 1). This would also account for the shallower CRC to phenylephrine in the control due to the imposition of slightly offset CRCs to the two receptors (Fig. 2B).

Tail artery.
Comparison of WT vs. KO phenylephrine response curves revealed no differences in EC50 (6.2 vs. 6.2) or maximum response (1.36 ± 0.1 vs. 1.37 ± 0.2 g). Strong rhythmic contractions often developed following initial exposure to agonist. This activity was abolished in the presence of nifedipine (1 µM). In the presence of nifedipine, WT and KO EC50 values were shifted to the right (to 5.9 and 5.8, respectively); maximum responses to phenylephrine were reduced under these conditions, and the maximum was smaller in KO (WT, 0.94 ± 0.06 g vs. KO, 0.77 ± 0.06 g) (Fig. 2C). Two-way ANOVA with Bonferroni post test failed to detect a significant difference between WT and KO curves in the presence of nifedipine.

Antagonist affinities were measured in the presence of nifedipine (1 µM). In both controls and KO, BMY7378 was completely without effect at concentrations up to 1 µM, and therefore a pA2 could not be determined (Table 1).

In the KO the estimated affinity for 5MU lay between the values for aorta/carotid and the mesenteric artery; on this basis alone, categorization would have been difficult. However, since the {alpha}1B is not present and BMY7378 was without effect, the simplest conclusion is that responses are mediated by {alpha}1A-receptors with a relatively low affinity for 5MU. In the control mice the affinity for 5MU was slightly lower than in the KO, which would be consistent with a small contamination from {alpha} 1B.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The {alpha}1B-KO mouse clarifies the involvement of {alpha} 1-adrenoceptor subtypes in vasoconstriction relative to what has been achieved in other species with pharmacology alone. This study is the first to classify the {alpha} 1-adrenoceptor profile of a group of blood vessels in vitro for either normal (WT) or genetically altered mice. This provides an essential basis for further study, since there is a very limited literature concerning the pharmacology of the mouse vasculature.

The mice appear physically normal (both externally and internally) in every respect. During dissection and mounting of all four vessel types, no differences or difficulties were encountered by the investigators. Preliminary studies, using confocal microscopy, are now underway to establish the cellular arrangement of WT and KO vessels.

The {alpha}1-adrenoceptor pharmacology in the control mouse was essentially similar to the rat and subject to the same caveats (Table 1), validating the mouse as a platform for general analysis of {alpha} 1-adrenoceptor mechanisms. The pharmacology in the {alpha}1B-KO allowed a relatively straightforward interpretation, showing that in the case of each of the four vessels, either {alpha}1A or {alpha}1D was mainly responsible for the response to phenylephrine and that the difference from controls indicated a minor but definite role for {alpha}1B-adrenoceptors.

Comparison amongst the anatomically different vessels showed a clear separation into {alpha}1D-adrenoceptor dominance in the conducting arteries of aorta and carotid and {alpha}1A-adrenoceptor dominance in the downstream "distributing" arteries of the tail and the first order mesenteric. However, the presence of {alpha} 1A in aorta remains difficult to discount due to the relatively high potency of 5MU. This clarifies the potential consensus that would have been possible in various species had it not been for the inability to exclude {alpha}1B-adrenoceptors. This inability stems from the lack of suitable selective {alpha}1B-antagonists. In some earlier studies CEC was believed to inactivate {alpha}1B-adrenoceptors selectively. However, the {alpha}1D/d-adrenoceptor has been shown to be capable of inactivation by CEC (18). Further to this, Hirasawa et al. (9) demonstrated that CEC is capable of inactivating {alpha}1A- and {alpha} 1B-adrenoceptor membrane preparations, whereas in whole cells it can inactivate only the {alpha}1B-adrenoceptor with any significance. Therefore, we cannot rely on the effects of CEC as an indicator of {alpha}1B-subtype function. We endeavored to employ it, but our observations reinforced the concept of its lack of selectivity.

Overall, the results with aorta and carotid indicate an increase in {alpha} 1D-adrenoceptor affinity following {alpha}1B-KO.

It would be inaccurate to define the mouse tail and first-order mesenteric arteries as "resistance" arteries. The size of the species causes these distributing arteries to be of equivalent size (~200 µM) to small arteries described as "resistance" in other species. However, when arteries of this size have been analyzed by pharmacological means in dog (2), rabbit (20), rat (12, 21), and human (10), the analysis has regularly concluded that either {alpha}1A or {alpha}1B are present but that conclusive definition is impossible. A recent study of vessels taken from mice overexpressing the {alpha}1B-adrenoceptor provided no support for a major vasoconstrictor role (24). However, as indicated previously, analysis of vessels expressing multiple subtypes is complex. It is uncertain what effect the presence of an overabundance of one subtype would have on the expected antagonist pharmacology. In this respect we believe a reductionist approach may be more informative initially.

Knocking out the minor receptor ({alpha}1B) simplified identification by antagonist analysis of the major receptor in each vessel. This reinforces the concept that {alpha}1B-adrenoceptors do not play a dominant part in the physiological role of {alpha}1-adrenoceptors in vasoconstriction and therefore no major compensation results, as suggested by Cavalli et al. (4). On the other hand, there were changes in the basic responses to the agonist which require interpretation.

This was straightforward in the case of the tail and mesenteric arteries, where the loss of the {alpha}1B-adrenoceptor resulted in the removal of a small component of contraction. In tail, this was manifest as a reduction in the maximum sustained contraction, suggesting an overlapping and summating concentration-related contraction from the two receptors. In mesenteric artery, the KO lost a component with a slightly offset concentration relationship causing the steepening of the CRC toward that which would be expected from a single receptor.

In carotid artery there was an increase in sensitivity to phenylephrine, whereas in aorta the same trend failed to reach statistical significance. There is no anticipated pharmacological reason for the increased sensitivity in carotid, so this must be seen as a phenotype of unknown origin related to the loss of the {alpha}1B-adrenoceptor. The lower potency of BMY7378 in the WT aorta compared with KO would be consistent with contamination by {alpha}1B. High affinity for prazosin has prompted the use of {alpha}1H (H = high) as a possible terminology (8). In KO, aorta and carotid, prazosin sensitivity appears to move toward the high-affinity state. BMY7378 is already very high in both WT (9.7) and KO (9.6) carotid. However, the increased affinity of the KO aorta for BMY7378 may indicate a high-affinity state of the {alpha}1D-subtype that may exist under certain conditions. The conclusion for aorta is, therefore: {alpha}1D with a small vasoconstrictor contribution from {alpha}1B.

In each of the four vessels tested, the presence (or not) of the {alpha} 1B-adrenoceptor changes the response to phenylephrine in a different way. Thus the response to agonists alone could not unravel the complicated consequences of the KO, but genetics plus antagonist pharmacology could. This emphasizes that analysis of agonist action alone in a receptor KO mouse sheds little light without additional antagonist information.

The strengthening of the pharmacological analysis gained from the KO aids understanding of analogous situations in human tissue. In human skeletal muscle resistance arteries (10) the {alpha}1-adrenoceptor pharmacology is very similar to mouse mesenteric and tail arteries in the present study (Table 1). In that case, as in control mouse vessels in the absence of the knowledge from the KO, categorical classification was not possible. Taking our current observations into account, analysis should be simpler in future.

Does this clarification of which subtype is effective in which vessel tell us anything about the physiological function of this family of receptors? The pattern suggests that {alpha}1D is the major candidate for mediator of smooth muscle contraction in large conducting "windkessel" arteries, where the function of contraction is to stiffen the vessel. On the other hand in distributing arteries (and by the similarity of the pharmacology also resistance arteries) it is {alpha}1A which mediates contraction, whose function is (directly) to alter the flow of blood and (indirectly) to regulate peripheral resistance and hence blood pressure. This would be in harmony with the localization of the genes for these subtypes on separate chromosomes. Although they might both utilize catecholamines as physiological activators, the physiological consequences are quite distinct, with one protecting the efficient function of the heart ({alpha}1D, compliance), the other ensuring adequate distribution of blood ({alpha}1A, resistance).

There may be a developmental aspect, since the aorta and carotid arise from the same embryonic origin but the smaller distributing vessels develop later along with the host organ. This may limit the expression of {alpha}1D to the major arteries

In all vessels {alpha}1B can apparently contribute to contraction but not to an extent that makes much impact (except to confuse the pharmacology). This may point either to a more general role for {alpha}1B, or the effect may arise from a tolerable level of cross talk within the signaling pathways, with the major function of {alpha}1B being something other than smooth muscle contraction. There is evidence from cell culture that {alpha}1B may be involved in the regulation of cell growth (23). This is further supported by degeneration of parts of the central nervous system in mice harboring a constitutively active {alpha}1B-adrenoceptor, suggesting an effect on the regulation of the cell life/death cycle (24). The cellular arrangement of blood vessels taken from {alpha}1B-KO mice should now be studied in detail with a view to determining the role of {alpha}1B-adrenoceptors in vascular remodeling.

Other factors must be taken into account to arrive at an understanding of the likely physiological function of the receptor subtypes. For example, {alpha} 2-adrenoceptors can readily be shown to mediate pressor responses to catecholamines in vivo (6), but the relative contributions of {alpha}1- and {alpha}2-adrenoceptors to pressor responses are radically affected by blood gases (17). Furthermore, it proved very difficult to demonstrate {alpha} 2-adrenoceptor-mediated contraction of isolated arteries without the presence of a synergistic factor(s) (19a). By analogy, {alpha}1-subtypes causing minor or absent responses in vitro could come into their own in vivo.

Finally, it will be necessary to demonstrate the physiological activation of the receptor subtypes. From the sensitivities to phenylephrine, which, at {alpha} 1-adrenoceptors, is normally of a potency similar to that of noradrenaline and adrenaline, it seems likely that plasma levels of catecholamines [rarely above 10-8 M in mouse (24)] could activate the {alpha}1D-adrenoceptors of aorta and carotid. However, the higher threshold for {alpha}1A-adrenoceptors in tail and mesenteric arteries might take them out of the reach of circulating levels. Thus the {alpha} 1A-adrenoceptors might require the high local concentrations produced only by release of the catecholamines from perivascular nerves. The latter concept is consistent with the hypothesis of Stassen et al. (21) that {alpha}1A-adrenoceptors are present in blood vessels only when adrenergic nerves are present and might add the further idea that {alpha}1A-adrenoceptors are activated physiologically only by nerves.

Overall, this study has justified the hypothesis that the use of transgenic models coupled with classic pharmacological techniques will lead to a clearer understanding of receptor mechanisms in vascular (and nonvascular) tissues, provided that the phenotypic disturbance is minor. The data also open up a number of avenues for further investigation that would have been too risky with pharmacology alone.


    ACKNOWLEDGMENTS
 
We are grateful to Professor S. Cotecchia for kindly providing the knockout and wild-type mice.

We are coordinating partners of the EC-funded project VASCAN 2000 (QLG1-1999-00084). C. Deighan holds a British Heart Foundation postgraduate scholarship.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: C. J. Daly, Wolfson Bldg. (room 448), Univ. of Glasgow, Glasgow G12 8QQ, Scotland (E-mail: c.daly{at}bio.gla.ac.uk).

10.1152/ physiolgenomics.00065.2001.

1 Within this manuscript we have attempted wherever possible to be guided by the International Union of Pharmacology Societies (IUPHAR) guidelines for the naming of receptors and their subtypes. Recommendations for Nomenclature of Receptors Guideline No. 6 states " Recombinant receptors without well-defined functional characteristics and without evidence for localisation in tissues should be referred to by lower case letters. When the recombinant receptor is shown to be of functional relevance in whole tissues and is fully characterised, upper case letters should then be used. When there is strong pharmacological evidence for a new receptor, but the amino acid sequence has not been defined, the receptor should be referred to in upper case italics." (From The IUPHAR Compendium of Receptor Characterisation and Classification, compiled by the IUPHAR Committee on Receptor Nomenclature and Drug Classification 1998). In the case of {alpha}1-adrenoreceptor subtypes, we have assumed that all three subtypes have been shown to be of functional relevance in some tissue and should be expressed in upper case. This is not necessarily the case in the references cited, some of which predate, and some ignore, the guidelines. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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