This study provides new information about the relative importance of different α1-adrenoceptors during norepinephrine (NE) activation in rat renal resistance vessels. In Sprague-Dawley rats, we measured renal blood flow (RBF) using electromagnetic flowmetry in vivo and the intracellular free calcium concentration ([Ca2+]i) utilizing ratiometric photometry of fura 2 fluorescence in isolated afferent arterioles. Renal arterial bolus injection of NE produced a transient 46% decrease in RBF. In microdissected afferent arterioles, NE (1 μM) elicited an immediate square-shaped increase in [Ca2+]i, from 90 to 175 nM (P < 0.001). Chloroethylclonidine (CEC) (50 μM) had no chronic irreversible alkylating effect in vitro but exerted acute reversible blockade on norepinephrine (NE) responses both on [Ca2+]i in vitro and on RBF in vivo. The RBF response was attenuated by ∼50% by the putative α1A-adrenoceptor and α1D-adrenoceptor antagonists 5-methylurapidil (5-MU), and 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) (12.5 and 62.5 μg/h), respectively. The in vitro [Ca2+]i response to NE was blocked ∼25% and 50% by 5-MU (100 nM and 1 μM). BMY-7378 (100 nM and 1 μM) attenuated the NE-induced response by ∼40% and 100%. The degree of inhibition in vitro was similar to the in vivo experiments. In conclusion, 5-MU and BMY-7378 attenuated the NE-induced responses, although relatively high concentrations were required, suggesting involvement of both the α1A-adrenoceptor and α1D-adrenoceptor. Participation of the α1B-adrenoceptor is less likely, as we found no evidence for CEC-induced alkylation.
- renal circulation
- afferent arteriole
- vascular smooth muscle
it is well known that the sympathetic autonomic nervous system plays a crucial role in the control of renal hemodynamics and ultrafiltration and thereby in the short- and long-term regulation of the extracellular fluid volume and arterial blood pressure (10, 16). In addition, the release of renin from the granular cells located in the media of the distal end of the afferent arteriole is partly controlled via renal adrenoceptors (10). In renal resistance vessels, catecholamines released from nerve terminals and of humoral origin exert their effect by activation of cell surface adrenoceptors on smooth muscle cells to produce changes in cytosolic calcium concentration ([Ca2+]i) and subsequent contraction. The activation of the vascular adrenoceptors leads to the binding of Ca2+ to calmodulin followed by a change in calmodulin structure enabling subsequent activation of myosin light chain kinase that is responsible for an increase in smooth muscle cell tone (45). The increase in [Ca2+]i induced by receptor activation is thought to be mediated by recruitment of Ca2+from one or two major sources, i.e., mobilization from intracellular stores and entry from the extracellular space through voltage-dependent and/or receptor-activated calcium channels located in the cell membrane (27). The kidneys are endowed with a rich innervation of sympathetic origin with nerves extending primarily to the renal vasculature (1,10). In the rat renal vasculature, the α1-adrenoceptors have been shown to mediate the action of sympathetic activation (8, 11,40, 46). Furthermore, one group of investigators found, using assessment of renal blood flow (RBF) and glomerular filtration rate (GFR) and tubular pressure, that the renal vascular α1-adrenoceptor responsiveness was augmented in spontaneously hypertensive rats (SHR) rats compared with Wistar-Kyoto (WKY) rats (43). This indicates a possible role of these receptors in the development of hypertension.
The α1-adrenoceptors are pharmacologically subdivided in three subtypes, the α1A-, the α1B-, and the α1D-subtypes (4, 19, 25, 32). These receptors correspond to the cloned adrenoceptors α1a, α1b, and α1d, respectively (4). Originally it was believed that activation of α1A-receptors was coupled to influx of calcium from the extracellular space via voltage-gated channels and that α1B-activation caused mobilization of calcium from inositol triphosphate (IP3)-sensitive stores (18). This view, however, has been challenged over the years, and it has been shown that α1A-adrenoceptors can elicit calcium mobilization from IP3-sensitive intracellular stores in some vessels (3, 29, 32, 33, 41). Several reports indicate that different vascular beds exhibit a large heterogeneity with respect to adrenoceptor subtype expression. For example, α1B- and α1D-subtypes have been reported to be present in rat aorta (19, 31, 42). On the arteriolar side of rat skeletal muscle, the predominant subtype is α1D, whereas on the venular side it is the α1B-subtype (25). A predominance of the α1A-subtype is suggested for the mesenteric resistance vessels, whereas the α1B-adrenoceptor subtype appears to dominate in the superior mesenteric artery (23). The interpretation of the results is further complicated by the fact that the use of the different antagonists often gives a picture not in total agreement with any of the known α1-adrenoceptor subtypes, suggesting involvement of not yet described receptors (9, 25, 48). For the renal artery, α1A-, α1B-, and α1D-subtypes have been thought to mediate catecholamine-induced constriction (19, 44). Several reports suggest a major role for the α1A-subtype in the regulation of rat renal hemodynamics (2, 13, 37, 38). An apparent exception to this notion was reported in a study in which receptor binding and ribonuclease protection assay revealed expression of α1B-adrenoceptors in renal microvessels of WKY and SHR (5). Regarding the rat interlobar artery, the action of NE activation appears to be mediated by α1A-receptors (7). However, no direct studies have been performed to identify individual α1-adrenoceptor subtypes in isolated renal resistance vessels.
The present study was designed to provide insight into which α1-adrenoceptor subtype is present on the cell surface of smooth muscle cells in the rat renal microcirculation and is responsible for the chain of actions evoked by NE stimulation. This includes an increase in [Ca2+]i and a subsequent contraction and reduction in RBF. We employed the most selective agents available at the time of study. The adrenoceptor antagonists 5-methylurapidil (5-MU), chloroethylclonidine (CEC), and 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) were used to determine the relative importance of different α1-adrenoceptor subtypes. The [Ca2+]i was measured in vitro in isolated rat afferent arterioles using the ratiometric fluorescence of the indicator fura 2. Renal vascular reactivity was assessed in vivo in rats using electromagnetic flowmetry to measure RBF.
Measurements of cytosolic calcium concentration. Isolated afferent arterioles were microdissected from Sprague-Dawley rats (304 ± 11 g body wt). Several thin slices (thickness 0.5–1 mm) were cut from the midregion of the kidney. The slices were transferred to a dissection dish containing an ice-chilled physiological salt solution (PSS) solution with bovine serum albumin (Sigma) added to give a final concentration of 0.5 g/dl. The PSS solution had the following composition: 135 mM NaCl, 5.0 mM KCl, 1.0 mM CaCl2, 1.0 MgCl2, 10 mM HEPES, and 5.0 mM d-glucose. Sharpened forceps were used for the isolation procedure performed under microscopic visualization (magnification ×12–100). An interlobular artery was localized at its origin from an arcuate artery, and a wedge-shaped segment consisting of glomeruli, blood vessels, and tubular structures was removed. To obtain a single vessel, the tubular structures were stripped away one by one using forceps. Afferent arterioles were identified by their diameter and attachment to a glomerulus. A single afferent arteriole was cut as close as possible to the bifurcation arising from an interlobular artery using a sharp knife blade. To obtain a homogeneous population of arterioles, we used arterioles as superficial as possible from the outer half of the cortex. These were also usually the longest. If no satisfactory preparation was obtained during the first 60 min of dissection, then the kidney was discarded.
After completion of the dissection procedure, a vessel was loaded with fura 2 acetoxymethyl ester (fura 2-AM) for 45–60 min in the dark at room temperature as previously described (21, 35, 36). Fura 2-AM (Molecular Probes) was prepared as stock solution in DMSO (1 mM) and mixed with Pluronic F127 (Molecular Probes) (0.01%) and PSS to a final concentration of 2 μM immediately before use. The arteriole was then transferred to a chamber containing PSS on the stage of an inverted microscope (Olympus IX 70) using an Eppendorf micropipette. Thereafter, the proximal end of the arteriole and the glomerulus were aspirated into two concentric glass holding pipettes by using a syringe connected to the back of the pipettes to generate negative pressure. For measurements of [Ca2+]i, the arteriole was centered in the optical field of ×40 quartz oil-immersion objective (Olympus). The preparation was visualized by the use of video camera (Sony) and monitor. Variable shutters were adjusted to center an arteriole in the sampling window. This arrangement made possible continuous control of the position of the preparation throughout an experiment. The arteriole was excited alternatively with ultraviolet light of 340- and 380-nm wavelength from a dual-excitation wavelength DeltaScan equipped with dual monochromators and a light pathway chopper (Photon Technology International, Monmouth Junction, NJ). Fluorescent light was detected by a photometer after passing signals through a 510-nm band-pass filter. The fluorescent signal intensity was processed and stored by an IBM-compatible Pentium computer and Felix software (Photon Technology International). [Ca2+]i was calculated based on the ratio at 340/380 nm according to the equation described by Grynkiewicz et al. (17), as follows: [Ca2+]i =K d ⋅ [(R − Rmin)/(Rmax − R)] ⋅ (Sf/Sb), where K d is the dissociation constant of fura 2 for calcium; Sf and Sb are the 380-nm fluorescence at zero and saturating calcium concentrations, respectively; and Rmin and Rmax are values of R (fluorescence ratio 340/380) at low and at saturating calcium concentration, respectively. Values for K d, Rmin, Rmax, Sf, and Sb were determined with extracellular calibration as previously described (21). Previous results of extracellular calibration in our laboratory have been shown to agree within 14% of those obtained with intracellular calibration using ionomycin (21).
The volume of fluid in the experimental chamber was maintained constant by the use of a vacuum suction system. The experiments were performed at room temperature (26–29°C). The experimental solutions were added as a large volume that allowed total exchange of the composition in the experimental chamber several times. Previous studies revealed that 1 μM NE elicited a one-half of maximal response in [Ca2+]i (35); this concentration was used throughout the studies of isolated vessels. 5-MU was dissolved in 0.1 M HCl, and BMY-7378 and CEC were mixed in water to create stock solutions, which were frozen. On the day of an experiment, stock solutions were diluted in PSS to their final concentrations. The small amount of HCl in the 5-MU solution did not affect the pH of the final solution. The in vitro experiments were conducted as follows. Initially, the viability of each vascular preparation was tested by adding a short pulse (50 s) of NE to the bath solution to provide a final concentration of (1 μM). If there was no immediate [Ca2+]i response, then the preparation was discarded. In viable vessels, inhibitory agents were added before or after NE stimulation. Reproducibility of the response to NE served as an indicator for the viability of the vessel throughout the experiment. If the NE-induced response differed substantially from the initial response, then the preparation was discarded.
In the pretreatment series, arterioles were exposed to an adrenoceptor antagonist for 50 s before stimulation with NE, still in the presence of the antagonist. The [Ca2+]iresponse obtained during inhibitor treatment was compared with the response when NE alone was added directly to the PSS solution in the absence of inhibitor. To assess the possibility of irreversible alkylation (preferentially of α1B-adrenoceptors) caused by CEC, we also exposed vessels to 15–20 min pretreatment with this agent followed by subsequent washing before the second NE stimulation was performed. This treatment is supposed to block at least 50% of the action exerted by α1B-adrenoceptors (25, 47). Mean [Ca2+]i values for the control period were averaged between 10 and 15 s before addition of NE. In the experimental period, the initial peak and sustained plateau phases of the [Ca2+]i responses were averaged between 10–15 s and 30–35 s, respectively, after the stimulation was initiated. The experiments were performed in random order to establish reversibility and to exclude possible prolonged action of a particular pretreatment. In the poststimulation experiments, responses to NE were recorded for 50 s before a 50-s challenge with an adrenoceptor antagonist in the continued presence of NE. For this series, we present the plateau values of [Ca2+]i before and after inhibition.
RBF measurements. Experiments were performed on male Sprague-Dawley rats; body weight averaged 315 ± 25 g. The rats were fed standard rat laboratory chow and tap water ad libitum. Anesthesia was induced by intraperitoneal injection of pentobarbital sodium (65 mg/kg body wt), and the rats were placed on a servo-controlled heating table that maintained body temperature at 37°C. A tracheostomy was performed, and a tracheal catheter was inserted to facilitate breathing. The left carotid artery was cannulated to monitor mean arterial pressure (Statham P23 Db transducer) and to obtain blood samples for hematocrit measurements. The right jugular vein was cannulated for administration of supplemental addition of pentobarbital sodium and of isoncotic serum bovine albumin (47 g/l) to replace losses associated with surgery (1.25 ml/100 g body wt). Thereafter, isoncotic albumin (10 μl/min) was infused continuously for the duration of the experiment to maintain hematocrit and plasma protein concentration at presurgical levels. Midline and subcostal incisions were made to expose the abdominal aorta and the left kidney. A tapered and curved polyethylene PE-10 catheter was introduced into the left femoral artery and advanced through the abdominal aorta and ∼1 mm into the left renal artery (14, 34). This catheter was used to administer test agents directly into the renal artery before gaining access to extrarenal sites. The preparation was discarded if NE affected arterial pressure. Throughout the experiment, heparinized (30 U/ml) isotonic saline was infused (5 μl/min) via the renal arterial catheter. A noncannulating electromagnetic flow probe (Carolina Medical, King, NC) was placed around the left renal artery to measure RBF. Before starting the measurements, the animals were allowed to stabilize for 30–60 min after completion of the preparation.
The following drugs were used: NE (Whintrop Pharmaceuticals, New York, NY) was dissolved in water. 5-MU (Research Biochemicals International, Natick, MA) was dissolved in 0.1 M HCl. Control experiments established that the concentration of HCl in the final test solutions did not affect the RBF response to NE. BMY-7378 and CEC (Research Biochemicals International) were dissolved in water. Fresh solutions were prepared from frozen stock solution each day of an experiment and kept on ice until administration. A Cheminert sample injection valve was used to introduce a 10-μl bolus of NE into the renal artery infusion line (14, 34). One minute prior to administration of NE, the rate of renal artery infusion was increased to 144 μl/min. This rate of infusion allowed administration of the bolus within 5 s. After recovery of RBF to baseline level (usually within 1 min), the infusion rate was returned to 5 μl/min. In each rat, doses between 10 and 40 ng of NE were injected into the renal artery to produce a 30–50% reduction in RBF. The chosen dose was then used throughout a given experiment. The time interval between successive injections was 5–10 min. In preliminary studies, vehicle infusion or continuous infusion of the adrenoceptor antagonists was found to have no effect on basal RBF or arterial blood pressure. Furthermore, pilot studies established that repetitive administration of NE did not change basal steady-state RBF and arterial blood pressure during the course of an experiment. The antagonists were infused for 2 min before NE injection. Using saline solution, we established that the infusion did not cause a volume expansion of such a degree that it affected the NE-induced RBF response.
Data acquisition was performed as previously described (14, 34). Briefly, it consisted of an IBM-compatible Pentium computer and an analog-to-digital converter (Data Translation). The flow probe was interfaced to the data acquisition system by an electromagnetic flowmeter (Carolina Medical). A Hewlett-Packard model 8805 B carrier amplifier was used for the blood pressure sensor interface. The recordings were started when NE was introduced into the renal artery perfusion line and lasted for a period of 120 s, which was sufficient to allow blood flow to return to baseline values. The RBF values were normalized and expressed as a percentage of baseline values, which were calculated separately for each injection using the mean value observed during the 20-s time interval between introduction of NE and onset of the renal vascular response. The RBF values presented were averaged for 5 s during which the blood flow response to NE was maximal. The recordings obtained using the antagonists and NE were analyzed in the same fashion.
Statistical analysis. Data are presented as means ± SE. The Sigma Stat (SPSS) and Statistica (StatSoft Scandinavia) software were used for statistical analysis. Statistical significance was evaluated by analysis of variance for repeated measurements and Newman-Keuls test. Student's t-test was used for paired observations. P < 0.05 is considered statistically significant.
Measurements of afferent arteriolar [Ca2+]i. In 20 afferent arterioles from 19 rats, the baseline [Ca2+]i averaged 90 ± 7 nM. Previous studies established that 1 μM of NE elicited a one-half maximal response in [Ca2+]i (35), and this concentration was used in all subsequent studies on microdissected vessels. Addition of NE (1 μM) to the bath caused an abrupt and square-shaped increase in vascular smooth muscle [Ca2+]i (Fig.1, A and B). In most vessels, the response consisted of a sharp rise that was sustained at a near-maximum plateau level. In about 20–30% of the vessels, however, the transient initial peak was appreciably larger than the sustained plateau. Visible contraction of the afferent arteriole frequently correlated temporally with the recording of increased [Ca2+]i.
In the first vessel experiments, we investigated the alkylating effect of CEC on α1-adrenoceptors in rat afferent arterioles. Because of the irreversible nature of alkylation, we measured the control response to NE before CEC pretreatment. In these experiments, NE caused an increase to 189% of baseline [Ca2+]i, from 65 ± 9 to 117 ± 8 and 118 ± 9 nM at 10–15 and 30–35 s, respectively (n = 5, P < 0.001). After 15-min pretreatment with CEC (50 μM), the baseline [Ca2+]ivalue was 50 ± 7 nM; this latter value was, however, not significantly different from the previous control value. One minute after termination of the CEC treatment and thorough washout, NE administration caused an increase of [Ca2+]i to 171% of baseline at 10–15 s (from 49 ± 7 to 81 ± 13 nM) and to 196% at 30–35 s (to 91 ± 7 nM) (P < 0.003). NE administration 5 min after CEC washout caused an increase in [Ca2+]i to 202% of baseline at 10–15 s and to 195% at 30–35 s (from 55 ± 8 to 108 ± 21 and 103 ± 17 nM, respectively) (P < 0.01). These results are summarized in Fig. 2. It is clear that there was no indication of irreversible alkylation of NE-sensitive adrenoceptors in this preparation.
On the other hand, we found that CEC with pretreatment for 50 s but without CEC washout had an acute inhibitory effect, probably not attributable to alkylation, that was reversible. Upon short-term exposure to 50 μM CEC (50 s), the baseline levels of [Ca2+]i were unchanged (Fig. 1 A,right). In these experiments administration of NE alone elicited an increase in [Ca2+]ifrom 111 ± 18 to 164 ± 21 nM at 10–15 s and to 165 ± 17 nM at 30–35 s, an average increase to 152% of the baseline value (P < 0.02) (n = 3). In the experimental period, NE had no effect on [Ca2+]i in the presence of CEC (50 μM), indicating complete blockade (Fig.3 A). The average baseline [Ca2+]i value for these recordings was 93 ± 16 nM. Ten to fifteen seconds after NE stimulation in the presence of CEC it was 94 ± 16 nM, and between 30–35 s it averaged 97 ± 15 nM (n = 3). In other experiments, we found that a 10 times lower concentration of CEC also inhibited the NE-induced increase in [Ca2+]i. During the control period in these experiments, we found that NE increased [Ca2+]i from 78 ± 13 to 166 ± 18 nM at 10–15 s and to 156 ± 18 nM at 30–35 s after stimulation (P < 0.005) (Fig. 3 A). The change is normalized to 100% in Fig. 3. The low concentration of CEC (5 μM) almost completely prevented the NE-induced increase in [Ca2+]i at 10–15 s (76 ± 10 to 90 ± 13 nM) and at 30–35 s (94 ± 12 nM; n = 6) (Fig. 3 A).
We also tested the effect of pretreatment with 5-MU on the NE-induced [Ca2+]i response to investigate the involvement of the α1A-receptor and α1D-receptor. Neither dose of this antagonist affected baseline [Ca2+]i. After 5-MU (100 nM) pretreatment for 50 s, NE produced a rise in [Ca2+]i from 94 ± 16 to 158 ± 13 nM at 10–15 s, an increase that was sustained at 157 ± 11 nM at 30–35 s (P < 0.01, n = 7) (Fig. 3 B). In these vessels, in the absence of 5-MU, NE stimulated [Ca2+]i from 96 ± 16 to 181 ± 16 nM at 10–15 s and to 175 ± 17 nM at 30–35 s (P < 0.001). The increase at 10–15 s was significantly greater than the rise after 5-MU pretreatment (P < 0.02). The difference at 30–35 s, however, did not reach statistical significance (P > 0.1). After pretreatment with the higher 5-MU concentration (1 μM), NE elicited a [Ca2+]i change from 90 ± 12 to 131 ± 12 nM at 10–15 s and to 133 ± 15 nM at 30–35 s (P < 0.005) (n = 7). When the same preparations were exposed to NE in the absence of antagonist, the increase in [Ca2+]i was significantly greater, from 90 ± 11 to 181 ± 20 nM at 10–15 s and to 177 ± 19 nM at 30–35 s (P < 0.05).
The putative α1D-adrenoceptor antagonist BMY-7378 also significantly attenuated the NE-induced [Ca2+]i response in afferent arterioles. In the control period, NE increased [Ca2+]i from 102 ± 14 to 181 ± 22 nM at 10–15 s and to 171 ± 18 nM at 30–35 s after stimulation (P < 0.005). Pretreatment with BMY-7378 (100 nM or 1 μM) did not have any effect on baseline [Ca2+]i. In the presence of the lower BMY-7378 concentration, NE caused a [Ca2+]i increase from 98 ± 16 to 145 ± 19 nM at 10–15 s and to 148 ± 20 at 30–35 s (P < 0.01); both values are less than the control responses (P < 0.05, n = 5) (Fig. 3 C). After pretreatment with the higher BMY-7378 dose (1 μM), the NE-induced change in [Ca2+]i was abolished. The baseline [Ca2+]i value of 132 ± 41 nM was unchanged after 10–15 s (136 ± 44 nM) or 30–35 s (135 ± 42 nM) (n = 5). In the absence of BMY-7378, NE increased [Ca2+]i in the same vessels from 147 ± 44 to 285 ± 92 nM at 10–15 s and to 280 ± 92 nM at 30–35 s (P < 0.03).
The acute nonalkylating effect of CEC was confirmed by poststimulation exposure to CEC (5 μM). NE alone caused [Ca2+]i to rise from 76 ± 5 nM to the sustained plateau level of 151 ± 13 nM (P < 0.001) (n = 8). During continued NE stimulation, antagonism of adrenoceptors with CEC produced a sudden decrease in [Ca2+]i to a new level that averaged 95 ± 8 nM. This latter value was not significantly different from control baseline. In experiments using a higher concentration of CEC (50 μM), complete inhibition was produced. In these vessels, NE alone increased [Ca2+]ifrom 97 ± 24 to 180 ± 33 nM (P < 0.02). CEC reversed completely the response to NE, restoring [Ca2+]i to 85 ± 16 nM (n= 4) (Fig. 4 A).
In the same manner, we investigated the effect of 5-MU to evaluate the involvement of α1A- and α1D-receptors on the sustained [Ca2+]i plateau triggered by NE. These results are summarized in Fig. 4 B. NE alone elicited a rise in [Ca2+]ifrom 101 ± 12 to 175 ± 13 nM (P < 0.001) (n = 10). Upon addition of 5-MU (100 nM), during continued NE stimulation, the [Ca2+]i was slightly reduced to 150 ± 15 nM, a nonsignificant change (P = 0.1). The higher concentration of 5-MU (1 μM) attenuated the NE-induced response. NE stimulation increased [Ca2+]i from 118 ± 20 to 197 ± 37 nM in absence of antagonist (P < 0.005) (n = 7). During 5-MU treatment, [Ca2+]i averaged 140 ± 31 nM or 118% of control, which was not significantly different from baseline (P > 0.3).
We also found that posttreatment with BMY-7378, the α1D-receptor antagonist, attenuated the NE-induced [Ca2+]i plateau. BMY-7378 (100 nM) reduced the plateau slightly from 164 ± 11 to 143 ± 12 nM (P < 0.05), a value that is significantly higher than baseline (96 ± 7 nM; P < 0.001, n = 9). Poststimulation addition of the higher concentration of BMY-7378 (1 μM) almost completely reversed the NE-induced response (from 126 ± 28 to 218 ± 47 nM, P < 0.005), as [Ca2+]i fell to a value that is similar to baseline (143 ± 44 nM; P > 0.3,n = 7) (Fig. 4 C).
Measurements of RBF. Experiments were conducted to correlate responses of microdissected afferent arterioles to changes in renal vascular resistance in vivo. During euvolemic control conditions, RBF averaged 5.5 ± 0.5 ml ⋅ min− 1 ⋅ g kidney wt− 1 in 8 Sprague-Dawley rats. Arterial blood pressure and hematocrit averaged 112 ± 7 mmHg and 49 ± 0.4%, respectively. After establishing effects of NE on RBF, we determined the attenuation of the renal vascular response to adrenoceptor activation with NE using the adrenoceptor antagonists CEC, 5-MU, and BMY-7378. In these experiments, pretreatment consisted of infusion of an antagonist into the renal artery 2 min before bolus injection of NE (Fig. 5). NE alone produced, on the average, a transient 51 ± 6% maximum reduction in RBF (P < 0.001, n = 7) (Fig. 5 A). As in the in vitro experiments, the presence of CEC attenuated the NE-induced effect. Pretreatment with CEC infusion (2.5, 12.5, or 62.5 μg/h) for 2 min attenuated the vasoconstrictor effect of NE in dose-dependent manner. The lowest tested dose of CEC buffered the NE-induced decrease in RBF to 39 ± 8% of basal flow (P < 0.005 vs. NE). The intermediate dose of CEC attenuated the response so that NE caused a 31 ± 6% decrease of RBF (P < 0.05 vs. 2.5 μg/h). The highest dose of CEC inhibited the NE-induced vascular response, limiting the RBF reduction to 21 ± 6% of basal flow.
In the control period of the 5-MU experiments, NE alone reduced RBF by 41 ± 9% of baseline (P < 0.01, n = 5). During the experimental period, 5-MU (2.5, 12.5, or 62.5 μg/h) was infused into the renal artery for 2 min prior to injection of NE. These results summarized in Fig. 5 B show that the low dose of 5-MU tended to reduce the NE response (33 ± 7% of basal flow), but the attenuation was not statistically significant. The NE-induced decrease in RBF was reduced to 23 ± 6% of basal flow by the intermediate dose of 5-MU infusion (P < 0.01). The highest dose of 5-MU reduced the NE response to 19 ± 5% of basal RBF (P < 0.005). This degree of inhibition is similar to the value obtained upon intermediate dose of 5-MU infusion. The role of α1D-receptor assessed as BMY-7378 inhibition was examined in separate experiments (Fig. 5 C). Administration of NE in the control period transiently reduced the RBF by 43 ± 7% of the basal value (P < 0.001, n = 5). BMY-7378 (2.5, 12.5, or 62.5 μg/h) was infused into the renal artery for 2 min prior to NE injection. The low rate of BMY-7378 infusion did not alter the NE response (42 ± 7% of basal RBF). The intermediate dose of BMY-7378 attenuated the NE-induced reduction in RBF to 31 ± 5% of basal flow (P < 0.05). The high dose of BMY-7378 reduced the NE response to 21 ± 2% of basal RBF (P < 0.01), a value not significantly different from the value obtained with the intermediate BMY infusion rate. This overall response pattern with BMY-7378 was similar to that obtained using 5-MU.
The present study investigated the cellular mechanisms mediating adrenoceptor-induced activation of smooth muscle cells in the renal microcirculation. The aim was to evaluate the role of α1-adrenoceptor subtypes in calcium signaling and the contractile response of resistance arterioles. To this end, RBF was measured in vivo to complement determinations of [Ca2+]i in individual afferent arterioles isolated from Sprague-Dawley rats. An important aspect of this work is the combined in vivo and in vitro evaluation of vascular responses to a naturally occurring catecholamine, NE, and how NE-induced vasoconstriction is attenuated by pharmacological antagonists of α1-adrenoceptor subtypes. In pilot experiments, we established that injection of NE into the renal artery produces transient renal vasoconstriction in a dose-dependent manner, with total ischemia noted with high doses. To standardize the magnitude of NE-induced changes in renal vascular resistance, the amount of NE was adjusted in each animal to produce a 30–50% reduction in RBF, and then that particular dose was kept constant during subsequent administration of pharmacological inhibitors.
Earlier studies have demonstrated that NE is a potent constrictor of the renal vasculature (10, 24, 28, 35). Isolated vascular segments exposed to NE in vitro respond with reduced vessel diameter and an elevated smooth muscle [Ca2+]i (12,22, 24, 35, 36, 49, 50). Renal vasoconstriction, as a response to catecholamines, is mediated by α-adrenoceptors. Several in vivo and in vitro studies reveal that the α1-adrenoceptor predominates in renal resistance vessels (8, 11, 39, 46). On the basis of selectivity to different adrenoceptor antagonists, the α1-adrenoceptors are further divided into α1A-adrenoceptor and α1B-adrenoceptor subtypes (for reviews, see Refs. 4 and 32). The clonidine analog CEC is well known to alkylate and inactivate the α1B-adrenoceptor subtype (19, 25, 26). The adrenoceptor antagonists 5-MU and WB-4101 are thought to selectively antagonize the action of α1A-adrenoceptors (4, 25, 26, 32). More recently, a third subtype, the α1D-adrenoceptor, has been characterized by cloning and pharmacological studies (25, 30). The recombinant α1d-adrenoceptor (lower case letter in subscript indicates cloned receptor) has a high affinity for the antagonist BMY-7378 (15). These authors found in binding studies a pKi of 8.2 for the non-human recombinant α1d-adrenoceptor, giving a 126-fold selectivity over α1c-adrenoceptor (later studies have revealed that this receptor is most likely identical to the α1a-adrenoceptor) and a 100-fold selectivity over the α1b-adrenoceptor. It has also been shown that the α1D-adrenoceptor has a high affinity for both 5-MU and WB-4101, inhibitors that are less selective than the BMY compound because they also recognize the α1A-adrenoceptor subtype (25). K i values for these mixed antagonists reported from different laboratories exhibit some variation, ranging from 1–100 nM (25). Furthermore, it has recently been shown in vitro that CEC also alkylates the α1D-adrenoceptor subtype, almost at the same rate as the alkylation of α1B-adrenoceptors (47).
Regarding the renal resistance vessels, there is a paucity of information concerning the presence and action of particular α1-adrenoceptor subtypes. A dominant role for the α1A-adrenoceptor subtype has been suggested in the regulation of rat renal hemodynamics (2, 13, 37, 38). As for the relatively large rat interlobar artery, the action of NE appears to be mainly mediated by α1A-receptors (7). Accordingly, 5-MU is reported to counteract the NE-induced contraction of interlobar arteries from Wistar rats with high potency, whereas BMY-7378 and CEC exhibit a low potency attenuating effect (7). In contrast, receptor binding and ribonuclease protection assay indicate that the α1B-adrenoceptors are predominantly expressed in renal preglomerular microvessels of the rat (5).
In the present study, we report a unique combination of assessment of RBF in vivo and measurements of [Ca2+]i in isolated afferent arterioles. To our knowledge, this is the first functional study regarding α1-adrenoceptor subtypes in a defined segment of the renal microvasculature with a diameter <50 μm. In experiments where we pretreated an isolated preglomerular resistance vessel with CEC for 15 min with subsequent removal of CEC, there was no attenuating effect on the NE response; NE elicited the same increase in [Ca2+]i before and after the CEC treatment. These results imply that α1B-adrenoceptors most likely play a minor role in isolated rat afferent arterioles, since one would have predicted, if present, alkylation of at least 50% of a presumptive α1B-adrenoceptor under these conditions (47). Furthermore, since it has recently been suggested that α1D-adrenoceptor is alkylated by CEC to almost the same extent as the α1B-adrenoceptor (47), the absence of attenuation by CEC indicates limited participation of α1D-adrenoceptors.
Other investigators have reported that CEC has only minor effects on the renal circulation. After renal infusion of CEC, via a suprarenal artery, at a total dose of 3 mg/kg for 1 h followed by a 30-min recovery period, Elhawary et al. (13) found that the RBF dose-response curve to administration of phenylephrine had only undergone a threefold rightward shift. In contrast, these investigators found an almost total obliteration of the phenylephrine response by the irreversible α1A-adrenoceptor antagonist SZL-49 administered for 1 h at a total dose of 10 μg/kg. In isolated perfused kidneys, 100 μM CEC for 20 min is reported to have no effect on the renal vasoconstrictor response of nerve stimulation at 3 and 10 Hz (2). In the same study, it was found that 60-min perfusion with 30 nM of 5-MU blocked 80–90% of the renal vasoconstriction induced by 3 and 10 Hz nerve stimulation. These results implicate mediation of NE-induced vasoconstriction by the α1A-receptor or α1D-receptor. In another rat study, SZL-49 was found to antagonize the phenylephrine-induced reduction in GFR, urinary output, and Na+ excretion (6). The authors of these two latter studies concluded that the effect of SZL-49 was due to blocking of α1A-adrenoceptor in the renal vasculature. The specificity of SZL-49 as an α1A-adrenoceptor antagonist, however, has been questioned (33).
Regarding CEC, we observed that the calcium response to NE was totally abolished in the presence of this antagonist (5 or 50 μM after only 50-s pretreatment). Furthermore, we found that both concentrations of CEC immediately returned the [Ca2+]i to baseline in isolated afferent arterioles when CEC was administered during continued NE stimulation. At face value, these results indicate that the mechanism of action is too rapid to be due to alkylation. Since alkylation is thought to be the primary effect of CEC, the fast action and the reversibility of this compound may reflect an additional mechanism of action in the renal vasculature.
In in vivo experiments in the presence of the antagonist, we also found that CEC attenuated the NE-induced reduction in RBF. The NE-induced renal vasoconstriction was attenuated in a dose-dependent manner by pretreatment of the adrenoceptor antagonists CEC, 5-MU, and BMY-7378 at rates of 2.5, 12.5, and 62.5 μg/h for 2 min prior to a bolus injection of NE. Given a molecular weight of ∼400 for all of these drugs and the observed values for RBF and hematocrit, the approximate renal plasma concentrations of the drugs were calculated. For the infusion rates of 2.5, 12.5, and 62.5 μg/h, we estimate concentrations of 26, 130, and 650 nM, respectively. It is noteworthy that at the lowest infusion rate, only CEC displayed inhibition in vivo, attenuating the NE-induced reduction in RBF by 24%. The higher doses of CEC attenuated this response by 39 and 61%, respectively. Other investigators have reported that an even lower concentration of CEC (5 or 10 μg/kg injection to the abdominal aorta at the renal artery followed by continuous infusion at 1.25 or 2.5 μg ⋅ kg− 1 ⋅ h− 1) attenuates renal vasoconstriction induced in vivo by renal nerve stimulation in the rat (38). At 10-Hz nerve stimulation, CEC reduced the renal vascular response by 21%. The effect of such low concentrations of CEC might be explained by a nonalkylating action when CEC is present. However, in that study, the responses to phenylephrine and methoxamine were unaffected by CEC. The mechanism and significance of the acute (nonalkylating) action of CEC remain to be elucidated.
In previous studies, it has been established that 5-MU has an effect on whole kidney preparations (2, 37, 38). It is well known that 5-MU does not discriminate definitely between α1A- and α1D-adrenoceptors (25, 31); thus it is difficult to exclude the possibility that the blocking action found in the previous studies is due exclusively to an α1D effect. Earlier evidence supporting the existence of α1D-adrenoceptors in the renal vasculature is lacking.
To our knowledge, the present study is the first to investigate the effects of the more specific α1D-adrenoceptor antagonist BMY-7378 in the control of RBF and Ca2+ metabolism in renal resistance vessels. Infusion of BMY-7378 and 5-MU in vivo attenuated the NE-induced reduction in RBF. In the infusion experiments, the highest doses (∼130 and 650 nM) of both drugs inhibited 50% of the NE effect on RBF. In another study of rats in vivo, pretreatment with 5-MU for 15 min at rates of 1.25 and 2.5 μg/kg following bolus doses of 5 and 10 μg, respectively, attenuated the renal vasoconstrictor responses to phenylephrine or renal nerve stimulation by 40–50% (38). The drug was infused in the aorta, however, which makes it difficult to calculate drug concentration in the renal circulation and to exclude systemic effects. In this study, there was a reduction in arterial blood pressure with the highest infusion rate.
As mentioned earlier, we observed that the effects of 5-MU and BMY-7378 on NE-induced renal vasoconstriction were similar, suggesting the presence of α1D-adrenoceptors. However, an action of α1A-adrenoceptors cannot be excluded with any confidence. In favor of α1A-adrenoceptors is the absence of alkylating action of CEC, since the α1D-receptor is considered to be alkylated more rapidly than the α1A-adrenoceptors (47). On the other hand, evidence supporting involvement of α1D-adrenoceptors in [Ca2+]i changes in isolated rat afferent arterioles are the present results showing that pretreatment with BMY-7378 (100 nM and 1 μM) for 50 s reduced the NE-induced rise in [Ca2+]i in a dose-dependent fashion. Furthermore, in the posttreatment studies, BMY-7378 (1 μM) totally reversed NE-induced [Ca2+]iresponses. The effect of 5-MU was similar, although the dose dependency in the pretreatment studies exhibited a somewhat shallow pattern. It is, however, not possible to completely exclude a nonspecific effect of BMY-7378, as the concentrations are much higher than the pKi of 8.2 reported for BMY-7378 on the cloned rat α1d-adrenoceptor (15). On the other hand, our in vivo and in vitro results regarding BMY-7378 sensitivity are consistent with the apparent pKb of 6.86 found for BMY-7378 on putative α1D-adrenoceptors in rat skeletal muscle arterioles (25). These authors found a pKb of 7.35 for 5-MU. This indicates that the receptor(s) in our preparation is more sensitive to BMY-7378, since 5-MU (1 μM) blocked no more than one-half of the NE-induced [Ca2+]i elevation in the pretreatment studies.
Nevertheless, pharmacological agents presently available and employed in the this study make it difficult to definitively identify α1-adrenoceptor subtypes present in rat renal resistance vessels on the basis of the current classification of these receptors. In fact, the existence of α1-adrenoceptors that do not entirely share the characteristics of known α1-adrenoceptor subtypes is emphasized by several investigators on the basis of data similar to ours (9, 25, 48). On the other hand, the relative accessibility of the antagonists to receptors might also be of importance. For example, recently it has been suggested that CEC preferentially inactivates adrenoceptors on the cell surface irrespective of the subtype and that localization is a more important factor for the degree of CEC inactivation than the subtype per se (20). Future studies, such as radioligand binding experiments and localization of mRNA for the receptor subtypes, may provide a clearer picture that fully characterizes the α1-adrenoceptor(s) responsible for catecholamine-induced control of renal hemodynamics.
In summary, our study presents a unique combination of in vitro and in vivo evidence that provides insight into activation of adrenergic receptors in renal resistance vessels. There is excellent general agreement between these two different preparations with respect to the action of NE and the blocking of its action with adrenoceptor antagonists. Both the NE-induced [Ca2+]i response in isolated afferent arterioles and the constriction of the renal vasculature in vivo were attenuated by BMY-7378 and 5-MU, which implicates the presence of both α1A-adrenoceptors and α1D-adrenoceptors. A major role of α1B-adrenoceptors is not very likely, as there was no discernible alkylating effect of CEC. On the other hand, the adrenoceptor(s) exhibited a pronounced CEC sensitivity in the presence of CEC that was observed both in vivo and in vitro. However, the relative importance of the α1A-adrenoceptor, α1D-adrenoceptor, and perhaps an unique receptor in rat renal preglomerular resistance vessels cannot be fully characterized using the current pharmacological classification of α1-adrenoceptors.
The technical assistance of J. J. Feng is gratefully acknowledged.
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:).
These studies were supported by National Heart, Lung, and Blood Institute Research Grant HL-02334. The visit of M. Salomonsson was sponsored in part by the Swedish Medical Research Council, Medical Faculty Lund University, Maggie Stephens Foundation, and the Berth von Kantzow's Foundation.
Present address of M. Salomonsson: Dept. of Medical Physiology, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
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. §1734 solely to indicate this fact.
- Copyright © 2000 the American Physiological Society