INTRODUCTION

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AN EPISODE OF EXPOSURE to a G protein-linked receptor stimulus not only causes an immediate response but also may induce desensitization, causing the tissue to subsequently display decreased reactivity to receptor stimuli (26). The classic explanation for this attenuation of cell responsiveness to a ligand is receptor desensitization (19, 26). However, several lines of evidence suggest that reductions in postreceptor signaling systems may also play a role (3, 9, 16, 22, 30).

Homologous and heterologous desensitization are the terms currently used to describe the overall phenomenon of postactivation attenuation of cell responsiveness (19, 26). Homologous receptor desensitization involves a reduction in same-receptor responsiveness. The mechanisms behind this effect have been rigorously studied by Lefkowitz and others (19), who show, for example, that -adrenergic receptor stimulation can activate receptor kinases, leading to -adrenergic receptor desensitization and ultimately to a reduction in -adrenergic receptor numbers. Heterologous desensitization is, by definition, a more complex modulatory mechanism consisting of desensitization of receptors other than that stimulated to cause the desensitization or desensitization resulting from modulation of postreceptor signaling systems (26).

Desensitization of vascular smooth muscle contractions is a well-known phenomenon, but the precise subcellular mechanisms responsible for this adaptive response remain to be determined. Homologous -adrenergic receptor desensitization occurs in cultured vascular smooth muscle cells, but to induce this response, cells appear to require stimulation periods in excess of several hours (3, 11). Interestingly, stimulation of intact rat arterial muscle with epinephrine for 12 h does not reduce -adrenergic receptor numbers or affinity, but -receptor-induced contractile activity is greatly diminished, implying that postreceptor desensitization is activated by the prolonged receptor stimulation period (16).

Recent evidence shows that shorter episodes of receptor activation (<1 h) of rabbit femoral artery (22), hog carotid artery (30), or rabbit detrusor (28) reduce KCl-induced contractile activity. Because KCl produces contractions by causing membrane depolarization rather than acting as a receptor stimulus, these results indicate that short periods of receptor stimulation can desensitize signaling systems downstream from receptor activation in smooth muscle.

If receptor stimulation can reduce reactivity of smooth muscle to KCl by activating a postreceptor desensitizing mechanism, then stimulation of a receptor type other than that used to produce desensitization may also be affected by this mechanism. One modulatory mechanism that plays a key role in regulation of smooth muscle contractile reactivity involves changes in the Ca²⁺ sensitivity of contractions (29). In particular, receptor agonists increase Ca²⁺ sensitivity, whereas agents that increase cGMP decrease Ca²⁺ sensitivity (12, 13). In short, by changing Ca²⁺ sensitivity, the level of contraction may be greatly increased or decreased without considerable changes in the concentration of cytosolic free Ca²⁺ ([Ca²⁺]_i). The goal of the present study was to test the hypothesis that heterologous postreceptor desensitization involves, at least in part, a reduction in Ca²⁺ sensitivity of receptor-induced contractions.

	METHODS

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Tissue preparation. Tissues were prepared as described previously (22). Renal arteries obtained from female New Zealand White rabbits were cleaned of adhering tissue and stored for 48 h in cold (0-4°C) physiological saline solution (PSS) composed of (in mM) 140 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.6 CaCl₂, 1.2 Na₂HPO₄, 2.0 MOPS (adjusted to pH 7.4 at either 0 or 37°C, as appropriate), 0.02 Na₂-EDTA (to chelate trace heavy metals), and 5.6 D-glucose. High-purity (>17 M distilled and deionized) water was used throughout. Before arterial muscle rings were secured between a micrometer and an isometric force transducer (model 52, Harvard Apparatus, South Natick, MA) in water-jacketed muscle chambers (Radnoti Glass Technology, Monrovia, CA), the endothelium was removed by gently rubbing the intimal surface with a metal rod, and the adventitia was removed by microdissection.

Isometric force. Contractile force (F) was measured as described previously (22). Tissues were allowed to equilibrate at 37°C for 1 h, and the muscle length at which active force was maximum (L_o) was then determined for each tissue with K⁺ as agonist (110 mM KCl substituted isosmotically for NaCl) using an abbreviated length-tension curve (8, 22). Once tissues were stretched to L_o, no further length changes were imposed. For each tissue, the degree of steady-state F produced at L_o by incubation for 5-10 min in 110 mM KCl was equal to the optimal force for muscle contraction (F_o), and subsequent contractions were calculated as F/F_o. Tissues contracted with phenylephrine (PE) were relaxed by PE washout in the presence of 1 µM phentolamine (-adrenergic receptor antagonist). In tissues contracted with histamine (HA), 1 µM cimetidine (H₂-receptor blocker) was added 10 min before the addition of HA. In tissues contracted with KCl, 1 µM phentolamine was added to block potential -adrenergic receptor activation caused by the release of norepinephrine from periarterial nerves. To perform concentration-response curves, HA or KCl was added cumulatively every 7-10 min.

Intracellular free Ca²⁺ concentration. [Ca²⁺]_i was measured as described previously (24). Arterial rings secured between a micrometer and an isometric force transducer and positioned in front of a quartz window in a custom-fabricated water-jacketed muscle chamber (Radnoti Glass Technology) were loaded for 2.5 h with 10 µM fura 2-AM (Molecular Probes, Eugene, OR) plus Pluronic F-127 (0.01% wt/vol) to enhance solubility. Tissues were washed for 30 min and, in response to alternate excitation at 340 and 380 nm with the use of a rotating filter wheel, fluorescence emissions at 500 nm for each excitation wavelength (F₃₄₀ and F₃₈₀) were collected by a photomultiplier tube and amplified (model 1642, Ithaco), digitized (Data Translations DT2811), and visualized on a computer screen (CODAS software package, DATAQ Instruments). [Ca²⁺]_i (in nM) was calculated using the formula [Ca²⁺]_i = [(R  R_min)/(R_max  R)] × (S_f/S_b) × K_d, where R_min and R_max are the ratios produced, respectively, in a Ca²⁺-free solution (PSS containing 0 mM CaCl₂, 5 mM EGTA, 110 mM KCl substituted for NaCl, and 20 µM ionomycin) and in the presence of excess Ca²⁺ (PSS containing 3.6 mM CaCl₂, 110 mM KCl substituted for NaCl, and 20 µM ionomycin); S_f/S_b is the ratio of F₃₈₀ in the Ca²⁺-free solution to F₃₈₀ in the presence of excess Ca²⁺; and K_d is the dissociation constant (224 nM) (7). All fluorescence measurements were corrected for tissue background fluorescence by adding 4 mM MnCl₂ to the R_max solution to quench the fura 2 signal at the end of the experiment. Fluorescence ratios (F₃₄₀/F₃₈₀) taken in resting (0.36 ± 0.01) and fully contracted (0.36 ± 0.01, n = 21, P > 0.05) tissues before being loaded with fura 2-AM were not different, indicating that contractions, per se, had no effect on ratiometric tissue fluorescence. Furthermore, a comparison of F₃₄₀ and F₃₈₀ fluorescence values taken after the 4 mM MnCl₂ quench at the end of the experiment (0.35 ± 0.03 and 1.06 ± 0.11, respectively) with those values taken before the fura 2-AM loading (0.33 ± 0.03 and 0.97 ± 0.11, respectively, n = 21, P > 0.05) were not different, suggesting that signal interference from fluorescent intermediates potentially produced by partial hydrolysis of fura 2-AM was negligible.

Statistics. For analyses of concentration-response data for each tissue, a curve-fitting program (GraphPad) was used to obtain the four parameters of a sigmoidal curve [maximum, minimum, slope, and concentration producting half-maximum contraction (EC₅₀)]. For statistical analyses of logarithmic data (i.e., EC₅₀ values), geometric means were used (5). For convenience, log EC₅₀ values were converted to EC₅₀ values in micromoles per liter in the text. To construct Fig. 5, Ca²⁺ values for each tissue were converted so that values ranged between 0 and 1. This was accomplished by dividing the difference between the agonist-stimulated value and the curve-fitted minimum value (in nM) by the difference between the curve-fitted maximum and minimum values (in nM), represented as Ca/Ca_max.

	RESULTS

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Effect of PE pretreatment on HA-induced contractions. An HA-induced concentration-contraction curve produced in rabbit renal artery not loaded with the Ca²⁺ indicator fura 2 (Fig. 1) was characterized by an EC₅₀ of 0.30 ± 0.06 µM, a slope of 4.75 ± 0.70, and a maximum response of 1.01 ± 0.01 F/F_o (n = 3). In tissues pretreated for 30 min with 10 µM PE and washed for 10 min before addition of the first HA concentration, the HA-induced concentration-contraction curve was characterized by an ~1/2 log rightward shift in the EC₅₀ to 0.93 ± 0.08 µM (P < 0.05 compared with control) without a change in the slope (3.94 ± 0.69) or maximum response (0.96 ± 0.04 F/F_o, n = 3; Fig. 1).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Histamine (HA) concentration-contraction curves produced in control tissues and tissues pretreated for 30 min with 10 µM phenylephrine (PE). Duration of time between washout of PE and addition of the first HA concentration ([Histamine]) was 10 min. A dotted vertical line was drawn at 0.56 µM HA to highlight large difference between contractile force (F/Fo) responses produced at this HA concentration by control and PE-pretreated tissues. Data are means ± SE; n = 3 rabbits.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Dependency of degree of desensitization produced by PE on 1) concentration of PE and 2) duration of PE pretreatment. Dependencies were quantified by measuring relative effectiveness of 0.56 µM HA to produce contractions in control tissues and tissues pretreated with 10, 1, or 0.1 µM PE for 30 min or with 10 µM PE for 10 min. Duration of time between washout of PE and addition of HA was 10 min. Data are means ± SE; n = 4-6 rabbits. * P < 0.05 compared with control.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Reversibility of desensitization produced by PE pretreatment. Reversibility was quantified by measuring the degree of contraction produced by 0.56 µM HA in control tissues compared with that produced by tissues 10, 30, 60, and 90 min after pretreatment for 30 min with 10 µM PE. Data are means ± SE; n = 4-5 rabbits. * P < 0.05 compared with control.

Effect of PE pretreatment on HA-induced increases in [Ca²⁺]_i. In tissues loaded with the Ca²⁺ indicator fura 2, HA produced a concentration-contraction curve that was similar to that produced by tissues not loaded with fura 2 (compare Figs. 4A and 1). In fura 2-loaded tissues, PE pretreatment (30 min at 10 µM followed by 10-min PE washout and relaxation) caused a rightward shift in the HA concentration-contraction curve as it did in tissues not loaded with fura 2 (EC₅₀: control = 0.32 ± 0.03 µM, PE pretreated = 0.75 ± 0.05 µM, n = 4, P < 0.05; Fig. 4A). Likewise, as in tissues not fura 2 loaded, both the slope of the curve and the maximum response were not different from the control values (slope: control = 5.41 ± 0.56, PE pretreated = 4.47 ± 0.74; maximum response: control = 1.04 ± 0.02 F/F_o, PE pretreated = 1.01 ± 0.05 F/F_o). This PE pretreatment also produced a rightward shift in the HA concentration-[Ca²⁺]_i curve (EC₅₀: control = 0.31 ± 0.03 µM, PE pretreated = 0.54 ± 0.07 µM, n = 4, P < 0.05; Fig. 4B). However, the slope of the curve produced by tissues that had been PE pretreated was approximately twofold greater than that of the control response (slope: control = 3.71 ± 0.55, PE pretreated = 6.27 ± 0.81, n = 4, P < 0.05; Fig. 4B). The minimum and maximum [Ca²⁺]_i responses produced by control tissues and tissues that had been pretreated with PE were identical (minimum: control = 61 ± 10 nM, PE pretreated = 62 ± 6 nM; maximum: control = 278 ± 30, PE pretreated = 266 ± 33 nM; Fig. 4B).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effect of 10 µM PE pretreatment for 30 min on HA concentration-contraction (A; ) and HA concentration-[Ca2+]i (B; ) curves produced in tissues loaded with the Ca2+ indicator fura 2. Responses of control tissues that were not pretreated with PE are represented by open symbols. As in Fig. 1, a dotted vertical line was drawn at 0.56 µM HA. Note that 0.78 µM HA produced an increase in [Ca2+]i comparable to the control value (B), but even at 1 µM, HA did not produce an increase in contractile force that was comparable to the control value (A). Data are means ± SE; n = 4 rabbits. * P < 0.05 compared with control.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Contractile force (represented as F/Fo) and cytosolic free Ca2+ (normalized as Ca/Camax) data from Fig. 4 were plotted together for control (A) and 10 µM PE-pretreated tissues (B). Data are means ± SE. Force and Ca2+ displayed identical sensitivities to HA in control (A), but Ca2+ was more sensitive to HA than was force in PE-pretreatment group (B). Thus PE pretreatment produced a greater rightward shift in the HA concentration-contraction curve than in the HA concentration-Ca2+ curve.

Relationship between HA-induced increases in contraction and [Ca²⁺]_i. To examine the dependency of steady-state force on [Ca²⁺]_i in tissues activated by HA, data from Fig. 5 were plotted as shown in Fig. 6A. The relationship between force and Ca/Ca_max for control tissues was linear, with a slope just slightly greater than unity (1.18, Fig. 6A); that is, nearly a one-to-one relationship existed between HA-induced increases in Ca²⁺ and force. However, in tissues that were pretreated for 30 min with 10 µM PE, the relationship between increases in Ca²⁺ and force was different from that produced in control tissues. When Ca²⁺ data were considered that showed an increase from the lowest point to the earliest point achieving near-maximum [Ca²⁺]_i (i.e., ~250 nM [Ca²⁺]_i produced by 0.78 µM HA; see Fig. 4B), the slope of the relationship between Ca/Ca_max and force was significantly less than that of the control (0.62, P < 0.05), and the maximum increase in Ca/Ca_max produced at 0.78 µM HA corresponded with an increase in force of 0.6 ± 0.07 F/F_o (Fig. 6A, shaded square). This force value was significantly less than the control force value (1.03 ± 0.04) that produced an identical increase in Ca/Ca_max of 0.92-fold Ca_max (Fig. 6A, shaded circle). Thus PE-pretreated tissues displayed a reduced sensitivity of contractions to increases in [Ca²⁺]_i. Further increases in HA concentration above 0.78 µM produced additional increases in force without an additional increase in [Ca²⁺]_i (see Fig. 4B), suggesting that tissues regained normal sensitivity at higher HA concentrations or with a combination of higher HA concentrations and time. These data were similar to those produced when tissues were stimulated with increasing KCl concentrations rather than increasing HA concentrations (Fig. 6B); that is, after pretreatment of tissues for 30 min with 10 µM PE followed by a 10-min relaxation period, 25 mM KCl (Fig. 6B, shaded square) produced an increase in Ca/Ca_max statistically identical to that produced by control tissues stimulated with 40 mM KCl (Fig. 6B, shaded circle), but produced a much weaker increase in force (0.20 ± 0.08 vs. 0.73 ± 0.06 F/F_o, P < 0.05) (Fig. 6B). The relationship between Ca²⁺ and force for KCl-stimulated tissues fit an exponential better than a linear curve; that is, r² values for exponential and linear curve fits were, respectively, 0.92 and 0.83 for control data and 0.85 and 0.53 for PE-pretreatment data. The reason for the apparent difference in the form of the relationship between force and Ca²⁺ when HA and KCl were compared was not pursued further. The Ca²⁺-force curve produced by tissues stimulated with KCl and pretreated with PE was significantly different from the KCl-stimulated control curve (P < 0.05). Interestingly, when these four plots were compared together, for a given moderate increase in Ca²⁺ (e.g., 0.7-fold Ca_max; Fig. 7, dotted lines), tissues from the HA control group produced the highest force (~0.7-fold F_o), KCl after PE-pretreatment produced the lowest force (~0.2-fold F_o), and HA after PE pretreatment or KCl control fell in between these extremes (~0.4-fold F_o) (Fig. 7).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Ca2+ desensitization demonstrated by analyses of dependencies of force on Ca2+ in control tissues (open symbols) compared with tissues pretreated with 10 µM PE (filled symbols) and stimulated 10 min after PE washout with increasing concentrations of HA (A) or KCl (B). In PE-pretreated tissues, low concentrations of both HA and KCl produced increases in Ca2+ equivalent to those produced in control tissues, but these increases in Ca2+ produced significantly weaker increases in force (shaded symbols, means ± SE; see text). Thus PE-pretreated renal arterial muscle was desensitized to increases in Ca2+. m, Slope.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Summary of dependencies of contractile force on Ca2+ showing relative relationships in tissues stimulated with HA and KCl in naive tissues (not PE pretreated) and in tissues with a history of PE pretreatment (10 µM PE for 30 min followed by 10-min PE washout). Dotted lines indicate that a wide range of forces may be produced by a given increase in Ca2+ (see text for further description).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The degree of reactivity of renal artery to HA was found to be dependent on the recent history of receptor activation. For example, stimulation of rabbit renal artery ₁-adrenergic receptors with 10 µM PE for a short duration (30 min) reduced the ability of this tissue to subsequently respond to stimulation by another receptor type, H₁-histaminergic receptors, for 90 min after cessation of the initial PE stimulation. Reduced reactivity to HA may have been caused by reductions in H₁-receptor numbers, receptor-G protein coupling, or other components of stimulus-contraction coupling beyond receptor activation, such as Ca²⁺ mobilization or regulation of Ca²⁺ sensitivity of contractions. This is the first study to provide evidence in support of the hypothesis that the degree of receptor-stimulated Ca²⁺ sensitization of contractions can be reversibly attenuated by a prior short episode of receptor activation.

One important regulatory mechanism that operates downstream from receptor activation involves changes in the degree of Ca²⁺ sensitivity of contractions (12, 17, 29). Receptor stimulation immediately increases the Ca²⁺ sensitivity of contractions by activation of the low-molecular-weight G protein RhoA (6), resulting in a force-to-[Ca²⁺]_i ratio greater than that produced by K⁺-depolarization (12, 29); that is, the level of force produced for a given level of [Ca²⁺]_i is greater during receptor activation than during K⁺-depolarization (12). Moreover, the two major physiological mechanisms causing smooth muscle relaxation, namely, increases in cGMP and cAMP, cause reductions in Ca²⁺ sensitivity (12, 13, 14). However, contractile receptor ligands and agents that elevate cellular levels of cyclic nucleotides also alter [Ca²⁺]_i (12, 23). Thus smooth muscle utilizes at least two mechanisms to regulate the level of contraction, namely, changes in [Ca²⁺]_i and changes in Ca²⁺ sensitivity.

In the present study, prior exposure of rabbit renal artery to 10 µM PE for only 30 min caused a greater reduction in the sensitivity of HA-induced contractions than of HA-induced increases in [Ca²⁺]_i; that is, PE pretreatment reduced the ability of HA to increase force more than it reduced the ability of HA to increase [Ca²⁺]_i. Thus the most novel aspect of the work presented here is the evidence suggesting that ₁-adrenergic receptors can induce a mechanism that temporarily reduces the ability of receptor stimuli to subsequently produce increases in Ca²⁺ sensitivity of contractions. The significance of this finding is that it adds a new dimension to receptor-induced alterations in Ca²⁺ sensitivity. These data support the hypothesis that the degree of increase in Ca²⁺ sensitivity is a variable dependent on the activation history of the vascular smooth muscle cell. In this scenario, naive cells would produce greater increases in Ca²⁺ sensitivity than cells that had recently been stimulated with a maximum concentration of contractile receptor agonist.

Attenuation of cell responsiveness to extracellular ligands is a well-known cellular regulatory mechanism that may function to maintain target cell function within normal, physiological limits (19, 26). Currently, the primary explanation for receptor-induced attenuation of cell responsiveness is receptor-response uncoupling and subsequent reductions in receptor numbers (19). However, studies of smooth muscle show that a reduction in the numbers or affinity of -adrenergic receptors to agonists is not sufficient to explain reductions in agonist reactivity (3, 16). An alternative explanation is that some agonists not only stimulate contractions but also may induce desensitization of signaling systems downstream from the receptor. Perhaps the strongest argument that postreceptor mechanisms play a role in short-term receptor-induced attenuation of responsiveness is that the strength of KCl-induced contractions can be reduced in arteries with a history of receptor activation (22, 30). KCl bypasses receptors, producing contractions by causing membrane depolarization. Thus the subsequent reduction in tissue reactivity to KCl caused by pretreatment with a receptor ligand could not be the result of receptor downregulation but must be due to some postreceptor desensitization of cell signaling.

If receptor activation is required to activate RhoA to increase Ca²⁺ sensitivity, then in cells activated by KCl, RhoA should not be activated, and Ca²⁺ sensitivity should be low. However, a recent study (25) and data from the present study show that PE pretreatment reduces the Ca²⁺ sensitivity of KCl-induced contractions, suggesting that RhoA is activated by KCl, as well as by receptor ligands, or that RhoA is basally active. A recent study suggests that the former case is more likely because incubation of portal vein with DC3B, a cell-permeable inhibitor of RhoA, significantly inhibited KCl-induced contractions but had no effect on the pCa-tension curve in -toxin-permeabilized tissues (6). Moreover, Yanagisawa and Okada (31) have shown that K⁺ depolarization increases Ca²⁺ sensitivity of arterial contractions. These data taken together support the hypothesis that RhoA is not active in resting vascular smooth muscle but that both receptor activation and membrane depolarization induce RhoA activation leading to increases in Ca²⁺ sensitivity and that the ability of either a receptor ligand or KCl to activate RhoA may be reduced by prior strong receptor activation. Moreover, data from the present study support the hypothesis that only in naive arteries do receptor agonists increase Ca²⁺ sensitivity above that produced by KCl, whereas in arteries with a recent history of strong receptor activation, agonist-induced Ca²⁺ sensitization may not necessarily exceed that produced by KCl. For example, in arteries pretreated with 10 µM PE for 30 min and then relaxed for 10 min (i.e., in arteries with a history of ₁-adrenergic receptor activation), HA-induced Ca²⁺ sensitization was roughly equivalent to that induced by KCl (see Fig. 7).

Activation of the modulatory mechanism resulting in reduced reactivity to HA was dependent on both the concentration of PE and duration of ₁-receptor stimulation. Moreover, the modulatory mechanism was completely reversible, suggesting that it was a physiologically relevant mechanism and not due to rundown of the tissue. Reduced reactivity to HA was evident when tissues were pretreated with PE for as a short a duration as 10 min, but the concentration of PE required to reduce subsequent HA reactivity was greater than that required to produce half-maximum contractions. Thus, compared with -adrenergic receptor downregulation in vascular smooth muscle, which appears to take many hours to occur (3, 11, 16), this modulatory mechanism was "switched on" very rapidly. Moreover, although desensitization did not occur at low levels of receptor activation, very high concentrations were also not required. PE is less potent than norepinephrine at causing contractions in rabbit renal arteries. For example, 10 µM PE and 0.32 µM norepinephrine produce nearly equivalent activation of arteries (18). Because intra- and perisynaptic norepinephrine concentrations in the rabbit ear artery range from 0.5 to 0.7 µM, and those in guinea pig uterine artery and rat portal vein are ~10 µM (1), muscle activation by 10 µM PE may be regarded as reflecting levels of ₁-adrenergic stimulation within the moderate-to-high physiological range.

Vascular smooth muscle cells contain many more receptors than are required to produce maximum responses (i.e., the receptors are spare in number; Ref. 27). For example, to produce half-maximum contractions in rabbit ear artery requires activation of as little as 1% of the total -adrenergic receptor pool (20), and maximum contractions are achieved in rabbit renal arteries when only 23% of the total -adrenergic receptors are occupied (21). The precise role that spare receptors play in normal physiology is a matter of speculation. However, it is clear that spare receptors are not functionally distinct receptors but are simply "extra" receptors that, when stimulated, do not contribute further to the particular response measured because the response is already maximal (2). Results from the present study support the notion that, in rabbit arterial smooth muscle, one function of spare receptors is to "notify" the cell that enough receptor ligand exists in the extracellular milieu to produce maximum contractions. This "notification" involves, in part, activation of a signaling system targeting Ca²⁺ sensitivity of the contractile apparatus such that the ability of receptor stimuli to increase Ca²⁺ sensitivity is temporarily attenuated.

PE pretreatment caused a rightward shift in both HA-contraction and HA-[Ca²⁺]_i curves. The finding that the HA-contraction curve was shifter farther to the right than the HA-[Ca²⁺]_i curve indicated that PE-pretreatment induced a decrease in Ca²⁺ sensitivity. However, the fact that the HA-[Ca²⁺]_i curve was shifted to the right compared with that for control tissues suggests that PE pretreatment also induced a dissociation between receptor activation and mobilization of [Ca²⁺]_i. Additional studies are required to determine whether this effect involved alterations in the type or efficacy of linkage between activated receptors and G proteins or in regulation of Ca²⁺ influx, release, uptake, or efflux. Indeed, there are several exciting possibilities to be examined on the basis of recent literature. With the advent of the three-state model of receptor activation (15), changes in agonist potency may be explained by changes in active conformation and G protein coupling, and it may be proposed that PE pretreatment reduced the effectiveness of receptor-G protein coupling. Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis of G subunits and have been implicated as effector antagonists (10). Thus enhanced RGS activity produced by PE pretreatment could theoretically have reduced the potency of HA-induced increases in [Ca²⁺]_i. Lastly, Ca²⁺ channels and pumps are known to be regulated, and PE-induced downregulation of Ca²⁺ influx through L-type Ca²⁺ channels has been shown to occur in vascular smooth muscle (4).

In summary, the present study indicates that ₁-adrenergic receptor activation in rabbit renal artery not only caused strong contractions but also initiated a mechanism causing subsequent attenuation of reactivity to HA, as reflected by a large (~1/2 log) rightward shift in the HA-contraction curve and a moderate (~1/4 log) rightward shift in the HA-[Ca²⁺]_i curve. These dramatic effects were produced after only a 30-min stimulation with 10 µM PE, but the reduced reactivity completely reversed within 90 min. Because the rightward shift in the HA-[Ca²⁺]_i curve was significantly less than the rightward shift in the HA-contraction curve, these data suggest that the reduced reactivity involved, in part, a reduction in the ability of HA to increase the Ca²⁺ sensitivity of contractions. These data support the hypothesis that the degree of stimulus-induced Ca²⁺ sensitization of contractions is dependent on the history of receptor activation.