alpha 1-Adrenoceptor subtypes on rat afferent arterioles assessed by radioligand binding and RT-PCR

Max Salomonsson, Melinda Oker, Susan Kim, Hua Zhang, James E. Faber, and William J. Arendshorst

Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We utilized [3H]prazosin saturation and competition radioligand binding studies to characterize the expression of alpha 1-adrenoceptors in preglomerular vessels. mRNA for adrenoceptor subtypes was assayed using RT-PCR. The vessels were isolated using an iron oxide-sieving method. [3H]prazosin bound to a single class of binding sites (Kd 0.087 ± 0.012 nM, Bmax 326 ± 56 fmol/mg protein). Phentolamine displaced [3H]prazosin (0.2 nM) with a pKi of 8.37 ± 0.09. Competition with the selective alpha 1A-adrenoceptor antagonist 5-methylurapidil fit a two-site model (pKi 9.38 ± 0.21 and 7.04 ± 0.15); 59 ± 3% of the sites were high-affinity, and 41 ± 3% were low-affinity binding sites. Competition with the alpha 1D-adrenoceptor antagonist 8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) fit a one-site model with low affinity (pKi 6.83 ± 0.03). The relative contents of alpha 1A-, alpha 1B-, and alpha 1D-adrenoceptor mRNAs were 64 ± 5, 25 ± 5, and 11 ± 1%, respectively. Thus there was a very good correlation between mRNA and receptor binding for the subtypes. These data indicate a predominance of the alpha 1A-adrenoceptor subtype in rat renal resistance vessels, with smaller densities of alpha 1B- and alpha 1D-adrenoceptors.

renal circulation; vascular smooth muscle; reverse transcription-polymerase chain reaction; mRNA; renal nerve; catecholamine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE IMPORTANCE OF THE SYMPATHETIC nerves in the control of the cardiovascular system is well known. Catecholamines of nervous and humoral origin exert their effect by reversible activation of cell surface adrenoceptors on vascular smooth muscle cells which produces changes in cytosolic calcium concentration ([Ca2+]i). This in turn leads to binding of Ca2+ to calmodulin followed by a change in calmodulin structure, causing the activation of myosin light chain kinase that is responsible for the increased tone in the smooth muscle cells (38). The adrenoceptors are subdivided in several major classes, namely, the alpha 1-, alpha 2-, and beta -adrenoceptor subtypes (42). The renal resistance vessels are richly innervated by nerves of sympathetic origin (1). In the rat renal vasculature the alpha 1-adrenoceptors have been shown to mediate the action of sympathetic control of renal hemodynamics, glomerular ultrafiltration, and both short- and long-term regulation of extracellular fluid volume and arterial blood pressure (11). In addition, the release of renin from the granular cells at the distal end of the afferent arteriole may be partly controlled via renal alpha - as well as beta -adrenoceptors (11). A possible role of the alpha 1-adrenoceptors in the generation of hypertension is suggested by the observation that the responsiveness of these adrenoceptors in the kidney is augmented in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto rats (WKY) (36).

According to current criteria, the vascular alpha 1-adrenoceptors are pharmacologically further subdivided into three known subtypes, the alpha 1A-, alpha 1B-, and the alpha 1D-subtypes (5, 20, 26, 31), all of which have been cloned. Initially it was thought that activation of alpha 1A-adrenoceptors elicits entry of Ca2+ from the extracellular space via voltage-gated channels and that activation of alpha 1B-adrenoceptors causes mobilization of Ca2+ from intracellular inositol trisphosphate (IP3)-sensitive stores (21). However, this notion has been challenged by more recent studies. For example, activation of alpha 1A-adrenoceptors is known to trigger recruitment of Ca2+ from IP3-sensitive intracellular stores, at least in some vessels (4). Little is known about signaling pathways mediating effects of different alpha 1-adrenoceptors in renal resistance vessels.

Reports from various laboratories reveal heterogeneity with respect to relative alpha 1-adrenoceptor subtype expression in different peripheral vascular beds. For example, alpha 1B- and alpha 1D-receptors appear to be present in rat aorta (21, 25, 29). At least two alpha 1-adrenoceptors have been reported to mediate catecholamine-induced constriction of the main renal artery in vitro (21, 37). Regarding the physiological regulation of the renal hemodynamics in vivo, a major role for the alpha 1A-adrenoceptors has been suggested by most (2, 14, 33) but not all studies (6). In the rat interlobar artery, norepinephrine (NE) activation seems to be mediated by alpha 1A-adrenoceptors (8). With regard to a major resistance vessel, we recently found that the NE-induced [Ca2+]i response in isolated afferent arterioles and the constriction of the renal vasculature in vivo are attenuated by 8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378), an adrenoceptor antagonist selective for the alpha 1D-adrenoceptor subtype (18), and by 5-methylurapidil (5-MU), which preferentially blocks the alpha 1A-adrenoceptor subtype (5, 30, 32). These observations implicate mediation by the alpha 1A-adrenoceptor or a combination of alpha 1A- and alpha 1D-adrenoceptors in catecholamine-induced vasoconstriction in the renal microcirculation. Furthermore, we did not find any indications for a major role of alpha 1B-adrenoceptors based on the observation that there was no discernible long-term inhibition by chloroethylclonidine (CEC), an agent that preferentially alkylates the alpha 1B-adrenoceptors (21, 26, 27).

In the present study, we extended our investigation of alpha 1-adrenoceptors in renal resistance vessels to determine directly which subtypes are present in isolated rat afferent arterioles. Relative reverse transcription-polymerase chain reaction (RT-PCR) and radioligand ([3H]prazosin) binding were used to characterize the message and expression of the alpha 1-adrenoceptors. Pharmacological agents were used to identify alpha 1A- or the alpha 1D-adrenoceptor subtypes in competitive binding studies. Receptor density and mRNA abundance correlate well; both data sets indicate a predominance of the alpha 1A-adrenoceptor subtype in renal resistance arterioles with alpha 1B- and alpha 1D-adrenoceptor present in lesser abundance.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Radioligand Binding Studies

Isolation of preglomerular vessels. Preglomerular resistance arterioles (diameter < 50 µm) were isolated from kidneys of 250- to 300-g male Sprague-Dawley rats (Harlan, Indianapolis, IN) using the iron oxide-sieving technique previously described by Chatziantoniou and Arendshorst (7). Briefly, renal vascular tissue was obtained from two to three rats for each experiment. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg body wt), and the aorta was cannulated below the renal arteries through a midline abdominal incision. The aorta was ligated above the renal arteries, the left renal vein was cut, and the kidneys were slowly perfused with cold (4°C) PBS (in mM: 125 NaCl, 17 K2HPO4, 3 NaH2PO4, and 5 MgCl2, pH 7.4) solution until the renal venous effluent was free of blood. Thereafter, the kidneys were perfused with ice-cold magnetized iron oxide suspension (1% Fe2O3) for 10-15 s, excised, and placed in cold PBS. All subsequent steps of isolation were performed at 4°C. After decapsulation of the kidneys and dissection of the cortex from the medulla, the cortical tissue was minced with a razor blade and homogenized in 5 ml of PBS using a Polytron Tissuemizer. Preglomerular vessels, glomeruli, and the surrounding connective tissue were separated from the homogenate using a magnet. The iron oxide-loaded tissue was passed through needles of decreasing size (22-23 gauge) and sieved through a 125-µm mesh sieve to detach connective tissue, remove iron oxide from the large vessels, and to separate afferent arterioles from glomeruli. The vascular tissue was recovered from the top of the sieve and resuspended in PBS. A magnet was used to remove the microvessels and separate them from tubular fragments. The recovered vascular tissue consisted of interlobular arteries and afferent arterioles >95% free of glomerular and tubular tissue. The microvessels were incubated with collagenase (0.1 mg/ml PBS, type 1A, Sigma) for 30 min with constant shaking at 37°C. After enzymatic treatment, the preglomerular arterioles were removed from the digested tissue with the aid of a magnet and resuspended in PBS. This purified suspension was shaken vigorously (to help disperse iron oxide from the vessels) while being passed through a 27-gauge needle and sieved through a 125-µm mesh. The recovered supernatant was sonicated for two 30-s intervals to disperse any remaining iron oxide from the arterioles; a magnet was used to remove the free iron oxide. The purified solution was transferred to a 50-ml polyethylene centrifuge tube and centrifuged at 6,000 g for 30 min in a free-angle rotor. The final pellet of vascular tissue was resuspended in assay buffer and used for the binding and RT-PCR studies.

[3H]prazosin binding assays. Aliquots of microvessels (25 µg of protein) were incubated in a final volume of 250 µl, 10 mM Tris buffer (pH 7.4) with multiple [3H]prazosin concentrations up to 1 nM under continuous shaking for 60 min at room temperature. Protein concentrations, as well as binding conditions, and time for equilibrium were determined in preliminary studies (3). Bound ligand was separated from the free ligand using centrifugation in a sucrose gradient: 0.18 ml of the samples was gently layered over 0.2 ml of assay-binding buffer containing 20% sucrose in 0.5-ml polyethylene microcentrifuge tubes and were centrifuged at 5,000 g for 30 min in a free-angle rotor. Thereafter, the tubes were rapidly frozen in dry ice, the tips and tops of the tubes were separated, and the tips, containing the bound ligands, and the tops, containing the free ligands, were assayed in a liquid scintillation counter (Wallace Win Spectral 1414). Nonspecific binding, defined as binding in the presence of 10 µM phentolamine, was less than 10% of total binding. In competition studies, the alpha 1-adrenoceptor antagonists 5-MU and BMY-7378 were used to compete against 0.20 nM [3H]prazosin. 5-MU is considered to be selective for alpha 1A- and alpha 1D-adrenoceptors; BMY-7378 is a selective alpha 1D-adrenoceptor antagonist (18). Phentolamine was used as a nonselective alpha 1-adrenoceptor agonist. All assays were performed in triplicate.

Reverse Transcriptase-Polymerase Chain Reaction

Isolation of preglomerular smooth muscle cells and RNA extraction. The vessels were isolated from Sprague-Dawley (SD), WKY, and SHR rats (200-250 g) using the iron oxide-sieving method as described above. However, in this series of experiments, the vascular tissue was recovered from the top of the sieve and homogenized in the RNA STAT-60 solution. Thus no enzymatic collagenase treatment was used in these preparations. Smooth muscle RNA was extracted using the RNA STAT-60 method (Tel-Test, Friendswood, TX) according to the product manual and as described elsewhere (41). Total RNA from tissues was subjected to a second round of RNA extraction with the guanadinium method (41), followed by treatment with RQ1 RNase-free DNase (1 unit per 10 µg RNA) for 45 min at 37°C to prevent genomic DNA contamination. RNA concentration was determined spectrophotometrically at 260 nm. Purity was assessed according to absorbance ratio at 260/280 nm and was found to be >1.8. Quality was evaluated by electrophoresis on a 1.2% agarose gel after formaldehyde treatment.

Oligonucleotide primers. The following oligonucleotides were used (BRL; Life Technologies, Grand Island, NY): alpha 1A-sense, 5'-CGAGTCTACGTAGTAGCC-3'; alpha 1A-antisense, 5'-GTCTTGGCAGCTTTCTTC-3'; alpha 1B-sense, 5'-ATCGTGGCCAAGAGGACC-3'; alpha 1B-antisense, 5'-TTTGGCTGCTTTCTTTTC-3'; alpha 1D-sense, 5'-CGCGTGTACGTGGTCGCAC-3'; alpha 1D-antisense, 5'-CTTGGCAGCCTTTTTC-3'; cyclophilin sense, 5'-ATCCTGAAGCATACAGGTC-3'; and cyclophilin antisense, 5'-AGTGAGAACAGAGATTAC-3'; which amplify 204-, 201-, 218-, and 340-bp fragments of the alpha 1A, alpha 1B, alpha 1D, and cyclophilin genes, respectively. The single-tube method with rTth DNA polymerase (Promega, Madison, WI) was used for both reverse transcription and PCR amplification as described by the manufacturer. A quantity of 100 ng total RNA was used for alpha 1A, alpha 1B, and alpha 1D detection, and 20 ng total RNA was used for cyclophilin as an internal control. Reverse transcription of RNA was performed in a 20-µl volume consisting of 200 µM dNTP, 1 mM MnCl2, 10 mM Tris · HCl, pH 8.3, 90 mM KCl, 75 µM antisense primer, and 5 U of rTth DNA polymerase. The reactions were performed in a GeneAmp model 2400 thermocycler amplifier for 20 min at 60°C utilizing thin-walled MicroAmp reaction tubes (Perkin-Elmer) without mineral oil overlay. The reaction was stopped by placing the tubes on ice. PCR amplification was carried out in the same tube in a volume of 100 µl containing 1.5 mM MgCl2, 10 mM Tris · HCl, pH 8.3, 100 mM KCl, 0.05% (wt/vol) Tween 20, 0.75 mM EGTA, 5% (vol/vol) glycerol, and 15 µM sense primer. PCR was preceded with one step at 95°C for 60 s, followed by 35 cycles for alpha 1-adrenoceptors (30 cycles for cyclophilin) at 95°C for 60 s and 62°C for 30 s, and a final extension step of 8 min at 62°C. Each 20 µl of PCR product was subjected to electrophoresis in 1.5% agarose gel containing 0.5 µg/ml ethidium bromide for electrophoresis at 120 V. The ethidium bromide-stained products were then photographed and analyzed with densitometry. Adrenoceptor mRNA values were normalized to the RT-PCR product of cyclophilin, which was amplified for each RNA sample as an internal control. The absence of genomic DNA contamination was demonstrated by control samples without reverse transcriptase.

Data analysis. The LIGAND program was used to estimate maximum specific binding (Bmax) and dissociation constant (Kd) under equilibrium conditions. Competition binding data were analyzed with Graph-Pad PRISM for best fit to a one- or two-site model. P < 0.05 was considered statistically significant. pKi values were determined according to Cheng and Prusoff (9).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Radioligand Binding Data

Saturation binding of [3H]prazosin. [3H]prazosin bound to isolated renal preglomerular vessels in a concentration-dependent manner between 0.03 to 1 nM (Fig. 1). Nonspecific binding in the presence of excess phentolamine (10 µM) was less than 10% of total binding. Specific binding increased until the radioligand concentration reached a saturating value of ~0.25 nM. [3H]prazosin bound to a single class of binding sites, with a Kd of 0.087 ± 0.012 nM and a Bmax of 326 ± 56 fmol/mg protein within the concentration range examined (n = 6).


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Fig. 1.   A: representative experiment showing saturation of binding [3H]prazosin to preglomerular resistance vessels obtained from a 6-wk-old Sprague-Dawley rat. B: Scatchard plot of data from the same experiment.

Effects of competitive antagonists on [3H]prazosin binding. To examine subtypes of alpha 1-receptors, competition binding studies were performed initially using the nonselective alpha 1-adrenoceptor antagonist phentolamine. Subsequent studies were conducted using the alpha 1A- and alpha 1D-subtype-specific antagonist 5-MU and the alpha 1D-adrenoceptor subtype-selective antagonist BMY-7378, to displace the binding of 0.2 nM [3H]prazosin. As is shown in Fig. 2, all three compounds displaced [3H]prazosin in a dose-dependent manner. Phentolamine competed with [3H]prazosin binding in a manner consistent with a one-site model; the pKi was 8.37 ± 0.09 (n = 5). In contrast, 5-MU evidenced significant two-site competition, with pKi values of 9.38 ± 0.21 and 7.04 ± 0.15, respectively (n = 7). The high-affinity binding sites amounted to 59 ± 3% of the total, with 41 ± 3% representing the low-affinity site. On the other hand, the alpha 1D-receptor antagonist BMY-7378 displayed one-site competition and a lower potency [pKi 6.83 ± 0.03 (n = 6)], indicative of a small population of alpha 1D-receptors.


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Fig. 2.   Competitive inhibition of specific binding of [3H]prazosin by phentolamine, 8-(2-[4-(2- methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378), and 5-methylurapidil (5-MU) in rat preglomerular vessel preparations. Data are means ± SE of at least 5 separate experiments (at least 2 rats/experiment).

RT-PCR Data

Rat renal afferent arterioles expressed mRNA for all three known alpha 1-adrenoceptor subtypes. Since the densitometric data of the products from samples of the different rat strains (Sprague-Dawley, WKY, and SHR) were not markedly different, the data were pooled. The ethidium bromide-stained alpha 1-adrenoceptor mRNA products are shown in Fig. 3 for each individual experiment. The relative contents of mRNA for alpha 1A-, alpha 1B-, and alpha 1D-adrenoceptors were 64 ± 5, 25 ± 5, and 11 ± 1%, respectively (Fig. 3, bottom). These values are consistent with PCR data from rat whole kidney preparations reported previously (35).


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Fig. 3.   Top: electrophoresis gel of RT-PCR products. Lane 1 ("M"): BRL 100-bp ladder with 600-, 1,500-, and 2,072-bp bands intensified. Lanes 2-4 are controls [no reverse transcriptase (RT)] of depicted strain preparations [Sprague-Dawley (SD), Wistar-Kyoto (WKY), and spontaneously hypertensive rats (SHR)] demonstrating absence of genomic DNA. Lane 5 is positive control RNA samples for the different alpha 1-adrenoceptor subtypes: Rat1 fibroblasts stably transfected with either the alpha 1A or alpha 1B gene are indicated by alpha 1A and alpha 1B, respectively; alpha 1D-positive control is rat whole kidney RNA. Lanes 6-12 are independent RNA samples each pooled from 2-4 kidneys from 1-2 rats. RNA was loaded at 100 ng/lane for adrenoceptors and at 20 ng/lane for cyclophilin (CP). Bottom: average relative scanning optical density (OD) values for gene products of each alpha 1-adrenoceptor subtype for rats of SD, WKY, and SHR strains (n = 7). Each product was subjected to background subtraction, normalized to cyclophilin, and corrected for differences in product length.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Catecholamine-induced vasoconstriction of the renal microcirculation is mediated by alpha -adrenoceptor adrenoceptors. Several in vivo and in vitro studies have established that function of the alpha 1-adrenoceptor predominates over alpha 2-adrenoceptors in renal resistance vessels (10, 12, 34, 39). Despite the considerable amount of work that has been done regarding expression of alpha 1-adrenoceptor subtypes in different vascular beds, there is no clear general consensus (for reviews see Refs. 5 and 30). In the large renal artery, all three subtypes appear to be present (21, 29, 37). Our previous study of rat preglomerular alpha 1-adrenoceptor subtypes suggested that NE-induced vasoconstriction most likely is mediated by alpha 1A- or alpha 1D-adrenoceptors, but not alpha 1B-adrenoceptors (32). We observed that an NE-induced increase in isolated afferent arteriolar [Ca2+]i is resistant to CEC, which is well known to preferentially alkylate and inactivate this subtype (21, 26, 27). Furthermore, we found excellent agreement between both the NE-induced [Ca2+]i response and increases in renal vascular resistance in vivo. Both responses were attenuated by BMY-7378 and 5-MU, indicating contributions of both alpha 1A- and alpha 1D-adrenoceptors. From the results of our previous study, however, it was not possible to fully delineate the relative contribution of these two subtypes. In the present study, we therefore sought to define the relative abundance of alpha 1-adrenoceptor subtypes utilizing two different in vitro techniques, namely, radioligand binding and densitometric evaluation of the RT-PCR product of alpha 1-adrenoceptor mRNA extracted from isolated rat preglomerular vessels.

For renal resistance vessels, a dominant role for the alpha 1A-adrenoceptor subtype has been suggested in the regulation of vasomotor tone (2, 14, 33). Likewise, in the larger interlobar artery, which might contribute slightly to the regulation of renal hemodynamics, 5-MU inhibited NE-induced contraction with high potency, whereas the alpha 1D-adrenoceptor-selective antagonist BMY-7378 and CEC had minor attenuating effects (8). An apparent exception to this notion is a study in which receptor binding and RNase protection assay suggest a predominance of the alpha 1B-adrenoceptor in rat renal preglomerular microvessels (6). In the present study, the high sensitivity to prazosin in the saturation studies confirmed the presence of alpha 1-adrenoceptors. The Kd value for prazosin obtained in our studies agrees well with those reported for kidney cortex (16) and cloned alpha 1-adrenoceptors (23). Furthermore, the relatively high sensitivity to 5-MU and absence of CEC-induced alkylation in our previous study utilizing [Ca2+]i measurements and renal blood flow are consistent with these results (32).

Using [3H]prazosin binding in preparations from different locations in the rat kidney, Feng et al. (16) concluded that alpha 1A- and alpha 1B-adrenoceptors are equally expressed in renal cortex, largely based on the observation that CEC decreased Bmax roughly 50% from 186 to 75 fmol/mg (16). It should be noted that their cortical tissue consisted of both vascular and tubular structures. Relevant is the observation of Gopalakrishnan et al., that 50% of the [3H]prazosin binding to a pure proximal tubule preparation is sensitive to CEC (19). Thus it is reasonable to conclude that the large fraction of renal cortex sensitive to CEC is primarily due to the predominance of tubular receptors.

The pKi value of the high-affinity 5-MU site in the present study (9.38), representative of 59% of the total number of alpha 1-adrenoceptors, and the pKi for phentolamine (8.37) are in agreement with values previously reported for the cloned alpha 1A-adrenoceptor (17, 23, 22, 24). This indicates a predominance of the alpha 1A-adrenoceptor, which is in agreement with our RT-PCR results establishing that 64% of the total alpha 1-adrenoceptor mRNA codes for alpha 1A-adrenoceptors. This value is similar to the 67% observed for whole rat kidney mRNA (35). There is also a predominance of the alpha 1A-adrenoceptor mRNA in human renal artery and in branches thereof as assessed by RNase protection assay and in situ hybridization (28). The pKi value (7.04) of the low-affinity site, representative of 41% of the total receptor population, approximates average values for cloned alpha 1B- (6.87 ± 0.1) and alpha 1D-adrenoceptors (7.46 ± 0.2) (15). The pKi value of 6.83 for BMY-7378 in our afferent arterioles is considerably lower than values for cloned alpha 1D-adrenoceptors (8.44 ± 0.2) but in the same range as the average value for cloned alpha 1B-adrenoceptors (6.51 ± 0.2) (15). Taken together, these results suggest that alpha 1D-adrenoceptor protein is weakly expressed in isolated rat preglomerular vessels. This conclusion is reinforced by our PCR data which show that alpha 1D-adrenoceptor mRNA constitutes only 11% of the total alpha 1-adrenoceptor mRNA. The average pKi value of 6.28 ± 0.1 of BMY-7378 for the cloned alpha 1A-adrenoceptor also supports the idea of alpha 1A-adrenoceptor dominance in the afferent arterioles (15). Furthermore, our semi-quantitative RT-PCR results demonstrate that alpha 1B-adrenoceptor mRNA constitutes 25% of the total alpha 1-adrenoceptor mRNA. The functional role for the alpha 1B adrenoceptor in the renal vasculature is, however, uncertain. In this regard, we found in a previous study that pretreatment with CEC (50 µM) for 15 min at 28°C had no effect on the ability of NE to elicit an increase in [Ca2+]i in isolated preglomerular resistance vessels (32). If alpha 1B-adrenoceptors were present and functional, then we would expect at least 50% effect of alkylation of alpha 1B-adrenoceptors under the prevailing conditions (40). Also, since it has recently been suggested that the alpha 1D-adrenoceptor may be alkylated by CEC almost as rapidly as the alpha 1B-adrenoceptor (40), the absence of attenuation by CEC might indicate limited participation of alpha 1D-adrenoceptors (32). Other investigators have also reported only minor effects of CEC on the renal circulation (2, 14).

Our analysis of mRNA clearly shows that there is potential for synthesis of all known alpha 1-adrenoceptor subtypes in the renal vasculature. It might be that some receptors are expressed only during certain conditions. Support for this notion derives from the finding that alpha 1B-adrenoceptor expression is increased by chronic hypoxia (13). Therefore, a contribution of other alpha 1-adrenoceptor subtypes cannot be excluded even if it seems obvious from this study, as well as a majority of studies in this area, to conclude that the alpha 1A-adrenoceptors predominate in the renal microvasculature. It is possible in certain developmental stages, conditions, or diseases that other alpha 1-adrenoceptor subtypes may play a role in the control of renal microcirculation.

In summary, our study presents the results of [3H]prazosin binding and competition inhibition by agents known to possess different selectivity for different alpha 1-adrenoceptor subtypes (i.e., BMY-7378 and 5-MU) in renal preglomerular resistance vessels. In addition, we present results from an evaluation of the gene product expression for alpha 1-adrenoceptor subtypes using RT-PCR. In agreement with earlier observations by us and by others, the present results indicate that the predominant alpha 1-adrenoceptor subtype in the rat preglomerular vasculature is the alpha 1A-subtype, although to a smaller extent, the alpha 1B and alpha 1D-adrenoceptors are also expressed at the mRNA and protein levels. The predominance of the alpha 1A-adrenoceptor is consistent with this dominance as the major subtype controlling renal vascular resistance.


    ACKNOWLEDGEMENTS

These studies were supported by National Heart, Lung, and Blood Institute Research Grants HL-02334 (to W. J. Arendshorst) and HL-62584 (to J. E. Faber). M. Salomonsson's visit was sponsored in part by the Swedish Medical Research Council, Medical Faculty Lund University, Maggie Stephens Foundation, and the Berth von Kantzow's Foundation.


    FOOTNOTES

Present address of M. Salomonsson: Dept. of Medical Physiology, The Panum Institute, Blegdamsvej 3, Copenhagen N 2200, Denmark.

Address for reprint requests and other correspondence: W. J. Arendshorst, Dept. of Cell and Molecular Physiology, CB #7545 School of Medicine, Rm. 152, Medical Sciences Research Bldg., Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: arends{at}med.unc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 December 2000; accepted in final form 19 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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