Interaction between α₁- and α₂-adrenoreceptors contributes to enhanced constrictor effects of norepinephrine in mesenteric veins compared to arteries

Abstract

Mesenteric veins are more sensitive than arteries to the constrictor effects of sympathetic nerve stimulation and α-adrenoceptor agonists. We tested the hypothesis that α₁- and α₂-adrenoceptors interact to enhance adrenergic reactivity of mesenteric veins. We studied neurogenic and agonist-induced constrictions of mesenteric veins and arteries in vitro. Norepinephrine concentration-response curves were left-shifted in veins compared to arteries. UK 14,304 (0.01–1 μM, α₂-adrenoceptor receptor agonist) did not constrict arteries or veins but enhanced constriction and Ca²⁺ signals mediated by α₁-adrenoceptor stimulation in veins. Yohimbine (α₂-adrenoceptor receptor antagonist) and MK912 (α_2C-adrenoceptor receptor antagonist), but not α_2A- or α_2B- adrenoceptor antagonists, produced rightward shifts in norepinephrine concentration-response curves in veins. Pharmacological studies revealed that α_1D-adrenoceptors mediate venous constrictions. Norepinephrine responses in veins from α_2C-adrenoceptor knock-out (KO) mice were not different from wild type veins. Yohimbine inhibited norepinephrine constrictions in α_2C-adrenoceptor KO veins suggesting that there is upregulation of other α₂-adrenoceptors in α_2C-KO mice. These data indicate that α_1D- and α_2C-adrenoceptors interact in veins but not arteries. This interaction enhances venous adrenergic reactivity. Mesenteric vein-specific α₂-adrenoceptor linked Ca²⁺ and perhaps other signaling pathways account for enhanced venous adrenergic reactivity.

1. Introduction

Sympathetic nerves regulate blood pressure, in part, by regulating arterial and venous tone (Anderson, et al., 1989, Guyenet, 2006). Small arteries are the main determinants of total peripheral resistance while veins are capacitance vessels (Martin et al. 1998). Sympathetic nerves are the major regulator of venous tone and capacitance (Pang, 2001) which directly affect venous return to the heart and cardiac output (Guyton, 1955; Greenway and Lautt 1986). Because blood pressure is a product of total peripheral resistance and cardiac output, regulation of venous tone contributes to regulation of arterial pressure.

Veins have not been studied as extensively as arteries in relation to overall hemodynamics and most studies of veins have used large conduit veins (Gavin et al., 1997). The hemodynamic function of large conduit veins differs from that of splanchnic veins (including mesenteric veins) which are important due to their dense sympathetic nerve supply and high compliance (Pang, 2001). These characteristics impact the pathophysiology of hypertension where there is reduced venous capacitance (Ferrario, et al. 1970; Ricksten et al., 1981; London et al., 1985). When mesenteric capacitance is reduced, blood redistributes to the heart (Greenway and Lautt 1986) increasing cardiac output, which occurs in pre-hypertensive humans (Drukteinis and Roman, 2007). These findings point to the relevance of studies of the factors that regulate venous tone (Fink, 2009).

Mesenteric veins are more sensitive to adrenergic stimulation than mesenteric arteries (Hottenstein and Kreulen, 1987; Perez-Rivera et al., 2004, Luo et al., 2003). This might be due to artery-vein differences in α-adrenoceptor expression. α₂-Adrenoceptors potentiate constrictions mediated by α₁-adrenoceptors in mesenteric veins but not arteries (Perez-Rivera et al., 2007) confirming that α₂-adrenoceptors play a more prominent role constriction of veins than arteries (Flavahan et al., 1984, Ruffolo, 1986, Patel et al., 1981). A direct coupling between α₁- and α₂-adrenoceptors may mediate the functional interaction as occurs in the cauda epididymis (Haynes and Hill, 1996) and glial cells (Wilson and Minneman 1991). Interactions between α₁- and α₂ adrenoceptors are common in heterologous receptor expression systems (Reynen et al., 2000).

α-Adrenoceptors are G-protein coupled receptors and signaling via one receptor linked pathway may be affected by inputs from G-protein coupled receptors coupled to other signaling pathways (Milligan et al., 2006). In addition, α-adrenoceptors can form heterodimers (Hague et al., 2004; 2006 Uberti et al., 2005) that have functional properties different from monomeric receptors (Levac et al., 2002; Milligan et al., 2003). For example, α_1D-adrenoceptors, which mediate adrenergic constrictions in mesenteric veins (Daniel et al. 1997), can form heterodimers with other adrenoceptor subtypes (Hague et al., 2004, 2006; Uberti et al., 2005).

In the present study we tested the hypothesis that α_1D- and α₂-adrenoceptors interact in murine mesenteric veins but not arteries. We used a pharmacological approach to identify the α₂-adrenoceptor subtype that interacts with α_1D-adrenoceptors in mesenteric veins. This interaction contributes to the higher adrenergic reactivity of mesenteric veins compared to arteries.

2. Materials and methods

2.1 Animals

C57/Bl6 male mice (25–30g) were purchased from Charles River Breeding Laboratories (Portage, MI). In the animal care facility, mice were maintained according to the standards approved by the Institutional Animal Care and Use Committee at Michigan State University. Mice were housed individually in clear plastic cages with free access to standard chow (Harlan/Teklad 8640 Rodent Diet) and tap water.

Mice heterozygous for the neomycin-disrupted locus coding for the α_2C-adrenoceptor (http://jaxmice.jax.org/strain/002512.html) were purchased from Jackson Laboratories (Bar Harbor, ME). These mice have been described previously (Link et al., 1995). As the colony expanded, heterozygotes were bred and their offspring (9–12 weeks of age) were used. An established PCR protocol (http://jaxmice.jax.org/strain/003557.html) was followed to identify the genotype of individual pups.

2.2 In vitro preparation of mesenteric arteries and veins

Mice were euthanized with a lethal dose of pentobarbital (50 mg/kg, i.p.), and the small intestine with its associated mesentery was removed and placed in oxygenated (95% O₂, 5% CO₂) Krebs’ solution of the following composition (mM): 117, NaCl; 4.7, KCl; 2.5, CaCl₂; 1.2, MgCl₂; 1.2, NaH₂PO₄; 25 NaHCO₃ and 11, glucose. A piece of the intestine with associated vessels was removed and pinned flat in a silicone elastomer-lined (Sylgard; Dow Corning, Midland, MI) petri dish. A section of mesentery containing vessels close to the mesenteric border was cut out using fine scissors and forceps. The preparation was transferred to a smaller silicone elastomer-lined recording bath and blood vessels were isolated for study by carefully clearing away the surrounding fat. The recording bath containing the preparation was mounted on the stage of an inverted microscope (Olympus CK-2, Optical Analysis Corp. Nashua, NH) and superfused with warm (37°C) Krebs’ solution at a flow rate of 7 ml/min. All preparations were allowed a 20-min equilibration period during which the vessels relaxed to a stable resting diameter. The output of a black and white video camera (Hitachi model KP-111; Yokohama, Japan) attached to the microscope was fed to a frame grabber card (Picolo, Euresys Inc., TX, USA) mounted in a personal computer. The video images were analyzed real-time using Diamtrak Edge-tracking software (version 3.5, Diamtrak, Adelaide, Australia), which tracks the distance between the outer edges of blood vessel in the observation field. Changes in vessel diameter as small as 1 μm could be resolved.

2.3 Adrenoceptortor Antagonist studies

After tissue equilibration, adrenoceptor antagonists were applied for 20 minutes before norepinephrine concentration-response curves were generated. Norepinephrine, phenylephrine and UK 14,304 (5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline) were used as adrenoceptor agonists. Propranolol was used to block β-ardenoceptors. BRL44408 (2-[(4,5-Dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole), imiloxan and MK 912 ((2S, 12bS)1′,3′-dimethylspiro(1,3,4,5′,6,6′,7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinolizine)-2,4′-pyrimidin-2′-one) were used as α_2A- α_2B- and α_2C-adrenoceptor antagonists respectively. 5-methylurapadil (5-MU), L-765-314 (benzyl (2S)-4-(4-amino-6,7-dimethoxyquinazolin-2-yl)-2-(tert-butylcarbamoyl)piperazine-1-carboxylate) and BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride) were used as α_1A-α_1B- and α_1D-adrenoceptor antagonists respectively. pK_i and pK_B values of these antagonists for α-adrenoceptor subtypes are summarized in Table 1. Previous work has shown that there is good correlation between affinity estimates for these adrenoceptor antagonists obtained in radioligand binding (pK_i) and functional assays (pK_B)(Docherty, 1999). All drugs were purchased from Sigma-Aldrich (St. Louis, MO) and were added in known concentrations to the Krebs’ solution. A single adrenoceptor agonist concentration-response curve was obtained from each preparation. To avoid possible desensitization to norepinephrine, separate preparations were used to construct concentration response curves in the absence and presence of α-adrenoceptor antagonists. However, a control curve and a paired curve in the presence of an α-adrenoceptor antagonist were always obtained on the same day using tissues from the same mouse.

Table 1.

pK_i values determined in radioligand binding assays and pK_B values obtained in functional assays for adrenoceptor (AR) antagonists used to characterize norepinephrine-induced constrictions of mesenteric veins.

Antagonist	pKi			pKB
	α2A-AR	α2B-AR	α2C-AR	α2A-AR	α2B-AR	α2C-AR
BRL44408	8.2	6.2	6.8	7.8a	---	5.7b
Imiloxan	5.8	6.9	6.0	---	---	---
MK912	8.9	8.9	10.2	8.9c	8.9	10c 10.1d
	α1A-AR	α1B-AR	α1D-AR	α1A-AR	α1B-AR	α1D-AR
5-MU	8.8	6.8	7.3	9.2e, 8.7g	7.1g	7.9h
L 765-314	6.3	8.3	7.3	-----	7.3i	-----
BMY 7378	6.6	7.2	9.4	6.7f, 6.5g	7.7f	9.0i

pKi values obtained from Hussain and Marshall, 1997; Patane et al., 1998. pKB values were obtained where available as follows:

^aProcine ciliary artery (Wikberg-Matsson and Simonsen, 2001);

^bHuman saphenous vein (Gavin et al., 1997);

^cguinea pig and rabbit cerbral cortex (Trendelenburg et al., 1996);

^dPorcine pulmonary vein (Görnemann et al., 2007);

^eRat perfused kidney (Blue et al., 1995);

^fRat spleen (α_1B) and rat vas deferens (α_1A)(Burt et al., 1995);

^gRat portal vein (α_1A)(Marshall et al., 1996);

^hRat aorta (α_1D)(Hussain and Marshall, 1997);

ⁱRat tail artery (α_1B)(Jahnichen et al., 2004);

2.4 Simultaneous measurement of diameter and intracellular Ca²⁺ in mesenteric veins

Venous smooth muscle cell intracellular Ca²⁺ was measured in vitro. Mesenteric veins were cannulated with glass micropipettes and pressurized to 6 mm Hg with no luminal flow, as described previously (Burns et al., 2004). Maximal outside diameter was obtained in 0 mM Ca²⁺ solution.

Emitted fluorescence of the ratiometric Ca²⁺-sensitive dye Fura 2 (Molecular Probes, Invitrogen, Carlsbad, CA) was used as an index of [Ca²⁺]_in. Fura 2-AM was added to the bath for 60 min at room temperature to load venous smooth muscle cells in already cannulated blood vessels. After Fura 2-AM was washed out of the vessel chamber, 30 min were allowed for dye de-esterification at 37°C. Fluorescence was measured using a microscope-based photometry system (Photon Technologies International, Birmingham, NJ, USA) as previously described (Burns et al. 2004). Fura-2 was excited with alternating 340 and 380nm wavelength light with a DeltaRam X high-speed multi-wavelength illuminator, and Fura 2 emission was measured at 510nm with a D-104 photomultiplier at 0.8Hz. The illuminator and photometer were mounted on a Nikon TE 300 inverted microscope (Nikon Instruments Inc., Melville, NY, USA) equipped with x20 (N.A. 0.5) and x40 (N.A. 0.75) Plan Fluor long working-distance objectives. FeliX software (Photon Technologies International) was used to control the illuminator and photometer and for data acquisition. After subtracting the background and normalizing the 340/380 ratio representing concentration of Ca²⁺, changes in global Ca²⁺ upon drug application are presented as % change from baseline. Veins were washed between drug applications until baseline diameter was restored.

2.5 Data analysis

Artery and vein constrictor responses are expressed as percent constriction from the resting diameter. Half-maximal effective α-adrenoceptor agonist concentration (EC₅₀) and maximum response (E_max) were calculated from a least-squares fit ofindividual α-adrenoceptor agonist concentration-response curves using the following logistic function (Origin 7.0 (Origin-Lab Corp., Northampton, MA):

$$Y=\{[{\text{E}}_{min}-E_{max}]/[1+{(\text{x}/{\text{EC}}_{50})}^{\text{n}}]+{\text{E}}_{max}$$

where E_min is the minimum response and was constrained to zero and n is the slope factor. All data are expressed as mean ± S.E.M. Statistical differences between group means for E_max and EC₅₀ values were assessed by Student’s two-tailed unpaired t test. A P value < 0.05 was considered statistically significant.

3. Results

3.1 Norepinephrine is a more potent constrictor of veins compared to arteries

We found that mesenteric veins were more sensitive the constrictor effects of norepinephrine compared to arteries (Fig. 1). The EC₅₀ for norepinephrine-induced constriction of veins was 0.02 ± 0.004 μM and for arteries this value was 4.0 ± 0.6 μM (P < 0.05, n=6).

Fig. 1.

Mesenteric veins are more sensitive than mesenteric arteries to the constrictor effects of norepinephrine. The norepinephrine concentration response curve is shifted to the left in mesenteric veins (n=5) relative to arteries (n=6). Data are mean ± S.E.M.

As norepinephrine can act at α₁- and α₂- and β-adrenoceptors in blood vessels, we next tested if any of these receptors were responsible for increased venous reactivity to norepinerphrine. The β-adrenoceptor antagonist propranolol did not affect norepinephrine concentration response curve in veins (Fig. 2A). However, yohimbine (0.3 μM), an α₂-adrenoceptor antagonist, caused a 8-fold rightward shift in the norepinephrine concentration response curve in mesenteric veins (Fig. 2B); the control EC₅₀ was 0.01 ± 0.002 μM while in the presence of yohimbine this value was 0.08 ± 0.01 μM (P < 0.05, n=6). Yohimbine did not affect norepinephrine responses in arteries (Fig. 2C).

Fig. 2.

α₂-adrenoceptors contribute to norepinephrine induced norepinephrine induced constrictions of mesenteric arteries but not veins. (A) Propranolol, a β-adrenoceptor antagonist, did not alter the norepinephrine concentration response curve in mesenteric veins (n=4). Yohimbine causes a rightward shift in the norepinephrine concentration response curve in mesenteric veins (n=6) (A) but not arteries (n=6) (B). Data are mean ± S.E.M.

3.2 An α₂-adrenoceptor agonist, UK 14,304 increases Ca²⁺ in mesenteric veins

The data presented above suggest that α₂-adrenoceptors contribute indirectly to norepinephrine-induced constriction of mesenteric veins. In order to begin to probe the mechanism responsible for this facilitation, we simultaneously measured constriction and intracellular Ca²⁺ in veins when low concentrations of the α₁-adrenoceptor agonist, phenylephrine (0.1 μM) and UK 14,304 (0.1 μM) were applied individually and together. When applied individually, phenylephrine and UK 14,304 produced little or no constriction of veins (Fig. 3A). However, combined drug application caused a constriction that was significantly greater than the sum of the individual responses (P < 0.05, Fig. 3A). While UK14,304 did not constrict veins, it produced a 13 ± 4% increase in intracellular Ca²⁺ over baseline (Fig. 3B). Phenylephrine increased intracellular Ca²⁺ by 18 ± 1% over baseline. Combined application of phenylephrine and UK 14,304 produced an additive increase in intracellular Ca²⁺ (Fig. 3B).

Fig. 3.

UK 14,304 potentiated constrictions of mesenteric veins caused by phenylephrine. (A) Low concentrations of UK 14,304 and phenylephrine applied individually caused little venoconstriction. However when the same concentrations were applied simultaneously they caused a more than additive constriction. (B) Fura-2 based Ca²⁺ imaging in mesenteric veins revealed, that phenylephrine or UK14,304 applied alone produced modest increases in intracellular Ca²⁺; agonist co-application caused an additive response. *indicates significantly different from responses caused by individual agonist application (P < 0.05, n = 5). Data are mean ± S.E.M.

3.3 α-adrenoceptor subtypes in murine mesenteric veins

We next used a pharmacological approach to identify functional α₁- and α₂-adrenoceptor subtypes in venous smooth muscle. Phenylephrine concentration-response curves were obtained in the absence and presence of α_1A-, α_1B- and α_1D-adrenoceptor antagonists (Fig. 4; Table 1). Only the α_1D-adrenoceptor antagonist, BMY 7378, caused a rightward shift in the phenylephrine concentration response curve (Fig. 4C, Table 2).

Fig. 4.

α_1D-Adrenoceptors mediate phenylephrine-induced constrictions of mesenteric veins. Phenylephrine concentration response curves were unaffected by the α_1A-adrenoceptor antagonist 5-methylurapadil (5-MU)(A) or the α_1B-adrenoceptor antagonist, L-765-314 (B). The α_1D-adrenoceptor antagonist, BMY-7378 produced rightward shift in the phenylephrine concentration-response curve (C). Data are mean ± S.E.M.

Table 2.

Phenylephrine constriction of mesenteric veins in the absence or presence of antagonists for the α_1A-, α_1B- and the α_1D-adrenoceptors.

	Emax (%)	EC50 (− log M)
5-methylurapidil (α1A-adrenoceptor antagonist)
PE (control)	36.5 ± 2.8 (9)	6.5 ± 0.1 (9)
PE/5-MU (0.01 μM)	37.2 ± 3.4 (6)	6.5 ± 0.1 (6)
PE/5-MU (0.1 μM)	40.2 ± 1.4 (8)	6.2 ± 0.1 (8)
L-765,314 (α1B-adrenoceptor antagonist)
PE (control)	41.6 ± 3.0 (7)	6.5 ± 0.1 (7)
PE/L-765,314 (0.1 μM)	34.3 ± 4.6 (5)	6.4 ± 0.2 (5)
BMY-7378 (α1D-adrenoceptor antagonist)
PE (control)	37.4 ± 2.7 (11)	6.1 ± 0.1 (11)
PE/BMY-7378 (0.1 μM)	32.7 ± 3.7 (4)	5.8 ± 0.2a (4)
PE/BMY-7378 (0.3 μM)	35.9 ± 1.4 (8)	5.3 ± 0.1a (8)

Data are mean ± S.E.M. Numbers in parentheses are the number of animals from which data were obtained. E_max is the maximum constriction. EC₅₀ is the negative logarithm of the molar concentration of agonist producing half maximal constriction.

^aP < 0.05 vs control.

We next attempted to identify the α₂-adrenoceptor subtype that contributes to norepinephrine-induced constriction of veins using α-adrenoceptor subtype selective antagonists. BRL44408, an α_2A-adrenoceptor antagonist (Fig. 5A), and imiloxan, an α_2B-adrenoceptor antagonist (Fig. 5B), did not change norepinephrine concentration response curves. Only the α_2C-adrenoceptor antagonist, MK 912, caused a significant rightward shift in the norepinephrine concentration response curve (Fig. 5C). The control norepinephrine EC₅₀ was 0.007 ± 0.001 μM while in the presence of MK 912 (0.01 μM) this value was 0.04 ± 0.009 μM (P < 0.05, n=6).

Fig. 5.

α_2C-Adrenoceptors contribute to norepinephrine induced constrictions of mesenteric veins. The α_2A-adrenoceptor antagonist BRL44408 (A) and the α_2B-adrenoceptor antagonist imiloxan (B) did not change the norepinephrine concentration response curves in mesenteric veins. (C) MK912 (α_2C-adrenoceptor antagonist) caused a significant rightward shift in the norepinephrine concentration response curve (n=6). Data are mean ± S.E.M.

3.4 Studies in mesenteric veins from α_2C-adrenoceptor knockout (KO) mice

To test the hypothesis that α_2C-adrenoceptors contribute to norepinephrine-induced constrictions of mesenteric veins, we used tissues from α_2C-adrenoceptor KO mice. There were no differences in norepinephrine concentration-response curves in veins from wild type and α_2C-adrenoceptor KO mice (Fig. 6A). The E_max and EC₅₀ values in wild type mesenteric veins were 53 ± 3% and 21 ± 6 nM (n=10) respectively. The E_max and EC₅₀ values in α_2C-adrenoceptor KO mesenteric veins were 52 ± 2% and 29 ± 2 nM (n=14); these values were not different from those in wild type veins (P > 0.05). In addition, yohimbine (0.3 μM) produced a 6-fold rightward shift in the norepinephrine concentration-response curve in veins from α_2C-adrenoceptor KO mice (Fig. 6B); the control and yohimbine treated EC₅₀ values were 44 ± 13 (n=6) and 264 ± 95 (n=5) nM, respectively (P < 0.05). This was similar to the rightward shift caused by yohimbine in veins from wild type mice. Yohimbine did not change the E_max value (Fig. 6B). To test the possibility that other α₂-adrenoceptor subtypes compensated for the loss of a functional α_2C-adrenoceptor, we used veins from the KO mice and tested the effects α_2A- and α_2B-adrenoceptor antagonists on norepinephrine constrictions. The α_2B-adrenoceptor antagonist, imiloxan (1 μM) did not change the norepinephrine concentration response curve, while the α_2A-adrenoceptor antagonist, BRL44408 (0.3 μM), reduced the maximum norepinephrine-induced constrictions of veins from α_2C-adrenoceptor KO mice (Fig. 6C). The control E_max value was 47 ± 3% (n=5) while in the presence of BRL44408 this value was 34 ± 3% (n=8; P < 0.05). There were no differences in the norepinephrine EC₅₀ value in the absence (44 ± 11 nM) or presence (100 ± 41 nM; P > 0.05) of BRL44408.

Fig. 6.

Norepinephrine concentration response curves in mesenteric veins from α_2C-adrenoceptor KO mice did not differ from curves obtained in veins from wild type mice (A). Yohimbine caused a rightward shift in norepinephrine concentration response curve in vein from KO mice (B). In mesenteric veins from α_2C-adrenoceptor KO mice, the α_2A-adrenoceptor antagonist, BRL44408, but not the α_2B-adrenoceptor antagonist imiloxan inhibited constrictions caused by norepinephrine (C). Data are mean ± S.E.M.

4. Discussion

4.1 Indirect contribution of α₂-adrenoceptors to norepinephrine-induced constrictions of mesenteric veins

Mesenteric veins are more sensitive to the constrictor effects of sympathetic nerve stimulation and to norepinephrine than mesenteric arteries (Hottenstein and Kreulen, 1987; Smyth et al., 2000; Luo et al., 2003; Perez Rivera et al., 2004; Park et al., 2007). Therefore, we studied postjunctional mechanisms that could contribute to the enhanced adrenergic reactivity of mesenteric veins in vitro. Previous work suggested that α₂-adrenoceptors contribute to norepinephrine-induced constrictions of murine mesenteric veins but not arteries (Perez-Rivera et al., 2007). This was confirmed in the present study where we showed that yohimbine inhibits norepinephrine-mediated constriction of veins but not arteries. However, the α_2-adrenoceptor agonist, UK 14,304, did not constrict mesenteric veins. Although UK 14,304 did not directly constrict veins, it produced a more than additive constriction when co-applied with phenylephrine. This suggests that there is a synergistic interaction between constrictor pathways activated by α₁- and α₂-adrenoceptors in mesenteric veins. Facilitation of α₁-adrenoceptor coupled Ca²⁺ signaling by α₂-adrenoceptors is one mechanism by which this interaction could occur. However, combined application of UK 14,304 and phenylephrine produced only an additive increase in intracellular Ca²⁺ so other mechanisms must account for the synergistic interaction between α₁- and α₂-adrenoceptors in mesenteric veins. It is possible that there is a functional interaction between α₁- and α₂-adrenoceptors in MV as occurs in the cauda epididymis (Haynes and Hill, 1996) and primary glial cell cultures (Wilson and Minneman 1991). Functional interactions between α₁-adrenoceptors and α₂-adrenoceptors occur in heterologous receptor expression systems (Reynen et al., 2000). There may also be artery vs. vein differences in signaling mechanisms downstream from increases in intracellular Ca²⁺

4.2 α₁- and α₂-adrenoceptor subtypes in mesenteric veins

We used a pharmacological approach to identify the α₁-adrenoceptor subtype mediating α-adrenoceptor agonist induced constriction of mesenteric veins. We found that the α_1D-adrenoceptor antagonist, BMY 7378, inhibited constrictions caused by phenylephrine while α_1A- and α_1B-adrenoceptor antagonist did not affect these responses. We conclude that α_1D-adrenoceptors mediate constriction of murine mesenteric veins as shown previously for the canine mesenteric vein (Daniel et al. 1997).

We next identified the α₂-adrenoceptor subtype expressed by mesenteric veins. Previous work showed that the α_2C-adrenoceptor contributes to constrictions of the porcine pulmonary vein (Görnemann et al., 2007). Our pharmacological studies using α₂-adrenoceptor subtype selective antagonists indicated that α_2C-adrenoceptors contribute to norepinephrine-induced venoconstriction. BRL44408 has 25 and 100-fold higher affinity for the α_2A- over the α_2B- and α_2C-adrenoceptors respectively making this drug useful for identifying responses mediated by α_2A-adrenoceptors. However, antagonists for the α_2B-adrenoceptor (imiloxan) and the α_2C-adrenoceptor (MK912) are only 10–20-fold more selective over other subtypes. This modest selectivity limits their usefulness for receptor identification. Nonetheless, the rightward shift of the norepinephrine concentration-response curve caused by MK912 closely resembles the shift produced by yohimbine. Furthermore, BRL44408 and imiloxan did not change norepinephrine concentration response curves. Taken together, our data and those published previously (Gavin et al., 1997) indicate that α_2C-adrenoceptors contribute to norepinephrine-induced constriction of mesenteric veins.

Because of the limited selectivity of imiloxan and MK912, we used α_2C-adrenoceptor KO mice to verify the contribution of this receptor to norepinephrine-induced constriction of mesenteric veins. We predicted that veins from α_2C-adrenoceptor KO mice would be less sensitive to norepinephrine and that yohimbine would not change the norepinephrine concentration response curve. However, veins from the α_2C-adrenoceptor KO animals did not exhibit attenuated sensitivity to norepinephrine and yohimbine produced a rightward shift in the concentration response curve in veins from these animals. This finding could be attributed to physiological compensation in the KO mice, where other α₂-adrenoceptor subtypes assume the function of the deleted α_2C-adrenoceptor. In our experiments, the α_2A-adrenoceptor compensated for the loss of functional α_2C-adrenoceptors because in α_2C-adrenoceptor KO veins, but not in wild type veins, BRL44408 inhibited norepinephrine responses. There are many examples where gene KO leads to upregulation of other proteins. For example, chloroethylclonidine, an antagonist of the α_1B-adrenoceptor, was more effective in inhibiting norepinephrine-mediated constrictions of femoral arteries from α_1D-adrenoceptor KO mice compared to arteries from wild type mice. These authors concluded that there was an increased functional role for the α_1B-adrenoceptor in the α_1D-adrenoceptor KO mice (Zacharia et al., 2005). α_1D-Adrenoceptors are the predominant receptor mediating α-adrenoceptor agonist-induced constriction of the carotid artery in mice (Deighan et al., 2005). However, in α_1D-adrenoceptor KO mice, α_1A-adrenoceptors mediate agonist induced constrictions of the carotid artery, a result that is consistent with compensatory upregulation of α_1A-adrenoceptors (Deighan et al., 2005). Another example of physiological compensation in the adrenoceptor family occurs in β₁/β₂-adrenoceptor KO mice that exhibit reduced muscarinic receptor density in the heart (Rohrer et al., 1999). In addition, these mice exhibit an exaggerated hypotensive response to a β₃-adrenoceptor agonist suggesting upregulation of β₃ receptor function in these mice. These studies demonstrate that genetic deletion of one adrenoceptor subtype can lead to upregulation of other adrenoceptors.

4.3 Implications for understanding differential adrenergic reactivity in mesenteric arteries and veins

Our data indicate that differential α_1D- and α₂-adrenoceptor expression in mesenteric arteries and veins contributes to increased sensitivity of mesenteric veins to the constrictor effects of norepinephrine. In mesenteric veins, α₂-adrenoceptors link to a Ca²⁺-dependent signaling pathway which sensitizes mesenteric veins to the constrictor effects caused by α₁-adrenoceptor activation (Crowley et al., 2002).

Previous studies using tissues and cell lines have demonstrated a functional interaction between α₁- and α₂-adrenoceptors (Fukui et al., 2005). For example, direct coupling between endogenously expressed α₁-adrenoceptors and recombinant α_2A-adrenoceptors occurs in Chinese hamster lung cells (Reynen et al., 2000). In these studies, norepinephrine did not raise Ca²⁺ in cells only expressing native α₁-adrenoceptors but after transfection with α_2A-adrenoceptors, norepinephrine caused an increase in intracellular Ca²⁺ that was blocked by α₁- and α₂-adrenoceptor antagonists. This type of response also occurs in the cauda epidymis of guinea pig where α₂-adrenoceptor activation potentiates Ca²⁺ influx through L-type Ca²⁺ channels when α₁-adrenoceptors are activated simultaneously (Haynes and Hill 1996). α₂-Adrenoceptor activation alone did not cause Ca²⁺ entry. This mechanism is plausible as adrenergic responses of veins require Ca²⁺ influx (Aburto et al., 1993). Therefore, further functional studies in veins and venous smooth muscle cells are needed to identify pathways underlying the functional interaction between α_1D- and α₂-adrenoceptors.