(Hypertension. 1995;25:1294-1300.)
© 1995 American Heart Association, Inc.
Articles |
Tachyphylaxis of Human Forearm Vascular Responses Does Not Occur Rapidly After Exposure to Isoproterenol
From the Departments of Pharmacology and Medicine, Vanderbilt University School of Medicine, Nashville, Tenn.
Correspondence to Dr Alastair J.J. Wood, Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Medical Research Bldg, Room 546, Nashville, TN 37232-6602.
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Abstract In vitro and limited in vivo data suggest that rapid desensitization of ß-adrenoceptor responses occurs after exposure to agonist. Tachyphylaxis to a ß-adrenoceptor agonist would represent a potentially important mechanism for the short-term regulation of vascular tone. The effects of a 4-hour infusion of 400 ng/min intra-arterial isoproterenol on forearm blood flow and presynaptic ß-adrenoceptor–mediated norepinephrine release were determined in eight healthy volunteers. Intra-arterial isoproterenol at 400 ng/min resulted in a significant increase in forearm blood flow in all eight subjects at all time points, with no evidence of tachyphylaxis. In fact, forearm blood flow after 4 hours of the isoproterenol infusion (22.8±3.3 mL/100 mL per minute) was significantly greater than after 7 minutes (14.6±2.8 mL/100 mL per minute), 15 minutes (15.4±2.4 mL/100 mL per minute), and 30 minutes (17.4±3.0 mL/100 mL per minute) of the infusion (P<.05). Similarly, presynaptic ß-adrenoceptor responses showed no evidence of tachyphylaxis, so forearm norepinephrine spillover values after 7 minutes (6.6±0.94 ng/min), 15 minutes (7.6±1.5 ng/min), and 4 hours (8.8±1.1 ng/min) of isoproterenol infusion were increased and similar. Minimal systemic effects were observed, and there was no evidence of tolerance, there being no difference in heart rate after 7 minutes (70.7±2.7 beats per minute) and 4 hours (72.2±3.6 beats per minute) of isoproterenol infusion. Similarly, systemic norepinephrine spillover values at 7 minutes (422.6±60.6 ng/min), 15 minutes (436.3±48.7 ng/min), and 240 minutes (491.4±52.9 ng/min) all increased from a baseline value of 294±28.2 ng/min (P<.05) but were not significantly different from each other. Tachyphylaxis does not occur rapidly after exposure of forearm vascular smooth muscle to the ß-adrenoceptor agonist isoproterenol and is unlikely to play a major role in the rapid regulation of vascular response to a ß-adrenoceptor agonist.
Key Words: isoproterenol • norepinephrine • vasodilation • receptors, adrenergic, beta
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Introduction |
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Receptor desensitization occurs after exposure of ß-adrenoceptors to agonists by mechanisms that have been studied intensively1 and has been demonstrated in vitro in cultured cells, leukocytes, isolated organs, and isolated vascular smooth muscle.2 It is more difficult to demonstrate desensitization in vivo, but it has been studied in both humans3 4 and animals.5 6 7 In vivo studies have usually examined the hemodynamic changes occurring in response to a systemic infusion of isoproterenol7 8 ; consequently, it has been impossible to separate receptor desensitization from alterations in reflex sensitivity. Ideally, to limit the confounding effects of such reflex sympathetic activation, one should examine tolerance to a ß2-adrenoceptor agonist by administering the agonist directly into the vascular bed under study in doses that have minimal systemic effects.
We have recently demonstrated in the human dorsal hand vein that desensitization of postsynaptic ß-adrenoceptors occurred in vivo after prolonged (1-week) exposure to terbutaline, a ß-adrenoceptor agonist.9 Vincent and colleagues,10 also using the dorsal hand vein technique, demonstrated that short-term exposure to the ß-adrenoceptor agonist isoproterenol for 4 hours resulted in desensitization of subsequent venodilation in response to isoproterenol, suggesting that venous desensitization occurs rapidly in vivo. Stimulation of vascular ß2-adrenoceptors produces vasodilation, and regulation of vascular smooth muscle tone by ß-adrenoceptors may be clinically important because ß-adrenoceptor responsiveness has been found to be abnormal in hypertensive individuals11 and patients with heart failure12 and to be influenced by sodium intake.11 13 Regulation of vascular ß-adrenoceptors might also be expected to occur after exogenously administered agonists, such as occurs in the treatment of asthma, or after increased endogenous concentrations of the ß2-adrenoceptor agonist epinephrine, such as occurs in response to mental stress and in heart failure.14 15 If tolerance of vascular smooth muscle ß-receptor–mediated vasodilation occurs rapidly after exposure to an agonist, it would suggest that in addition to previously demonstrated long-term effects, tachyphylaxis may play a major role in the short-term homeostatic control of vascular smooth muscle response in health and disease.
Local ß-adrenoceptors may have actions, other than postsynaptic
receptor–mediated vasodilation, that could regulate vascular tone. The
release of norepinephrine from sympathetic nerves is subject to
presynaptic modulation by 
-adrenergic, ß-adrenergic, angiotensin,
and other receptors.16 17 We have recently demonstrated
that stimulation of presynaptic ß2-adrenoceptors by the
administration of low doses of isoproterenol directly into the brachial
artery in vivo in humans resulted in increased norepinephrine release
locally.18 The overall effect of a ß-adrenoceptor
agonist on forearm blood flow (FBF) therefore may be influenced not
only by postsynaptic ß2-adrenoceptors but also by
presynaptic ß-adrenoceptor response. Differential sensitivity of
presynaptic and postsynaptic ß2-adrenoceptors could
therefore result in the masking of alterations of
postsynaptic ß-adrenoceptor sensitivity by changes in presynaptic
response, or conversely, apparent alterations of postsynaptic
responses, as measured by FBF, could in fact be due to alterations in
presynaptic receptor responsiveness.
Therefore, the purpose of the present study was to determine whether short-term exposure to a locally administered ß2-adrenoceptor agonist for 4 hours resulted in tachyphylaxis of presynaptic and/or postsynaptic ß-adrenoceptor response in vascular smooth muscle in vivo.
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Subjects
Eight healthy, nonsmoking male volunteers aged 20 to 39 years (32.9±2.3 years) were studied. No subject had clinically significant abnormalities on history, physical examination, or routine laboratory tests, including complete blood count, prothrombin and partial thromboplastin times, renal and liver function tests, and electrocardiogram. Subjects did not take any medications for at least 2 weeks before the study and were maintained on a diet that was free of caffeine and alcohol for 24 hours before the study. All subjects provided written informed consent. The study protocol was approved by the Vanderbilt Committee for the Protection of Human Subjects, and procedures followed were in accordance with institutional guidelines.
Experimental Protocol
All experiments were performed in the morning with the subjects
resting supine in bed. An intravenous cannula was placed in an
antecubital vein of both arms. After subdermal administration of 1%
lidocaine, an 18-gauge polyethylene catheter (Cook Inc) was inserted
into the brachial artery of the nondominant arm for local infusions and
blood sampling. Arterial catheter patency was maintained with a saline
infusion of 30 mL/h. During intra-arterial administration of
isoproterenol, the total flow rate through the cannula was always
maintained at 30 mL/hr. Arterial blood pressure was measured by means
of a pressure transducer (Hewlett-Packard), and heart rate was recorded
from a continuous electrocardiographic monitor. After the arterial line
and intravenous catheters had been placed, subjects rested quietly for
30 minutes. [3H]Norepinephrine
(norepinephrine levo-[ring-2,5,6-3H], 43.7 to 56.9
Ci/mmol, New England Nuclear) was infused into the arm contralateral to
the arterial line. An initial loading dose of 25 µCi
[3H]norepinephrine was administered over 2 minutes
followed by a constant infusion of 0.90 µCi/min. The
[3H]norepinephrine was prepared for human
administration by the Vanderbilt Hospital Radiopharmacy, and
appropriate sterility and pyrogen testing were performed. Immediately
before use, [3H]norepinephrine was diluted to a
concentration of 2 µCi/mL in normal saline with 1 mg/mL ascorbic acid
added to the infusion solution. FBF was measured, and simultaneous
arterial and venous blood samples were drawn for determination of
baseline values after 30 and 40 minutes of
[3H]norepinephrine infusion. Isoproterenol (400 ng/min,
Isuprel, Winthrop Pharmaceuticals) was then infused intra-arterially by
an infusion pump (Harvard Apparatus) for 240 minutes. FBF was measured
as described below at baseline and after 7, 15, 30, 60, 90, 120, 150,
180, 210, and 240 minutes of isoproterenol infusion at 400 ng/min.
Simultaneous arterial and venous blood samples were drawn after
measurement of FBF at the 7-, 15-, and 240-minute time points. Blood
was collected in cooled tubes with EGTA and reduced glutathione
(Amersham Corp), placed on ice, and centrifuged at 4°C. Samples of
the [3H]nor- epinephrine infusion solution were
collected, stored, and later assayed in triplicate, as described for
the blood samples, to allow determination of the actual rate of
[3H]norepinephrine infusion. Plasma was stored at
-20°C until assayed.
Forearm Blood Flow
FBF was measured in the arm into which intra-arterial
isoproterenol was infused with mercury-in-Silastic strain-gauge
plethysmography.19 The wrist was supported in a sling that
raised the level of the forearm to above that of the atrium. The hand
was excluded from FBF measurement by inflation of a pediatric
sphygmomanometer cuff to 200 mm Hg around the wrist before and during
measurement. The volume of the forearm, excluding the hand and wrist,
was measured by water displacement.
Catecholamine Assay
Norepinephrine concentrations were measured by high-performance
liquid chromatography with the use of electrochemical detection and
3,4-dihydroxybenzylamine as the internal standard as described
previously.20 The chromatographic eluate coinciding with
the norepinephrine peak was collected and counted by liquid
scintillation. This allowed determination of plasma
[3H]norepinephrine concentration without interference
from tritiated metabolites. The intraday and interday coefficients of
variation were 7.8% and 7.6%, respectively.
For determination of whether isoproterenol induced endothelin release, endothelin-1 concentrations were measured by radioimmunoassay. Plasma samples were passed through a C8 Bond Elut column (Analytichem International), and endothelin-1 concentration was determined with 125I–endothelin-1 (Amersham), anti–endothelin-1 antibody, and endothelin-1 standard (Peninsula Laboratories). For further determination of whether isoproterenol induced an initial increase in the release of the vasoconstrictor angiotensin II (Ang II), six additional subjects were studied. Simultaneous arterial and venous blood samples were drawn at baseline and after isoproterenol (400 ng/min) had been infused into the brachial artery for 7 minutes. Samples were drawn into tubes containing 0.4 mL of a solution of 6.25 mmol/L 8-hydroxyquinoline, 30 mmol/L o-phenanthroline, 125 mmol/L EDTA, and 2 mmol/L captopril to prevent ex vivo Ang II generation. Ang II concentrations were determined by radioimmunoassay as previously described.21
Data Analysis
Prestimulation values obtained after 30 and 40 minutes of
[3H]norepinephrine infusion were similar, and their mean
was used as the baseline measurement. Calculations for the
determination of norepinephrine kinetics using the isotope dilution
method22 23 were performed as follows:
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where NE is norepinephrine, and A* and V* are arterial and venous concentrations of [3H]norepinephrine, respectively.
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where A and V are arterial and venous concentrations of endogenous norepinephrine, respectively, and Q is the forearm plasma flow derived from hematocrit, FBF, and forearm volume.
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Forearm norepinephrine plasma appearance rate and intrinsic clearance were obtained by dividing forearm norepinephrine spillover and forearm norepinephrine clearance by 1-FE.24
Norepinephrine plasma clearance from the whole body (systemic clearance) was calculated as ([3H]NE Infusion Rate)/A*. The rate at which norepinephrine entered plasma for the whole body (systemic spillover) was calculated as Systemic ClearancexA.
Data were analyzed by repeated-measures ANOVA, and the significance of differences between measures was determined by the Tukey-Kramer multiple comparisons test with a value of P<.05 accepted as the minimal level of significance.
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Results |
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Postsynaptic ß2-adrenoceptor stimulation by 400 ng/min intra-arterial isoproterenol resulted in a significant increase in FBF in all subjects at all time points (P<.001; Table 1 and Fig 1). There was no evidence of tachyphylaxis of the postsynaptic ß-adrenoceptor response; in fact, FBF after 4 hours of isoproterenol infusion (22.8±3.3 mL/100 mL per minute) was significantly greater than after 7 minutes (14.6±2.8 mL/100 mL per minute), 15 minutes (15.4±2.4 mL/100 mL per minute), and 30 minutes (17.4±3.0 mL/100 mL per minute) of the infusion (P<.05) but did not differ from FBF observed after 60 minutes of the infusion (21.8±3.5 mL/100 mL per minute). FBF at 7, 15, and 30 minutes was similar, but that at 60 minutes and subsequent time points was significantly greater (P<.05, Fig 1).
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To examine the possibility that the higher FBF in response to isoproterenol that we observed at the 60-minute and subsequent time points was due to differential sensitivity of presynaptic ß-adrenoceptor–mediated norepinephrine release or due to initial release and subsequent depletion or reduced release of a vasoconstrictor, we measured norepinephrine spillover across the forearm as well as the arterial and venous concentrations of endothelin and Ang II, two potent endogenous vasoconstrictors. Table 2 shows arterial and venous concentrations and the difference between venous and arterial concentrations of endothelin and Ang II. As shown by the arteriovenous difference, there was no evidence that either isoproterenol or increased blood flow increased production or release of Ang II (P=.63) or endothelin (P=.55). Forearm norepinephrine spillover increased significantly above baseline (P<.001) at all time points sampled during isoproterenol infusion, but spillover values after 7 minutes (6.6±0.94 ng/min), 15 minutes (7.6±1.5 ng/min), and 4 hours (8.8±1.1 ng/min) of isoproterenol infusion were not significantly different (Fig 2). After isoproterenol infusion, plasma norepinephrine appearance rate, a measure of forearm norepinephrine spillover that is independent of blood flow, also increased significantly above the baseline value of 1.09±0.17 ng/min, but the values after 7 minutes (9.8±1.7 ng/min), 15 minutes (12.9±3.3 ng/min), and 240 minutes (12.5±1.9 ng/min) were not significantly different. One subject was excluded from the analysis of the norepinephrine kinetics data because his norepinephrine spillover at the 7-minute time point was 20 SD from the group mean. Since values for this subject at baseline, 15, and 240 minutes were similar to those of the other subjects, this 7-minute time point result was thought to be due to a technical error, and all of his norepinephrine data were therefore excluded. This subject's data were included in the analysis of FBF, heart rate, and blood pressure.
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The isoproterenol dose infused had minimal systemic effects, with heart rate increasing by approximately 8 beats per minute from a baseline value of 62.8±2.1 beats per minute and with a small increase in systolic pressure and no change in diastolic pressure (Table 1). There was no evidence of tachyphylaxis, in that heart rate after 7 minutes of the isoproterenol infusion (70.7±2.7 beats per minute) was not different from that after 4 hours of the infusion (72.2±3.6 beats per minute). Similarly, systemic norepinephrine spillover at 7 minutes (422.6±60.6 ng/min), 15 minutes (436.3±48.7 ng/min), and 240 minutes (491.4±52.9 ng/min) all increased from a baseline value of 294.0±28.2 ng/min (P<.05) but were not significantly different.
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Discussion |
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Intra-arterial isoproterenol infusion for 4 hours did not result in tachyphylaxis of the FBF response to isoproterenol. In fact, during isoproterenol infusion, FBF after 60 minutes and at all subsequent time points was greater than after 7 and 15 minutes of the infusion (Fig 1). The reasons for this are unclear. The rapidity with which isoproterenol reached a steady state of vasodilation would depend on pharmacokinetic and pharmacodynamic variables. From existing data we expected that isoproterenol would have reached a steady-state level of vasodilation more rapidly. ß-Adrenoceptor–mediated responses have been reported to plateau rapidly and decrease within minutes after isoproterenol administration to isolated tissues in studies that have examined cAMP5 or vasodilator responses.25 These studies therefore suggested that the agonist associates rapidly with ß-adrenoceptors to produce a rapid physiological response. Furthermore, studies that have examined hemodynamic effects occurring after intravenous isoproterenol administration to humans confirmed these observations and found that the physiological responses occurring after isoproterenol administration were at steady state by 8 minutes.26 27 Isoproterenol infusion directly into the brachial artery, as was performed in the present study, allowed drug administration directly into the forearm vascular bed being studied, with a far smaller, rapidly equilibrating, relevant volume of distribution than that occurring after intravenous drug administration. Therefore, after intra-arterial isoproterenol administration, a rapid attainment of steady-state vasodilation was expected; therefore, the 7- and 15-minute time points were selected as representative of the effects of isoproterenol stimulation before any potential tachyphylaxis occurred. We are not aware of data showing that FBF responses to intra-arterial isoproterenol increase up to 60 minutes into the infusion.
We considered several possible explanations for these observations. Although diurnal variation in FBF occurs,28 these changes are small and would not account for changes of the magnitude observed. It is also unlikely that the delayed maximal response to isoproterenol was due to a delay in steady-state local concentrations being achieved because blood concentrations of isoproterenol in the forearm are likely to have achieved steady state rapidly within minutes after direct intra-arterial drug administration. The possibility that infused isoproterenol was taken up in nerve terminals and later rereleased is not likely because Goldstein and colleagues23 have shown that isoproterenol is not rereleased from nerve terminals. Another possibility is that recirculating isoproterenol in the systemic circulation resulted in an increased dose delivered to the forearm and that the time required for the systemic concentrations of isoproterenol to reach steady state accounted for the delayed maximal response to isoproterenol. To further address this possibility, we measured systemic venous isoproterenol concentrations in blood drawn from the arm opposite to the isoproterenol infusion in three subjects. The mean plasma concentration of isoproterenol after the 4-hour intra-arterial infusion of 400 ng/min isoproterenol was 72.7±5.0 pg/mL. From the FBF in these three subjects we calculated that these systemic isoproterenol concentrations would have resulted in the delivery of an additional 18.0±4.7 ng/min of isoproterenol to the forearm. It is unlikely that this 4.5% additional isoproterenol dose delivered to the forearm accounted for the observed increase in FBF at 60 minutes and subsequent time points because we have previously found FBF in response to 400 and 500 ng/min isoproterenol to be similar (unpublished data, 1994). Although we did not specifically measure local clearance of isoproterenol, the possibility of alterations in the local clearance of isoproterenol accounting for these findings is unlikely because the amount of isoproterenol appearing across the forearm (calculated from the venous isoproterenol concentrations and forearm plasma flow in the arm into which isoproterenol was infused in eight subjects) after 7 minutes of isoproterenol infusion (5.9±1.0 ng/100 mL forearm volume per minute) and after 480 minutes of isoproterenol infusion (7.0±1.1 ng/100 mL forearm volume per minute) was similar.
An alternative possibility is that isoproterenol, or the increase in FBF, facilitated the release of norepinephrine or increased the release of an endogenous vasoconstrictor and that this response decreased over time. We have recently demonstrated in the human forearm that presynaptic ß2-adrenoceptors regulate norepinephrine release.18 Consequently, in addition to examining the effect of prolonged isoproterenol infusion on FBF, we determined norepinephrine release by measuring norepinephrine spillover and norepinephrine plasma appearance rate, a measure of spillover that is independent of changes in flow.24 Using this technique we found, as we have previously demonstrated,18 an increase in local norepinephrine spillover during isoproterenol infusion, and this occurred at all time points. There was no evidence of tachyphylaxis of the presynaptic ß2-adrenoceptor response to isoproterenol in that norepinephrine spillover did not decrease during isoproterenol infusion. Forearm norepinephrine spillover after 7 minutes (6.6±0.9 ng/min), 15 minutes (7.6±1.5 ng/min), and 240 minutes (8.8±1.1 ng/min) was not different. Similarly, plasma norepinephrine appearance rate, a flow-independent measure of norepinephrine release, did not decrease during isoproterenol infusion. Therefore, rapid tachyphylaxis of presynaptic ß-adrenoceptor–mediated norepinephrine release does not account for the increased blood flow observed at the 60-minute and subsequent time points.
Isoproterenol has been shown to stimulate Ang II release in animals and hypertensive subjects,21 29 and shear stress mediated by increased flow has been shown to facilitate the release of endothelin,30 another potent endogenous vasoconstrictor. To address these possibilities, we measured arterial and venous endothelin concentrations in the subjects undergoing the 4-hour isoproterenol infusion and Ang II concentrations in an additional six volunteers before and after intra-arterial isoproterenol infusion. We found that 400 ng/min isoproterenol infused into the brachial artery resulted in a slight net negative production of Ang II across the forearm (Table 2). Taddei and colleagues,29 who demonstrated isoproterenol-induced Ang II production in the human forearm, studied hypertensive subjects and used higher doses of isoproterenol than we did, possible reasons for the differing results. Since 400 ng/min isoproterenol infused for 7 minutes did not induce Ang II production in the forearm in the present study, it is unlikely that initial Ang II production, and then a subsequent decrease in Ang II production during the prolonged isoproterenol infusion, accounted for the increase in FBF that we observed at later time points. Similarly, endothelin production does not explain this finding because overall endothelin production across the forearm as represented by differences between venous and arterial samples was not different after 15 and 240 minutes of isoproterenol infusion. Therefore, although we found no evidence that isoproterenol, or increased blood flow, resulted in the release of endothelin or Ang II, it is possible that the release of an endogenous vasodilator with consequent enhanced blood flow responses to isoproterenol could have occurred. Endothelium-derived relaxing factor is such an endogenous vasodilator, and its release is stimulated by increased shear stress.31 The present studies could not examine this possibility.
The rate at which desensitization of vascular responses to agonist occurs in vivo is unknown. We have previously shown in lymphocytes studied ex vivo that the proportion of ß-adrenoceptors in the high-affinity state decreased, as did ß-adrenoceptor–stimulated adenylate cyclase activity, when subjects were ambulant for 3 hours.4 Vincent and colleagues,10 using the dorsal hand vein model, showed that a 4-hour isoproterenol infusion resulted in the maximal vasodilator response to isoproterenol being reduced from 61±13% to 19±4%. In the present study we were unable to demonstrate tachyphylaxis of the arterial vascular smooth muscle response after 4 hours of isoproterenol infusion. There are several possible explanations for the differences between the findings of our study and those of Vincent and colleagues. Venous and arterial ß-adrenergic receptors may respond differently to exposure to agonist, with tolerance occurring at different rates. This possibility is difficult to exclude, and although Creager and colleagues,32 studying forearm vascular responses to isoproterenol, did not observe ß-receptor desensitization in patients with heart failure, the relative homogeneity of ß-receptor responses in tissues as disparate as lymphocytes and cardiac muscle argues that heterogeneity of ß-receptors in arteries and veins is not a likely explanation. A more plausible explanation is the different concentrations of isoproterenol that the vascular smooth muscle studied was exposed to during the 4-hour isoproterenol infusion in the two studies. Vincent and colleagues infused 271 ng/min isoproterenol, a lower dose than we used; however, this was infused via a butterfly needle directly into the dorsal hand vein, with measurements subsequently taken 1 cm upstream from the needle tip. In the arterial study we infused 400 ng/min isoproterenol and measured FBF. The forearm vasculature, because of dilutional effects, would have been exposed to lower concentrations of isoproterenol than the segment of vein immediately proximal to the needle tip in the dorsal hand vein model. Lower concentrations of ß-adrenoceptor agonist reflect physiological conditions more closely and suggest that rapid tolerance to a ß-adrenoceptor agonist does not occur in vivo after exposure of vascular smooth muscle to agonist.
The technique used in the present study therefore allowed the local intra-arterial infusion of the ß-adrenoceptor agonist isoproterenol to stimulate both presynaptic and postsynaptic ß2-adrenoceptors in the forearm and determination of their response. The isoproterenol dose used (400 ng/min) has previously been shown by ourselves13 18 and others33 to have minimal systemic effects and to produce no change in FBF in the contralateral forearm when infused intra-arterially in the same fashion as used in the present study. Although the effects of isoproterenol in the present study were largely limited to the forearm, the increase in forearm norepinephrine spillover was accompanied by a much smaller increase in systemic norepinephrine spillover and a small increase in heart rate. In contrast to the 10-fold or greater increase in forearm norepinephrine spillover, systemic norepinephrine spillover increased by less than 50%. This increase in systemic norepinephrine spillover may reflect either stimulation of presynaptic ß-adrenoceptors at tissue sites outside the forearm or reflex sympathetic stimulation caused by baroreceptor stimulation from undetected vasodepressor effects. These systemic effects provided the opportunity for us to examine the generalizability of the responses that we have described in the forearm. Neither of the systemic effects observed after isoproterenol infusion—increased heart rate and increased systemic norepinephrine spillover—attenuated during the isoproterenol infusion, suggesting that tolerance did not occur within 4 hours.
Receptor desensitization is defined as a waning of response in the face of continuous exposure to agonist; in fact, early in vitro studies that described ß-adrenoceptor desensitization demonstrated a reduced cAMP response to agonist in the continued presence of agonist.2 Vascular responses to a single dose of agonist, as used in the present study, have been successfully used in vitro to demonstrate desensitization.25 The use of either very high or very low doses of isoproterenol could obscure the detection of alterations in vascular sensitivity to isoproterenol in a study design examining vascular responses to a single dose of isoproterenol.25 The isoproterenol dose used in the present study (400 ng/min) was neither very low nor supramaximal, as an isoproterenol dose approximately twice the dose we administered resulted in 40% further vasodilation.33 Another possible reason for the failure of isoproterenol-mediated responses to decrease is that FBF responses may have been influenced by the corelease of other undetermined vasodilators, and it is possible that desensitization of the ß-adrenoceptor response may have occurred but been obscured by increased vasodilation as a result of the corelease of other vasodilators. Tachyphylaxis is a decreased response to a drug through mechanisms that do not necessarily involve only receptor desensitization. In the present study, although we cannot exclude the possibility that ß-adrenoceptor desensitization occurred and was masked by increased release of other vasodilators, responses to isoproterenol did not decrease during the 4-hour infusion and there was no tachyphylaxis. Performance of a dose-response experiment might be helpful in elucidating the mechanisms of desensitization had that occurred; however, since we did not observe tachyphylaxis, we did not perform this experiment. An alternative to the present study design in which vascular response to the continued infusion of a single dose of agonist was examined repeatedly would have been to allow a washout phase after the prolonged infusion of isoproterenol and before the second determination of response. This alternative design has the following disadvantages: undetected resensitization of ß-receptor–mediated responses could occur during the washout phase; activation of homeostatic mechanisms in response to the sudden cessation of profound increases in flow could occur; and the possible release of other vasodilators or vasoconstrictors as a result of the prolonged isoproterenol infusion need not depend on continued infusion and would therefore not be prevented by the washout phase.
Receptor desensitization has been extensively studied in vitro, and the mechanisms involved have been elucidated.34 35 36 Desensitization may occur because of loss of receptors,3 sequestration of the receptors away from the cell surface,37 or uncoupling of receptors from the guanine nucleotide regulatory protein (GS).38 These processes occur at different rates, and studies of isolated lymphocytes ex vivo have suggested that desensitization can occur rapidly, within minutes.39 40 The time course of receptor desensitization in vivo has not been well defined.38 In vascular tissue studied in vitro, desensitization to a ß-adrenoceptor agonist occurs within minutes25 and therefore complicates the detection of such desensitization in vivo because ß-adrenoceptor–mediated vasodilation, flow-mediated release of endogenous vasoconstrictors and vasodilators, and desensitization of the ß-adrenoceptor–mediated vasodilation all occur simultaneously. For these reasons we cannot exclude the possibility that ß-adrenoceptor desensitization occurred very rapidly or virtually instantaneously but was not detected; however, responses to isoproterenol did not decrease during the 4-hour infusion, and there was no tachyphylaxis. A kinetic model that describes both the relaxation and desensitization occurring after isoproterenol administration has been used to analyze vascular responses to isoproterenol in vitro.25 Such an analysis would require more data points than were obtained in the present study but may provide a useful tool in future studies addressing the complex issues of ß-adrenoceptor desensitization.
The importance of decreased responsiveness after short-term exposure to agonist would include not only decreased response to therapeutic agents but also decreased ß-adrenoceptor agonist–mediated vasodilation after relatively short-term increases in plasma epinephrine concentrations, such as occur after mental stress—a possible explanation for the association noted between blood pressure reactivity to mental stress and future hypertension.41 Our data suggest that in vascular smooth muscle, tachyphylaxis to a ß-adrenoceptor agonist does not occur rapidly in vivo, and therefore this mechanism is unlikely to play a major role in the short-term regulation of vascular tone. However, as we have shown previously,9 vascular desensitization does occur after long-term exposure to a ß-adrenoceptor agonist and may have a role in the long-term regulation of vascular tone.
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Acknowledgments |
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This work was supported in part by grants from the American Heart Association, Tennessee Affiliate, and US Public Health Service grants GM 31304, GM 46622, GM 07569, and GM 5M01-RR00095. C. Michael Stein was supported in part by a Merck Sharp & Dohme International Fellowship in Clinical Pharmacology.
Received March 18, 1994; first decision May 26, 1994; accepted February 16, 1995.
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- Harden TK. Agonist induced desensitization of the ß adrenergic receptor linked adenylate cyclase. Pharmacol Rev. 1983;35:5-32. [Medline] [Order article via Infotrieve]
- Fraser J, Nadeau J, Robertson D, Wood AJJ. Regulation of human leukocyte ß receptors by endogenous catecholamines: relationship of leukocyte ß receptor density to the cardiac sensitivity of isoproterenol. J Clin Invest. 1981;67:1777-1784.
- Feldman RD, Limbird LE, Naudeau J, Fitzgerald GA, Robertson D, Wood AJJ. Dynamic regulation of leukocyte ß adrenergic receptor-agonist interactions by physiological changes in circulating catecholamines. J Clin Invest. 1983;72:164-170.
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