Circadian Variation in Vascular Tone and Its Relation to α-Sympathetic Vasoconstrictor Activity

Study Population

We studied 12 normal subjects (7 men and 5 women). Their mean age (±SD) was 44±9 years. None had clinical or laboratory evidence of any past or present cardiovascular disease or any other systemic condition, and none were taking any medications. All the participants gave written informed consent for all procedures. This study was approved by the investigational review board of the National Institutes of Health.

Study Protocol

All studies were performed in a quiet room at a temperature of approximately 22°C (72°F). The participants were asked to refrain from smoking and from drinking alcohol or beverages containing caffeine for at least 24 hours before and during the study. Their diet was not otherwise restricted. Women were not studied during their menses.

Each subject underwent three identical studies at different times within the same 24-hour period. The studies were performed in the early morning (6 a.m. to 8 a.m.), in the afternoon (1 p.m. to 3 p.m.), and in the evening (8 p.m. to 10 p.m.). In order to avoid bias that might result if the study protocol were started at the same time of day for all subjects, the subjects were randomly assigned with respect to the time of day when the first study was performed. The first study was performed in the morning in five subjects, in the afternoon in three, and in the evening in four.

All the subjects were admitted to the cardiology ward of the National Heart, Lung, and Blood Institute 6 to 12 hours before the first study. Immediately after admission, a 20-gauge, 4.4-cm Teflon catheter was inserted into the brachial artery of the nondominant arm. The catheter remained in place throughout the study period, for approximately 24 hours. During this time, the arterial line was connected to a portable monitor equipped with sound alarms, to permit the immediate recognition and correction of catheter displacement. The subjects were allowed to have their regular meals, use the bathroom, watch television, and move about within the room. No breakfast or walking was permitted before the morning study; the afternoon and evening studies were performed at least 2 hours after meals and after at least 30 minutes of rest lying down.

Each study consisted of the infusion of drugs into the brachial artery and the measurement of the response of the vasculature (changes in regional blood flow) by means of plethysmography of the forearm. The arm into which the catheter was inserted was elevated slightly above the level of the right atrium, and a mercury-filled Silastic strain gauge was placed on the widest part of the forearm.^{21 , 22} This gauge was connected to a plethysmograph (model EC-4, D.E. Hokanson, Issaquah, Wash.)²³ calibrated to measure the percent change in volume; the plethysmograph in turn was connected to a chart recorder to record the flow measurements in the forearm. For each measurement, a cuff placed on the upper arm was inflated to 40 mm Hg with a rapid cuff inflator (model E-10, Hokanson) to occlude venous outflow from the extremity. A wrist cuff was inflated to suprasystolic pressures one minute before each measurement to exclude the hand circulation.²⁴ Flow measurements were recorded for approximately 7 seconds every 15 seconds; each mean value was calculated from seven readings.

Basal measurements were obtained after a three-minute infusion of 5 percent dextrose solution at 1 ml per minute. Sodium nitroprusside was then infused in doses of 0.8, 1.6, and 3.2 μg per minute (infusion rates, 0.25, 0.5, and 1 ml per minute, respectively), each for five minutes. Blood flow in the forearm was measured during the last two minutes of each dose. After a 30-minute rest period, another measurement was made to corroborate the return to base-line values, and phentolamine was then infused at a dose of 12 μg per minute per 100 ml of forearm volume for 10 minutes (infusion rate, 0.6 ml per minute). At these doses, phentolamine produces effective and complete α-sympathetic blockade.²⁵

The subjects did not know which drug was being infused. Blood pressure in the contralateral arm was measured by sphygmomanometry immediately before each measurement. Mean arterial pressure was calculated by adding one third of the pulse pressure to the diastolic pressure. Forearm vascular resistance was calculated as the mean arterial pressure divided by the forearm blood flow.

To rule out the possibility that changes in vascular tone on the day of the study were due to a long-lasting effect of phentolamine, 6 of the 12 subjects underwent additional studies on a different day. In these six subjects, phentolamine was infused into the brachial artery of the nondominant arm (12 μg per minute per 100 ml of forearm volume) for 10 minutes. With the uncatheterized arm used as a control, forearm blood flow was measured by strain-gauge plethysmography in both arms at base line, at the end of the 10-minute infusion of phentolamine, and every 5 minutes thereafter, until the flow in the catheterized arm had returned to values similar to those in the control arm for at least 15 minutes. These studies were done to demonstrate that the results of each subsequent study, performed at least seven hours after the previous infusion of phentolamine, were not influenced by the earlier infusion. The vasodilator action of phentolamine on the vasculature of the forearm, at the doses and by the route of administration used in our study of circadian changes, lasted approximately one hour. Forearm blood flow increased 58±28 percent during the infusion of phentolamine. After the infusion was discontinued, the flow gradually decreased in each subject, reaching values similar to those for the control arm an average of 64±10 minutes later (range, 45 to 80).

Statistical Analysis

Student's t-test was used to compare mean values. The responses to sodium nitroprusside and phentolamine were compared by analysis of variance. All calculated P values are two-tailed. All P values less than 0.05 were considered to indicate statistical significance. All group data are reported as means ±SD unless otherwise indicated.

Circadian Changes in Base-Line Vascular Tone

Figure 1. Figure 1. Mean Forearm Blood Flow and Vascular Resistance at Three Times of Day in 12 Healthy Subjects.

Values shown were obtained at base line (open circles) and after α-sympathetic blockade with the infusion of phentolamine (solid circles). The stippled areas indicate the vascular tone contributed by α-sympathetic vasoconstrictor forces. P values refer to the slope of the curve and were obtained by analysis of variance. Vertical bars indicate standard errors.

In these 12 normal subjects, the mean forearm vascular resistance in the morning (31±8 mm Hg per milliliter per minute per 100 ml of forearm volume) was significantly higher than in the afternoon and evening (25±6 and 22±7 mm Hg per milliliter per minute per 100 ml of forearm volume, respectively). Conversely, the blood flow was significantly lower in the morning than at other times of day (Fig. 1). This pattern of increased vascular resistance and reduced blood flow in the morning was found in 10 of the 12 subjects. The mean base-line resistance was also significantly higher, and the blood flow significantly lower, in the afternoon than in the evening (P<0.05). The pattern of lowest resistance and highest flow during the evening study was found in 8 of the 12 subjects.

Effect of α-Sympathetic Blockade

After the infusion of phentolamine, the forearm vascular resistance dropped and the blood flow increased significantly in each subject at each time of day when the studies were done. However, the mean phentolamine-induced decrease in resistance (38±6 percent) was significantly greater in the morning than in the afternoon (26±6 percent) and evening (21±7 percent) (Fig. 1 and 2). This pattern was found in 11 of the 12 subjects. The vasodilator effect of phentolamine was slightly but not significantly greater in the afternoon than in the evening (P = 0.08). As a consequence of this differing response at different times of day, no circadian variation was found in either vascular resistance or blood flow measured after vasodilation was achieved by α-sympathetic blockade with phentolamine (Fig. 1).

Response to Sodium Nitroprusside

Figure 3. Figure 3. Mean Vascular Response to Incremental Doses of Sodium Nitroprusside at Three Different Times of Day in 12 Healthy Subjects.

Vertical bars indicate standard errors.

Figure 4. Figure 4. Mean Forearm Blood Flow and Vascular Resistance at Three Different Times of Day in 12 Healthy Subjects at Base Line and after an Infusion of Sodium Nitroprusside at 0.8 μg per Minute.

The similar circadian pattern of both curves indicates that the magnitude of vasodilation produced by sodium nitroprusside (stippled areas) was similar at all three times of day. P values refer to the slope of the curve and were obtained by analysis of variance. Vertical bars indicate standard errors.

Sodium nitroprusside caused a dose-dependent vasodilation. The magnitude of the vasodilation with each of the three doses of sodium nitroprusside did not differ at different times of day (Fig. 3). Thus, the vascular resistance measured after the infusion of sodium nitroprusside was still significantly higher, and the blood flow significantly lower, in the morning than in the afternoon and evening for each dose of sodium nitroprusside used — i.e., there was a circadian variation in vascular resistance and blood flow after as well as before the administration of sodium nitroprusside (Fig. 4).

The results of our study demonstrate that vascular resistance is higher and blood flow lower in the morning than at other times of day. The elimination of this phenomenon by the administration of phentolamine indicates that a circadian change in the magnitude of α-sympathetic—mediated vasoconstrictor influences is a major determinant of the normally occurring circadian variation in arterial tone. The finding that sodium nitroprusside (a direct smooth-muscle dilator)^{26 27 28} did not eliminate the circadian pattern indicates that the effect of phentolamine was not due to a nonspecific vasodilator response but reflected circadian variation in α-adrenergic activity.

There is a circadian pattern in the risk of several cardiovascular events, such as myocardial infarction, sudden death, and stroke, in that they are more likely to occur during the morning hours than at other times of day.^{1 2 3 4 5 6 7 8 9 10} Our identification of increased α-sympathetic vasoconstrictor activity in the morning as a mechanism responsible for the increased vascular tone at this time of day suggests that this may be one factor triggering such serious events and may provide clues to therapeutic interventions designed to reduce their incidence. Our findings, of course, do not rule out the possibility that other physiologic processes with circadian variations, such as platelet aggregation and fibrinolytic activity, may be important in determining the circadian pattern of the risk of cardiovascular events.^{11 12 13 14}

Plasma levels of catecholamines are higher during the morning hours,^{13 , 15} suggesting increased sympathetic activity at this time of day. We demonstrated functional changes (i.e., the modulation of vascular tone throughout the day) that might be ascribed to such increased sympathetic activity and that may have physiologic importance. Thus, the existence of augmented α-adrenergic vasoconstrictor activity in the morning suggests that elevated plasma levels of norepinephrine, perhaps accompanied by increases in adrenergic neural activity, constitute an important mechanism for the morning increase in blood pressure.¹

Increased α-adrenergic vasoconstrictor activity has been demonstrated in a number of cardiovascular conditions, including hypertension,^{29 , 30} coronary stenosis,^{31 , 32} and effort-induced myocardial ischemia,^{33 , 34} and a circadian rhythm, with a worsening in the morning, has been described for each of these conditions.^{1 2 3 4 , 20} Our findings link these observations by demonstrating that the α-adrenergic influences that directly modulate vascular tone are themselves increased in the morning and therefore may account, at least in part, for some of the circadian rhythms in cardiovascular disease.

The observation that coronary vascular tone may also increase during the morning^{19 , 20} suggests that there is probably a general increase in vascular tone at this time, which may help precipitate vascular events. For example, in the presence of severe atherosclerotic narrowing of a coronary vessel, even relatively minor degrees of vasoconstriction may reduce blood flow critically.³⁵ Moreover, since endothelium-derived relaxing factor attenuates adrenergic-mediated vasoconstriction,^{36 37 38} the abnormal endothelial function of atherosclerotic³⁹ and hypertensive⁴⁰ arteries may permit the overexpression of vasoconstrictor forces imposed by α-sympathetic activity.^{41 , 42}

We do not believe that the intraarterial administration of small doses of phentolamine during the study influenced the natural circadian variation in vascular tone. First, the sequence in which the studies were performed was randomized to avoid the artifactual creation of circadian changes in vascular tone; under these conditions, any long-lasting effect of a vasoactive drug would tend to obscure rather than provoke a circadian rhythm. More important, the interval between two studies in the same subject was at least seven hours. The fact that the vasodilator effect of phentolamine, given at the doses and by the route of administration used, lasted approximately one hour demonstrates that the circadian changes in vascular tone that we found could not have been a consequence of the previous infusion of phentolamine. Finally, studies performed on ambulatory subjects who were not undergoing any pharmacologic intervention have shown reduced forearm blood flow in the early morning hours, with an increase in the afternoon and evening⁴³ — a rhythm identical to that which we found and one that supports further the concept that the circadian changes could not be ascribed to our study design and methods.

In conclusion, the results of this study demonstrate the presence of a circadian rhythm in vascular tone that is associated with and probably causally related to increased α-sympathetic activity in the morning. These observations provide further insight into the dynamic pathophysiologic processes that determine circadian variations in blood pressure and that may participate in triggering acute cardiovascular events.