Introduction

Top Abstract Introduction Methods Results Discussion References

Although sympathetic nerves are heterogeneously distributed along the vascular system (Bevan et al., 1980; Nilsson, 1984; Nilsson et al., 1986), exogenously supplied catecholamines constrict most peripheral blood vessels. In arterial vessels of the rat, except for the smaller arterioles (Faber, 1988; Ohyanagi et al., 1991), these adrenergic vasoconstrictor responses are primarily mediated by alpha-1 ARs (Vargas and Gorman, 1995). Arterial alpha-1 adrenergic vasoconstrictor responses can differ in many respects depending on the anatomical location of the vessel. For instance, in the poorly innervated rat aorta, contractile responses to alpha-1 adrenergic agonists resist inhibition by the alkylating agent phenoxybenzamine, removal of extracellular calcium, dihydropyridine calcium antagonists and pertussis toxin (Bognar and Enero, 1988; Trabizchi, 1994), whereas in the densely innervated rat mesenteric resistance arteries these interventions readily inhibit alpha-1 adrenergic responses (Boonen and De Mey, 1990a, b). Besides differences in receptor density, differences regarding the distribution of subtypes of alpha-1 AR may contribute to this heterogeneity. We hypothesized that this may be related to regional differences in periarterial innervation density.

Three subtypes of alpha-1 AR have been identified which share a comparably high affinity for prazosin and which are collectively referred to as alpha-1H AR (Bylund et al., 1994). The alpha-1A AR, alpha-1B AR and alpha-1D AR subtypes differ in their amino acid sequence, in their affinity for a growing list of synthetic ligands and with respect to irreversible blockade by certain alkylating agents (Bylund et al., 1994; Graham et al., 1996). They were reported to be heterogeneously distributed along the rat arterial tree (Piascik et al., 1994). The functional significance of this remains unclear because the affinity of alpha-1H AR subtypes for endogenous agonists does not differ (Ford et al., 1994) and because transfection studies indicated that each subtype can stimulate calcium influx and phospholipase activities (Esbenshade and Minneman, 1995). It was proposed that alpha-1H AR subtypes may differ primarily in terms of the mechanisms that govern their chronic control (Li et al., 1995; Rokosh et al., 1996). We hypothesized that these include long-term influences of sympathetic nerves. In this study we focused on the alpha-1A AR in rat arteries and evaluated whether the presence of this subtype is related to the presence of adrenergic nerves. To this end we determined the binding of [³H]prazosin and effects of the alpha-1A AR selective ligand (+)-niguldipine and the irreversible alpha-1B AR and alpha-1D AR antagonist CEC in eight types of rat artery that differ in terms of adrenergic nerve density. Also the presence of alpha-1A AR mRNA was evaluated in densely and poorly innervated rat arteries with the use of the RT-PCR technique. The arterial preparations included the thoracic aorta and some of its major side branches, three branching orders of mesenteric arteries and the mammary artery (a small muscular vessel with only sparse adrenergic innervation). To evaluate the influence of adrenergic nerves on the presence of the alpha-1A AR we repeated the experiments after chemical sympathectomy of the rats with 6-hydroxydopamine (Aprigliano and Hermsmeyer, 1977).

	Material and Methods

Top Abstract Introduction Methods Results Discussion References

Reagents. [7-methoxy-³H]Prazosin hydrochloride (79.2 Ci/mmol) was purchased from New England Nuclear ('s Hertogenbosch, the Netherlands). Phentolamine mesylate was obtained from Ciba Geigy (Basel, Switzerland), CEC from Research Biochemicals International (Natick, MA), and 6-hydroxydopamine hydrochloride, bovine serum albumin and calf thymus DNA from Sigma Chemical Co. (Saint Louis, MO). (+)-Niguldipine was a generous gift from Byk Gulden (Konstanz, FRG). All other reagents, which were of analytical grade, were obtained from Sigma or Merck (Darmstadt, FRG). All drug stock solutions were freshly prepared daily in bidistilled water except for (+)-niguldipine which was prepared at 0.01 M in dimethyl sulfoxide, diluted to 1 mM in ethanol and further diluted in water and for 6-hydroxydopamine which was dissolved in sterile saline at pH 4.7. Murine molony leukemia virus reverse transcriptase was obtained from Life Technologies (Gaithersburg, MD), random hexanucleotide primers from Promega (Leiden, the Netherlands) and dNTPs from Pharmacia (Uppsala, Sweden). AmpliTaq DNA polymerase was purchased from Perkin and Elmer (Norwalk, CT).

Animals. Twenty-week-old (n = 24) male inbred Wistar Kyoto rats (Central Animal Facility, Universiteit Maastricht, Maastricht, the Netherlands) were used. They were maintained on a 12-hr light/12-hr dark cycle and had free access to standard rat chow (Hope Farms, Woerden, The Netherlands) and drinking water. The experimental procedures were performed according to institutional guidelines and approved by the Ethics Committee for the Use of Experimental Animals (Universiteit Maastricht). Some of the rats (n = 12) were chemically sympathectomised with 6-hydroxydopamine (Aprigliano and Hermsmeyer, 1977). They received two i.p. injections of 50 mg/kg of the sympatholytic agent at 3-hr intervals. This was repeated 7 days later, and the animals were used 14 days after the first injection.

Tissue preparation. Rats were sacrificed by cervical dislocation and exsanguination. The thoracic aorta, carotid arteries, mammary arteries, superior mesenteric artery, renal arteries, femoral arteries and the mesentery were isolated and collected in KRB (composition in mM: NaCl, 118.5; KCl, 4.7; MgSO₄ · 7H₂O, 1.2; KH₂PO₄, 1.2; NaHCO₃, 25.0; CaCl₂, 2.5; glucose, 5.5) at room temperature and aerated with 5% CO₂ in oxygen. Adhering fat and connective tissue were removed by dissection from the large vessels, and segments approximately 3 mm in length were prepared. The adventitia was stripped off with fine watchmaker's forceps from the aortic preparations. The mesentery was pinned out in a dish coated with Sylgard (Dow Chemicals, Seneffe, Belgium) and filled with KRB. Fat and mesenteric veins were carefully removed and 5- to 10-mm-long segments were prepared from mesenteric small (first order side branch) and resistance-sized (third order side branch) artery. The segments of the eight types of artery were preincubated for 60 min in aerated KRB at 37°C. Part of the preparations were incubated for 30 min in 1 ml KRB containing 100 µM CEC and were then rinsed five times with 20 ml drug-free KRB. This treatment has been reported to result in irreversible blockade of alpha-1B AR and alpha-1D AR with little effect on alpha-1A AR (Michel et al., 1990).

Catecholamine content. Tissue NE content was measured as an indicator of the density of adrenergic nerves. Arterial segments were placed in 1 ml of 0.1 N HCl containing 3 g/l glutathione for 1 week, and the catecholamine content of the extract was determined by high-performance liquid chromatography and fluorescent detection (van der Hoorn et al., 1989). In preliminary experiments with mesenteric small arteries it was established that at least 48 hr were needed to extract all catecholamines and that these remained stable in the extract for at least 10 days. Unlike NE, the arterial contents of epinephrine and dopamine were below the detection limits. After extraction the preparations were solubilized in 1 ml of 1 N NaOH to determine their DNA content (Labarca and Paigen, 1980).

Ligand binding. Analysis of [³H]prazosin binding was performed essentially as described by Michel et al. (1993), but with use of intact arterial segments (Morel and Godfraind, 1989) instead of microsomes (Stassen et al., 1997). For saturation binding experiments, preparations were incubated for 60 min at 37°C in 250 µl of 50 mM Tris-HCl, 5 mM MgCl₂ (pH 7.4) (incubation buffer) containing 30 to 800 pM [³H]prazosin. Nonspecific binding was determined in parallel incubations in the continuous presence of phentolamine (25 µM). After incubation the arterial segments were gently blotted and rinsed during vortexing for 30 sec with 1 ml incubation buffer at 37°C to remove ligand trapped in the lumen of the arterial segments. They were then filtered over Whatman filters (GF/C) 5 times with 5 ml ice-cold incubation buffer. Arterial segments were recovered from the filters and solubilized in 200 µl of 1 N NaOH. Five milliliters of scintillation solution (Formula 989, Packard, Groningen, The Netherlands) was added to 100 µl of this preparation, and the radioactivity was determined in a liquid scintillation counter (Beckman, Fullerton, CA). To the remaining 100 µl, 900 µl H₂O was added and the protein content was determined as described by Bradford (1976), with bovine serum albumin as internal standard. Specific binding was calculated by subtracting nonspecific binding from total binding and for each individual experiment was analyzed in terms of density (B_max) and affinity (K_D) by Scatchard analysis (Graphpad Inplot, San Diego, CA). With respect to the Scatchard analysis, 5 to 7 data points were included for the thoracic aorta and 5 to 6 data points for mesenteric small arteries.

Determination of alpha-1A AR mRNA expression in mesenteric resistance arteries and thoracic aorta. Animals were anesthetized with ether and the thoracic aorta and the mesentery were rapidly removed and placed on ice-cold KRB. Connective tissue and adhering fat were removed from the aorta, whereas resistance-sized arteries were isolated from the mesentery. Both types of artery were then snap-frozen in liquid nitrogen and stored at 70°C until further processing. Total RNA was isolated with slight modifications of a method described previously (Auffray and Rougeon, 1980). Arterial segments (aorta, 10 mm; mesenteric resistance arteries, 20-30 mm) were homogenised in ice-cold 3 M LiCl/6 M urea solution (containing 0.1% sodium dodecyl sulfate and 0.3 volume of 2 M NaAc) by an Omni 2000 tissue homogenizer (Omni International, Waterbury, CT). After incubation for 16 hr at 4°C, RNA was pelleted by centrifugation (25 min, 14,000 × g, 4°C). The pellet was then dissolved in an ice-cold 4 M LiCl/8 M urea solution and centrifuged again (two times). After the third centrifugation, the pellet was dissolved in a 1% sodium dodecyl sulfate solution (500 µl containing 2.5 volumes of 2 M NaAc). After extracting the RNA solution with phenol (45 min), the RNA was precipitated for 16 hr with 3 M NaAc (0.1 volume) and ice-cold 100% ethanol (2 volumes). After centrifugation (14,000 × g, 25 min, 4°C) the pellet was washed with 70% ethanol, centrifuged again and the resulting pellet dissolved in 15 µl H₂O. The integrity of the RNA was checked by gel electrophoresis, whereas the total RNA content of each sample was determined by densitometric scanning of the 18 S band and comparing the signal with a RNA standard of 300 ng/µl. Between 0.75 and 1.5 µg of total RNA were isolated per mesenteric resistance artery segment, whereas aortic segments yielded 2 to 3 µg of total RNA.

Statistics. Findings are shown as mean ± S.E.M. Comparisons between types of vessel and analyses of the statistical significance of drug effects were performed by analysis of variance followed by Bonferroni's test for multiple comparisons (Wallenstein et al., 1980). P < .05 was considered to denote statistically significance.

	Results

Top Abstract Introduction Methods Results Discussion References

Intact rats. Table 1 summarizes NE content in eight types of artery of 20-week-old WKY rats. This index of adrenergic nerve density differed by 2 orders of magnitude between large elastic conduit arteries such as the thoracic aorta and carotid arteries (0.1 ng NE/µgDNA) and small and resistance-sized mesenteric artery side branches (10-25 ng NE/µgDNA). Intermediate innervation density (1-3 ng NE/µgDNA) was noted in muscular arteries such as the superior mesenteric, renal and femoral artery (table 1). The mammary artery, which is similar in size to the first order mesenteric artery side branches (lumen diameter, 400 µm), contained only 0.29 ± 0.02 ng NE/µgDNA. These marked regional differences in adrenergic nerve density were confirmed by glyoxylic acid-induced histofluorescence (Lindvall and Bjorklund, 1974) (not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Relation between arterial adrenergic innervation (expressed as ng NA/µg DNA) and the arterial alpha-1H AR density, indicated by the specific binding of [3H]prazosin (fmol/mg protein) in eight different arteries of the rat. No significant correlation was observed. Data are expressed as mean ± S.E.M.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Relation between arterial adrenergic innervation (expressed as ng NA/µg DNA) and the presence of specific binding sites for [3H]prazosin (expressed as percent of control) in eight different arteries of the rat after pretreatment with CEC (100 µM, A) or in the presence of (+)-niguldipine (30 nM, B). A significant positive correlation was found after pretreatment with CEC, whereas a significant negative correlation was observed in the presence of (+)-niguldipine. Data are expressed as mean ± S.E.M.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Typical experiments of amplification of cDNA of SM -actin (lane 2) and the alpha-1A AR (lane 3) by RT-PCR with total RNA from the thoracic aorta (left panel) or from mesenteric resistance arteries (right panel). Lane 1 shows the result of control experiments (RT-PCR without the addition of reverse transcriptase). Lane 4, 100-bp molecular weight marker. Representative 1.5% agarose gels stained with ethidium bromide are shown.

Sympathectomized rats. Chemical sympathectomy of 20-week-old WKY with 6-hydroxydopamine did not significantly modify the NE content in aorta, carotid and superior mesenteric arteries but reduced it by more than 75% in the other vessels (table 1). In both aorta and mesenteric artery side branches the density and affinity of [³H]prazosin binding was not significantly modified after chemical sympathectomy (table 2). Specific binding observed in the presence of 300 pM [³H]prazosin (normalized to the protein content of the preparations) was not significantly altered in the aorta and carotid artery but was reduced by approximately 30 to 50% in the femoral artery, superior mesenteric artery and mesenteric small arteries of sympathectomized rats (table 3). In large conduit and muscular arteries, the pharmacological properties of these binding sites were not modified. They were still blocked to a large extent by CEC and little affected by (+)-niguldipine (table 3). In small mesenteric artery side branches of sympathectomized rats, prazosin binding was now, in contrast to findings in intact rats, also profoundly reduced by CEC (intact, 40.4%; sympathectomized, 76.8%). (+)-Niguldipine, on the other hand, exhibited less effect than in small mesenteric vessels from the intact rats (intact, 76.8%; sympathectomized, 35.6%).

	Discussion

Top Abstract Introduction Methods Results Discussion References

We evaluated the relationship between perivascular adrenergic nerve density and the density and nature of prazosin binding sites in intact arterial segments. Only subnanomolar concentrations of prazosin were used to avoid interferences of alpha-2 AR (Bylund et al., 1994). Consequently, the analysis was limited to alpha-1 AR exhibiting a high affinity for the ligand (alpha-1H AR), although arterial responses to alpha-1 agonists may also involve subtypes of alpha-1 AR that exhibit a low affinity for prazosin (Muramatsu et al., 1990). These alpha-1L AR, which are poorly documented, were not addressed in the present study.

Because prazosin has been found not to modify NE release directly in isolated smooth muscle preparations, alpha-1H AR may be assumed to be located exclusively postjunctionally in rat arteries. Prolonged exposure of alpha-1 AR to agonists or antagonists, especially at 37°C, may result in internalization and sequestration of the receptors (Leeb-Lundberg et al., 1987; Cowlen and Toews, 1988). Because both prazosin and phentolamine have been claimed to be lipophilic (Cowlen and Toews, 1988), the specific binding sites which we measured can represent both membrane-bound and intracellular receptors and give an adequate indication of the actual cellular alpha-1 AR density. The dissociation constant of [³H]prazosin was similar in a large conduit vessel and small mesenteric arteries. It was similar to what has been reported for rat cerebral, cardiac and renal microsomes (Hanft and Gross, 1989; Hanft et al., 1989; Jackson et al., 1992) and for the cloned subtypes of alpha-1H AR (Michel and Insel, 1994). Alpha-1A AR, alpha-1B AR and alpha-1D AR have previously been found to exhibit identical affinity for prazosin (Hanft and Gross, 1989; Noguchi et al., 1993; Ford et al., 1994). This justifies the use of a single concentration of the ligand to compare alpha-1H AR density between tissues. Clearly a full saturation binding analysis is preferred, but the small size of some of the arteries that were included in the present study forced us to adopt this approach.

We confirmed that rat arteries differ considerably in adrenergic nerve density (Bevan et al., 1980; Nilsson, 1984; Nilsson et al., 1986). There does not seem to be a general pattern in this respect. Like the thoracic aorta, the carotid artery contained very little adrenergic neurotransmitter whereas other branches of the aorta such as the superior mesenteric, renal and femoral artery contained 20 times more NE. In the mesenteric arterial bed, abrupt changes in adrenergic nerve density were noted with branching. A marked increase in innervation density from the superior mesenteric artery to its first-order side branches is evident from both catecholamine content (table 1) and histochemistry (not shown). However, we also identified a small type of artery, the rat mammary artery, with very little innervation. Thus, size alone does not seem to determine perivascular innervation density. Differences in the production of nerve growth factor during development have been proposed to be responsible for regional differences in arterial innervation density in the adult (Falckh et al., 1992a, b).

Despite a marked difference in innervation density between rat thoracic aorta and mesenteric small arteries (150-fold), there was only a 2-fold difference in alpha-1 AR B_max values, with the densely innervated vessel exhibiting the highest receptor density. Also when comparing eight types of rat artery, the density of sites labeled by 300 pM prazosin differed much less than the periarterial nerve density. Moreover, differences in alpha-1H AR density between densely innervated arteries, such as the mesenteric artery side branches, were in the same order of magnitude as between densely and sparsely innervated arteries. Furthermore, no significant correlation could be found between alpha-1H AR density and arterial NE content. Alpha-1H AR consist of three subtypes, however (Hieble et al., 1995; Graham et al., 1996) and we used pharmacological tools to evaluate whether any of these could be closely related to the innervation. Because earlier observations indicated the presence of alpha-1A AR in densely innervated arterial systems such as the rat tail artery, renal and mesenteric vascular beds (Piascik et al., 1991; Blue et al., 1992; Kong et al., 1994; Williams and Clarke, 1995), we concentrated on this subtype. In aorta and carotid artery, an alpha-1A AR selective concentration of (+)-niguldipine did not significantly modify the specific [³H]prazosin binding, but the irreversible alpha-1B AR and alpha-1D AR antagonist CEC prevented all specific binding. In the densely innervated mesenteric artery branches (+)-niguldipine had a marked inhibitory effect, and a substantial fraction of the specific [³H]prazosin binding persisted after exposure to CEC. Intermediate findings were obtained in arteries with an innervation density ranging between that of the large elastic vessels and the densely innervated mesenteric arteries. Furthermore, a positive correlation was found between the alpha-1A AR density and adrenergic innervation. These findings indicate that the alpha-1A AR subtype predominates in arteries with a very high density of nerves, whereas in arteries with a low or intermediate innervation the alpha-1B AR and/or the alpha-1D AR seem to be the major subtype(s). We hypothesize that a high neurogenic input is required to induce and maintain the expression of the alpha-1A AR.

Comparison of eight types of rat artery suggests that in the rat arterial system alpha-1A AR predominates in vessels with a high innervation density. Presence of mRNA for this subtype in the mesenteric resistance-sized vessels and the virtual absence in the thoracic aorta supports the hypothesis that the expression of the alpha-1A AR subtype depends on a dense adrenergic innervation. Additional support was obtained in arteries of rats that had been chemically sympathectomized. After treatment with 6-hydroxydopamine, evaluation of the K_D in aorta and mesenteric small arteries did not reveal a significant modification of affinity for prazosin. No significant changes in binding of 300 pM [³H]prazosin were noted in aorta and carotid artery after sympathectomy, which is in line with these vessels being not or only sparsely innervated. In the other more densely innervated vessels binding of 300 pM [³H]prazosin was significantly reduced. After denervation, CEC blunted prazosin binding not only in large, poorly innervated arteries but also in mesenteric small arteries. Furthermore, (+)-niguldipine reduced binding in these vessels less markedly after sympathectomy than before. Altogether, these findings confirm that there is a positive relationship between the presence of adrenergic nerves and that of alpha-1A AR in rat arteries. They also suggest that this relationship is dynamic in nature because removal of the nerves was accompanied by a replacement of this receptor subtype by other alpha-1 AR subtype(s) (i.e., alpha-1B or alpha-1D AR). This is in line with observations by Hanft and Gross (1990); they demonstrated that reserpine treatment increased the proportion of alpha-1B ARs in rat cerebral cortex by decreasing the NE concentration in brain tissue. Desipramine, on the other hand, by inhibiting the neuronal uptake and thereby increasing the availability of the neurotransmitter, increased the 5-methylurapidil (an alpha-1A subtype selective tool) sensitive part of [³H]prazosin binding. This indicates that the tissue alpha-1 AR subtype composition is dynamic in nature and depends on the adrenergic input.

The nature of the signal that may link adrenergic nerves to the presence of alpha-1A AR on the arterial smooth muscle cells was not addressed in the present study. Besides NE itself, also sympathetic cotransmitters such as NPY and ATP (Burnstock, 1990) deserve future attention. Although sympathetic nerves may exert a trophic influence on the arterial wall during early development (Lee et al., 1987; Mangiarua and Lee, 1992), they help maintain vascular smooth muscle in a differentiated contractile state in the adult (Bevan and Tsuru, 1981; Branco et al., 1984). Similarly, the presence of sympathetic neurons delayed phenotypic modulation and growth in primary smooth muscle cultures of pig vas deferens and rabbit aorta and ear artery (Chamley et al., 1974, Chamley-Campbell et al., 1979). This has been suggested to be mediated by adenosine derived from neurogenically released ATP (Osswald, 1991). It may be worth addressing the possibility that nervous influences on postjunctional pharmacological properties may be rather nonselective in nature, i.e., not limited to alpha-1A AR, but part of a general influence of periarterial nerves on the phenotype of arterial smooth muscle cells. Future analysis of mechanisms and functional significance should ideally address expression, presence and efficacy of alpha-1 AR subtypes in the same tissues because possible discrepancies between expression, density, distribution and coupling of membrane receptors and differences between rat strains (Stassen et al., 1997).

In conclusion, we confirmed that rat arteries are innervated to a different extent by adrenergic nerves. Furthermore, the presence of the alpha-1A AR subtype seems to be positively related to the arterial innervation. Our findings indicate that this relationship is dynamic in nature and may involve a high neurogenic input because removal of the adrenergic nerves substantially changed the alpha-1 AR subtype composition in densely innervated arteries. This may be important in pathophysiological situations characterized by an increased sympathetic activity, such as hypertension (Lee et al., 1983; Mangiarua and Lee, 1990), or by depletion of sympathetic nerves as in congestive heart failure (Zelis et al., 1993).