General Pharmacology 34 (2000) 101–106
`
`Role of the postsynaptic ␣2-adrenergic receptor subtypes in
`catecholamine-induced vasoconstriction
`Irena Duka, Irene Gavras, Conrado Johns, Diane E. Handy, Haralambos Gavras*
`Hypertension and Atherosclerosis Section, Department of Medicine, Boston University School of Medicine, 715 Albany Street,
`Boston, MA, 02118, USA
`Received 28 January 2000; accepted 31 March 2000
`
`Abstract
`
`Catecholamines induce direct vasoconstriction mediated by postsynaptic ␣-adrenergic receptors (␣-ARs) of both the ␣1 and
`␣2 type. To evaluate the contribution of each ␣2-AR subtype (␣2A, ␣2B, and ␣2C) to this function, we used groups of genetically
`engineered mice deficient for the gene to each one of these subtypes and compared their blood pressure (BP) responses to their
`wild-type counterparts. Blood pressure responses to a bolus of norepinephrine (NE) were assessed before and after sequential
`blockade of ␣1-ARs with prazosin and ␣2-ARs with yohimbine. The first NE bolus elicited a brief 32 to 44 mm Hg BP rise (p ⬍
`0.001 from baseline) in all six groups. Prazosin decreased BP by 23 to 33 mm Hg in all groups, establishing a new lower baseline.
`Repeat NE at that point elicited lesser but still significant (p ⬍ 0.001) brief pressor responses between 32% and 45% of the
`previous BP rise in five of the six groups. Only the ␣2A-AR gene knockouts differed, responding instead with a 20-mm Hg fall
`in BP, a significant change from baseline (p ⬍ 0.001) and different from the pressor response of their wild-type counterparts
`(p ⬍ 0.001). The addition of yohimbine produced no further BP change in the five groups, but it did produce a small 7.5-mm
`Hg fall (p ⬍ 0.05) in the ␣2A-AR knockouts. Norepinephrine bolus during concurrent ␣1 and ␣2-AR blockade produced significant
`(p ⬍ 0.001) hypotensive responses in all subgroups, presumably attributable to unopposed stimulation of ␤2-vascular wall ARs.
`We conclude that the ␣2-AR-mediated vasoconstriction induced by catecholamines is attributable to the ␣2A-AR subtype because
`mice deficient in any one of the other subtypes retained the capacity for normal vasoconstrictive responses. However, the ␣1-ARs
`account for the major part (as much as 68%) of catecholamine-induced vasoconstriction. © 2000 Elsevier Science Inc. All rights
`reserved.
`
`Keywords: ␣2-Adrenergic receptor gene knockout mice; ␣1-Adrenergic blockade; ␣2-Adrenergic blockade; Norepinephrine
`
`1. Introduction
`␣2-Adrenergic receptors (␣2-ARs) are believed to play
`an important role in mediating the sympathetic nervous
`system (SNS) effects on blood pressure (BP). Three
`subtypes of ␣2-AR, designated as ␣2A, ␣2B, and ␣2C, were
`first suspected pharmacologically several years ago
`(Murphy and Bylund, 1988) and were subsequently con-
`firmed by molecular biology techniques (Bylund, 1992).
`They are activated to a variable extent by catechola-
`mines, exerting different effects depending of their lo-
`calization. Because of a lack of subtype-selective phar-
`macological compounds and radioligands, efforts to
`elucidate the exact function(s) of each ␣2-AR subtype
`have had limited success.
`
`Recently, the creation of genetically engineered mice
`deficient in each one of the ␣2-AR subtypes (Altman,
`et al., 1999; Link et al., 1996, 1995) became a valuable
`tool in dissecting the physiological effects of catechola-
`mines mediated by each subtype. Evidence from studies
`using such animals has shown that the ␣2A-AR subtype
`located in the central nervous system (CNS) and concen-
`trated in the brainstem (Tavares et al., 1996), which is
`known to be the center of cardiovascular control, is
`responsible for the tonic regulation of the SNS (Mac-
`Donald et al., 1997; MacMillan et al., 1996; Makaritsis
`et al., 1999a). The ␣2B-AR, thought to be the only one
`located in the vascular smooth muscle cells of the arte-
`rial wall, was proposed as having a role in the peripheral
`vasoconstrictor action (Altman et al., 1999; Link et al,
`1996; MacMillan et al., 1996). Recent data from our
`laboratory showed that the ␣2B-AR subtype is indeed
`* Corresponding author. Tel.: 617-638-4025; Fax: 617-638-4027.
`necessary in the hypertensive response to salt loading,
`E-mail address: hgavras@bu.edu
`0306-3623/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
`PII: S0306-3623(00)00051-3
`
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`I. Duka et al. / General Pharmacology 34 (2000) 101–106
`
`but no conclusion could be drawn from those studies
`about whether this role is central or peripheral (Mak-
`aritsisb et al., 1999b). No hemodynamic responses medi-
`ated by the ␣2C-AR are known so far (Link et al., 1996).
`The purpose of the current experiments was to evalu-
`ate the contribution of each ␣2-AR subtype in ␣2-AR-
`mediated peripheral vasoconstriction. To this aim, we
`studied the pressor effects of direct ␣2-AR stimulation
`by the administration of norepinephrine (NE) in ani-
`mals deficient for the ␣2A-AR (⫺/⫺), ␣2B-AR (⫹/⫺),
`or ␣2C-AR(⫺/⫺) gene compared with their wild-type
`controls. Because the pressor effects of NE that are
`mediated by the postsynaptic ␣1-AR are quantitatively
`much more important in peripheral vasoconstriction
`than those mediated by the postsynatic ␣2-AR (Ma-
`karitsis et al., 2000), we first blocked the ␣1-AR with
`prazosin, so that any changes in blood pressure elicited
`by NE should be attributed to activation of the vascular
`␣2-AR.
`
`2. Materials and methods
`
`2.1. Animals
`
`Six groups of male mice 7–11 weeks old and weighing
`22–31 g were used in this study: one group of each
`homozygous (⫺/⫺) knockout mice for the ␣2A-AR (n ⫽
`11), an ␣2C-AR (n⫽10) subtype, one group of heterozy-
`gous (⫹/⫺) ␣2B-AR subtype gene-deficient mice, and
`their their wild-type counterparts (n ⫽ 10 for each
`group). We used heterozygous ␣2B-AR gene-deficient
`mice because homozygous ␣2B (⫺/⫺) do not breed well
`to yield sufficient numbers. Heterozygous ␣2B-AR gene
`knockout mice have proved to be acceptable for such
`studies (Makaritsis et al., 1999a, 1999b, 2000), because
`they have been shown to have a very low level of expres-
`sion the ␣2B-AR protein (Link et al., 1996).
`Genotypes were determined by the polymerase chain
`reaction (PCR) from DNA isolated from the tail or
`spleen of the animals as described elsewhere (Makaritsis
`et al., 1999a, 1999b, 2000). In brief, to screen the ␣2A-AR
`line, MA.GF1, MAGB1, and PGK.2 primers were used
`to detect the intact ␣2A-AR gene (246 bp) or the inter-
`rupted ␣2A-AR gene (368 bp). To screen the ␣2B-AR lines,
`MB.GF2, MB.GB2, and PKG0.1 primers were used to
`detect
`the intact (365-bp) or interrupted (750-bp)
`␣2B-AR gene. Three others sets of primers (MC.GF1,
`MC.GB1, and PGK0.3) were used to detect the intact
`(377-bp) or interrupted (540-bp) ␣2C-AR gene. The pres-
`ence of the PGK.neoBpa insert was confirmed with the
`use of neo.F1 and neo.B3 primers to produce a 548-bp
`band by PCR. Each 25 ␮l PCR contained 0.2 ␮mol/l
`each primer, 0.2 mmol/l each dNTP, 2 mmol/l Mg2⫹, 10
`mmol/l Tris-HCl, pH 8.3, 50 mmol/l KCl, and 0.025
`U AmpliTaq Gold (Perkin Elmer). After incubation,
`samples were loaded on 3–4% Nusieve Agarose (FMC)
`gel and bands were separated.
`
`All animals were kept under a 12-h light/dark cycle
`in the animal facility of our institution and given free
`access to food (Purina Certified Rodent Chow, 5002)
`and distilled water. All experiments were conducted in
`accordance with guidelines for the Care and Use of
`Animals approved by the Boston University Medical
`Center.
`
`2.2. Surgical procedure
`
`Surgery was performed under anesthesia with intra-
`peritoneal sodium pentobarbital (50 mg/kg). A modified
`polyethylene catheter was introduced in the right iliac
`artery for BP recording, and a silastic tubing was placed
`in the right iliac vein for drug administration, as de-
`scribed elsewhere (Johns et al., 1996). After surgery,
`the animals were returned to their cages and allowed
`an overnight recovery period.
`
`2.3. Experimental protocol
`
`On the day after catheterization, the arterial line
`was connected to a BP transducer, and mean BP was
`recorded with a computerized data-acquisition system
`(Power Lab/400, AD Instruments Pty Ltd, Caste Hill,
`Australia). The venous line was connected to a Harvard
`infusion pump (Harvard Aparatus, Holliston, MA) for
`drug infusion. The baseline BP was recorded for at least
`30 min or until it became stable. At this point, a 100-␮l
`bolus of 1.2 ␮g/kg NE was injected, and BP changes
`were recorded. After BP had returned to baseline, a
`100-␮l bolus of prazosin (1.5 mg/kg) was injected, fol-
`lowed by a 1.5 mg/kg infusion. In previous experiments,
`we found this dose to completely block the response of
`␣1-AR to selective agonists (although it may have a
`weak affinity to ␣2-AR as well).
`When a new baseline was established for at least 30
`min, NE (1.2 ␮g/kg) was injected again, and BP changes
`were recorded. Then a 100-␮l bolus of the nonselective
`␣2-blocker yohimbine (2 mg/kg) was injected, and a 2-mg/
`kg infusion was started along with the continuing pra-
`zosin infusion to block all ␣2-AR as well. At least 30
`min later when BP was steady, NE was injected again,
`and ensuing changes in BP were recorded.
`
`2.4. Drugs
`
`The following drugs were used: norepinephrine (bitar-
`trate salt, Sigma Chemical Co., St. Louis, MO); prazosin
`hydrochloride (Sigma Chemical Co., St. Louis, MO);
`ascorbic acid (Sigma Chemical Co., St. Louis, MO); and
`yohimbine hydrochloride (RBI, Natick, MA). All drugs
`were dissolved in 0.9% saline with the exception of
`prazosin, which was dissolved in 5% dextrose. Ascorbic
`acid (1 mg/ml) was added to the NE solution to prevent
`oxidation. Bolus injections were given in a volume of
`100 ␮l, and the infusion rate was 100 ␮l/30 min.
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`103
`
`2.5. Statistical analysis
`
`All values are expressed as mean ⫾ SEM. Student’s
`t-tests for paired and unpaired data were used as appro-
`priate. The Mann-Whitney rank sum test was used for
`nonparametric data. Differences at p ⬍ 0.05 were con-
`sidered significant.
`
`3. Results
`
`The effects of sequential drug administration on BP
`in each group of ␣2-AR gene knockout mice and their
`wild-type counterparts are illustrated in Figure 1. Table
`1 gives in detail the numbers corresponding to these
`changes in BP after each manipulation.
`The first injection of NE elicited a significant (p ⬍
`0.001) hypertensive response in all animals. This hyper-
`tensive response to NE lasted only 1–2 min, after which
`BP returned to preinjection value. Then prazosin caused
`a significant fall in BP, which was similar in all groups
`of mice, and established a new baseline. Repeat adminis-
`tration of NE bolus produced a smaller, but still highly
`significant (p ⬍ 0.001), increase in BP in five of the six
`groups. In contrast with the other groups, the ␣2A-AR
`knockouts responded to this maneuver with a fall in
`BP, which was significant in regard to both their own
`baseline (p ⬍ 0.001) and to the response of their wild-
`type counterparts (p ⬍ 0.001; Table 1).
`The addition of a nonselective ␣2-AR blockade by
`yohimbine while the ␣1-AR blockade by prazosin con-
`tinued did not alter the BP in five of the six groups;
`however, in the ␣2A-AR knockouts, it further decreased
`the BP by an average 7.5 ⫾ 3.1 mm Hg (p ⫽ 0.034),
`thus establishing a new baseline in that group. At that
`point, another bolus of NE caused a significant fall in
`BP (p ⬍ 0.001) from the new baseline in all six groups,
`ranging from 15.2 mm Hg to 28 mm Hg and lasting
`for a couple of minutes, with no differences between
`knockouts and wild-type groups.
`
`4. Discussion
`
`Circulating catecholamines induce vasoconstriction
`mediated by postsynaptic ␣-AR (Aburto et al., 1995;
`Chen et al., 1988; Timmermans et al., 1987; Young et
`al., 1988). It has long been known that this is predomi-
`nantly an ␣1-AR function, with a lesser contribution
`from the ␣2-AR (Gavras et al., 1995). The purpose of
`the present experiments was to assess the contribution
`of each ␣2-AR receptor subtype to the direct vasocon-
`stricting effect elicited by bolus injections of norepi-
`nephrine. By using genetically altered mice deficient for
`the ␣2A-AR, ␣2B-AR, or ␣2C-AR genes and sequentially
`blocking the ␣1-AR and remaining ␣2-AR with succes-
`sive infusions of ␣1- and ␣2-blocking agents, we at-
`tempted to dissect the responses attributable to each
`
`subtype. The main finding of these experiments was
`that vasoconstriction mediated by direct activation of
`vascular ␣2-ARs is attributable to the ␣2A-AR subtype.
`Indeed, norepinephrine injected after ␣1-AR blockade
`with prazosin (which has a 1000-fold greater affinity
`for ␣1 than ␣2-AR) elicited in all, except the ␣2A-AR
`knockouts, a BP rise amounting to 32–45% of that pro-
`duced by the same injection before prazosin, indicating
`that 55–68% of this hypertensive response was due to
`the ␣1-AR. More importantly, the ␣2A-AR gene knock-
`outs under prazosin blockade responded to norepineph-
`rine with a transient hypotensive reaction. This hypoten-
`sive response was similar to the one elicited subsequently
`by norepinephrine in all subgroups after pretreatment
`with both yohimbine and prazosin, to ensure concurrent
`blockade of all ␣2-AR types.
`The data indicate that as much as 68% of adrener-
`gically induced vasoconstriction is mediated by periph-
`eral postsynaptic ␣1-AR, confirming previously existing
`knowledge (Gavras et al., 1995). The remainder would
`be due to stimulation of ␣2-AR located on the vascular
`wall. Because both the ␣2B-AR and the ␣2C-AR gene-
`deficient mice exhibited the same degree of hyperten-
`sive response as did their wild-type counterparts,
`whereas the ␣2A-AR gene knockouts were unable to
`raise their BP in response to NE, we had to conclude
`that the peripheral postsynaptic ␣2A-AR is the ␣2-AR
`subtype mediating vasoconstriction. Given the capacity
`of prazosin for partial ␣2B-AR blockade in addition to
`␣1-AR blockade, the fact that NE elicited comparable
`pressor responses in ␣2B-AR ⫹/⫹ and ␣2B-AR ⫹/⫺ mice
`would not be sufficient evidence that the ␣2B-AR does
`not contribute to the pressor response. However, the
`fact remains that all five groups (except the ␣2A ⫺/⫺)
`exhibited vasoconstriction in response to NE under pra-
`zosin infusion. Indeed, only after the addition of yohim-
`bine was all ␣2-AR-mediated vasoconstriction com-
`pletely abolished. These data indicate that the presence
`of the ␣2A-AR but not the ␣2B-AR subtype is necessary
`for this vasoconstrictive response to NE. This is consis-
`tent with our earlier studies, where only ␣2A-AR but not
`␣2B-AR mRNA could be detected on the arterial wall
`of rabbits (Handy et al., 1998). This interpretation, how-
`ever, is opposite that given by other authors (Link et
`al., 1996; MacDonald et al., 1997) of their data, for
`which they concluded that the ␣2B-AR has a role in the
`peripheral vasoconstrictive effect elicited by adrenergic
`agonists. Nevertheless, their data are not in conflict with
`ours, because the pressor action attributed to ␣2B-AR
`stimulation in their studies could in fact be attributable
`to CNS rather than peripheral ␣2B-ARs. Indeed, our
`subsequent series of studies on salt-induced hyperten-
`sion in mice deficient in each one of these ␣2-AR sub-
`types (Makaritsis et al., 1999a, 1999b, 2000) led us to
`conclude that the central presynaptic ␣2B-AR appears
`to mediate all systemic hypertensive reactions; whereas
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`Fig. 1. Mean blood pressure responses to sequential IV administration of ␣2-adrenergic agonists and antagonists in mice deficient for the gene
`of each ␣2-AR subtype, compared with their wild-type counterparts. Key: NE, norepinephrine; 䉬, wild type; 䊐, ␣2-AR subtype deficient. Values
`are expressed as mean ⫾ SEM, symbols denote significant changes in blood pressure in each group of mice versus their own baseline (** p ⬍
`0.001, * p ⬍ 0.05) and in knockout animals versus their wild-type controls (## p ⬍ 0.001).
`
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`105
`
`Table 1
`Effects of various manipulations on BP in six groups of animals
`⌬BP (mm Hg) in response to each manipulation
`NE after ␣1
`blockade**
`17.8 ⫾ 1.53
`⫺20.5 ⫾ 2#
`13.1 ⫾ 1.9
`12.9 ⫾ 1.9
`11.6 ⫾ 1.5
`13.2 ⫾ 1.6
`
`Group
`␣2A (⫹/⫹) (n ⫽ 10)
`␣2A (⫺/⫺) (n ⫽ 11)
`␣2B (⫹/⫹) (n ⫽ 10)
`␣2B (⫹/⫺) (n ⫽ 15)
`␣2C (⫹/⫹) (n ⫽ 10)
`␣2C (⫺/⫺) (n ⫽ 10)
`
`Mean BP
`(mm Hg)
`Baseline
`116.2 ⫾ 0.6
`114.6 ⫾ 1.4
`101.8 ⫾ 2.1
`103.1 ⫾ 1
`104.1 ⫾ 2
`100.7 ⫾ 2
`
`After NE**
`37.6 ⫾ 2.77
`44.2 ⫾ 5.5
`40.3 ⫾ 6.1
`36.4 ⫾ 3.9
`31.4 ⫾ 2.54
`34.4 ⫾ 2.7
`
`After ␣1 blockade**
`⫺30.1 ⫾ 2.02
`⫺33.6 ⫾ 2
`⫺24.4 ⫾ 2
`⫺23 ⫾ 2
`⫺25.3 ⫾ 2.2
`⫺22.8 ⫾ 2.3
`
`After ␣1 ⫹ ␣2
`blockade
`⫺3.9 ⫾ 2.75
`⫺7.5 ⫾ 2*
`2.1 ⫾ 2.7
`⫺1.0 ⫾ 1.2
`⫺1.8 ⫾ 1.6
`1.7 ⫾ 2.5
`
`NE after ␣1 ⫹ ␣2
`blockade**
`⫺28.6 ⫾ 3.18
`⫺20.7 ⫾ 2.8
`⫺16.1 ⫾ 2.3
`⫺21.1 ⫾ 2.5
`⫺15.2 ⫾ 3.23
`⫺17.4 ⫾ 3.5
`
`Note: Values are expressed as mean ⫾ SEM. Changes in BP are compared with the baseline before each drug administration. # p ⬍ 0.001
`between ␣2A ⫺ AR knockout mice and their wild-type counterparts. * p ⬍ 0.05 for ␣2A ⫺/⫺ mice versus their previous baseline. ** p ⬍ 0.001
`from baseline for all values in column.
`
`the central ␣2A-AR (which is the predominant presynap-
`tic ␣2-AR in the CNS) is indeed responsible for the
`hypotensive sympathoinhibitory effects attributed to
`presynaptic ␣2-AR stimulation, as concluded by these
`investigators (Altman et al., 1999; MacDonald et al.,
`1997; MacMillan et al., 1996) and by our own studies
`(Makaritsis et al., 1999a, 2000).
`It is possible that different postsynaptic ␣2-AR sub-
`types might mediate direct vasoconstriction to different
`extents in various vascular beds (Phillips, et al., 1997;
`Ping and Faber, 1993). It is notable in this respect that,
`in the study by MacMillan et al. (1996) in D79N mice,
`which carry a point mutation in the ␣2A-AR gene, when
`the site of injection was the femoral instead of the ca-
`rotid artery, the data could suggest that the ␣2A-AR
`should be considered to be responsible for direct vaso-
`constriction. Furthermore, some of these differences
`could be species related, inasmuch as various studies
`have used mice, rats, or rabbits.
`Why did norepinephrine produce a hypotensive re-
`sponse in the ␣2A-AR knockouts (after ␣1-AR blockade
`with prazosin) rather than no response at all? One expla-
`nation could be that, in the absence of any postsynaptic
`␣-AR capable of eliciting vasoconstriction, the nonse-
`lective adrenergic agonist NE would stimulate only the
`vasodilatory ␤2-adrenergic receptors on arterial smooth
`muscle. The same explanation could be given for the
`uniformly hypotensive responses elicited by NE in all
`six groups after combined blockade with prazosin and
`yohimbine. Yohimbine itself produced no change in BP
`baseline in the five groups, probably because its central
`presynaptic hypertensive effects in this case were bal-
`anced by the peripheral postsynaptic hypotensive ones.
`However, in the ␣2A-AR knockouts, which lack the ca-
`pacity for a central presynaptic hypertensive reaction
`to ␣2-AR antagonists (Altman et al., 1999), a small hypo-
`tensive response due to blockade of either central
`␣2B-AR or any residual postsynaptic ␣2-AR, although
`weak, became apparent.
`In summary, these studies, by using sequential phar-
`macologic blockade and stimulation of adreneric recep-
`
`tors in genetically altered mice deficient for each one
`of the ␣2-AR subtypes, produced evidence suggesting
`that the ␣2A-AR subtype is the one responsible for pe-
`ripheral ␣2-AR-mediated vasoconstictive responses.
`However, they also confirm that postsynaptic ␣-AR-
`mediated vasoconstriction is mainly (as much as 68%)
`a function of the ␣1-AR.
`
`Acknowledgments
`
`This work was supported by NIH Grant No. 1P50HL-
`55001. The authors are indebted to Dr. Brian Kobilka
`for providing the breeders for the mice used in these
`experiments.
`
`References
`
`Aburto, T., Jinsi, A., Zhu, Q., Deth, R.C., 1995. Involvement of protein
`kinase C activation in alpha2 adrenoceptor mediated contractions
`of rabbit saphenous vein. Eur J Pharmac 277, 35–44.
`Altman, J.D, Trendelenburg, A.U., MacMillan, L., Bernstein, D., Lim-
`bird, L., Starke, K., Kobilka, B.K., Hein, L., 1999. Abnormal regu-
`lation of the sympathetic nervous system in ␣2A adrenergic receptor
`knockout mice. Mol Pharmac 56, 154–161.
`Bylund, D.B., 1992. Subtypes of ␣1 and ␣2-adrenergic receptors. FA-
`SEB J 6, 832–839.
`Chen, D.G., Dai, X.Z., Buche, R.J., 1988. Postsynaptic adrenoceptor-
`mediated vasoconstriction in coronary and femoral vascular beds.
`Am J Physiol 258, H984–H992.
`Gavras, H., Handy, D.E., Gavras, I., 1995. Alpha-adrenergic receptors
`in hypertension. In: Laragh, J.H., Brenner, B.M. (Eds.), Hyperten-
`sion, pathophysiology, diagnosis and management, 2nd ed., Raven
`Press, New York, pp. 853–861.
`Handy, D.E., Johns, C., Bresnahan, M.R., Tavares, A., Bursztyn, M.,
`Gavras, H., 1998. Expression of ␣2-adrenergic receptors in normal
`and atherosclerotic rabbit aorta. Hypertension 32, 311–317.
`Johns, C., Gavras, I., Handy, D.E., Salomao, A., Gavras, H., 1996.
`Models of experimental hypertension in mice. Hypertension 28,
`1064–1069.
`Link, R.E., Desai, K., Hein, L., Stevens, M.E., Chruscinski, A., Bern-
`stein, D., Barsh, G.S., Kobilka, B.K., 1996. Cardiovascular regula-
`tion in mice lacking ␣2-adrenergic receptor subtypes b and c. Sci-
`ence 273, 803–805.
`Link, R.E., Stevens, M.S., Katalunga, M., Scheinin, M., Barsh, G.S.,
`
`Slayback Exhibit 1091, Page 5 of 6
`Slayback v. Eye Therapies - IPR2022-00142
`
`

`

`106
`
`I. Duka et al. / General Pharmacology 34 (2000) 101–106
`
`Kobilka, B.K., 1995. Targeted inactivation of the gene encoding
`the mouse ␣2C-adrenoceptor homology. Mol Pharmac 48, 48–55.
`MacDonald, E., Kobilka, B.K, Scheinin, M., 1997. Gene-targeting
`homing in on alpha2-adrenoceptor subtype function. Trends Phar-
`mac Sci 18, 211–219.
`MacMillan, L.B., Hein, L., Smith, M.S., Piascik, M.T., Limbird, L.E.,
`1996. Central hypotensive effect of the ␣2A-adrenergic receptor
`subtype. Science 273, 801–803.
`Makaritsis, K.P., Handy, D.E., Johns, C., Kobilka, B.K., Gavras, I.,
`Gavras, H., 1999a. Role of the ␣2B-adrenergic receptor in the devel-
`opment of salt-induced hypertension. Hypertension 33, 14–17.
`Makaritsis, K.P., Johns, C., Gavras, I., Altman, J.D., Handy, D.E.,
`Breshanan, M.R., Gavras, H., 1999b. Sympathoinhibitory function
`of the ␣2A-AR receptor subtype. Hypertension 34, 403–407.
`Makaritsis, K.P., Johns, C., Gavras, I., Gavras, H., 2000. Role of ␣2-
`adrenergic receptor subtypes in the acute hypertensive response
`to hypertonic saline infusion in anephric mice. Hypertension 35,
`609–613.
`
`Murphy, T.J., Bylund, D.B., 1988. Comparison of (H) clonidine ⫹ (3H)
`yohimbine binding: possible subtypes of ␣2-adrenergic receptors. J
`Pharmac Exp Ther 244, 571–578.
`Phillips, J.K., Vidovic, M., Hill, C.E., 1997. Variation in mRNA expres-
`sion of alpha-adrenergic, neurokinin and muscarinic receptors
`among four arteries of the rat. J Auton Nerv Syst 62(1–2), 85–93.
`Ping P., Faber, J.E., 1993. Characterization of alpha-adrenoceptor
`gene expression in arterial and venous smooth muscle. Am J Phys-
`iol 257, H1501–H1509.
`Tavares, A., Handy, D.E., Bogdanova, N.N., Rosene, D.L., Gavras,
`H., 1996. Localization of ␣2A- and ␣2B-adrenergic receptor subtypes
`in brain. Hypertension 27, 449–455.
`Timmermans, P.B.M.W.M., Chiu, A.T., Thoolen, M.M.M.C., 1987.
`Calcium handling in vasoconstriction to stimulation of alpha1 and
`alpha2 AR. Can J Physiol 65, 1649–1657.
`Young, M.A., Vatner, D.E., Knight, D.R., Graham, R.M., Homcy,
`C.J., Vatner, S.F., 1988. Alpha-adrenergic vasoconstriction and
`receptor subtypes in large coronary arteries of calves. Am J Physiol
`255, H1452–H1459.
`
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