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Am J Physiol Regul Integr Comp Physiol 280: R968-R975, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 4, R968-R975, April 2001

Expression of myogenic constrictor tone in the aorta of hypertensive rats

Michelle Rapacon-Baker, Fan Zhang, Michael L. Pucci, Hui Guan, and Alberto Nasjletti

Department of Pharmacology, New York Medical College, Valhalla, New York 10595


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the effect of intraluminal pressure or stretch on the development of tone in the descending thoracic aorta from rats with aortic coarctation-induced hypertension of 7-14 days duration. Increments of pressure >100 mmHg decreased the diameter of thoracic aortas from hypertensive but not from normotensive rats. The pressure-induced constriction was not demonstrable in vessels superfused with calcium-free buffer. Stretched rings of aorta from hypertensive rats exhibited a calcium-dependent constrictor tone accompanied by elevated calcium influx that varied in relation to the degree of stretch. Blockers of L-type calcium channels and inhibitors of protein kinase C reduced both basal tone and calcium influx in aortic rings of hypertensive rats. Hence, the thoracic aorta of hypertensive rats expresses a pressure- and stretch-activated constrictor mechanism that relies on increased calcium influx through L-type calcium channels via a protein kinase C-regulated pathway. The expression of such a constrictor mechanism is suggestive of acquired myogenic behavior.

myogenic tone; protein kinase C; calcium influx; aortic smooth muscle; hypertension


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EXPOSURE TO CALCIUM-FREE MEDIA decreases resting tension in stretched segments of descending thoracic aorta taken from spontaneously hypertensive rats (23), or from rats with aortic coarctation-induced hypertension (26), but not from normotensive rats. The relaxing effect of calcium-free media in the aorta of hypertensive rats is rapidly reversed upon reintroduction of calcium into the bathing media. Thus the aortic smooth muscle of hypertensive rats appears to display a calcium-dependent basal tone that is absent from aortic smooth muscle of normotensive rats.

Protein kinase C (PKC) inhibitors were shown to decrease the basal tone of rings of thoracic aorta from rats with aortic coarctation-induced hypertension (26). Accordingly, the calcium-dependent basal tone displayed by the aortic rings of hypertensive rats may rely on mechanisms involving PKC. In this respect, it is known that PKC-dependent mechanisms enhance the sensitivity of the contractile apparatus of vascular smooth muscle to calcium (12, 17, 19, 28) and that, in some vascular preparations, PKC-regulated mechanisms potentiate voltage-gated calcium entry (10) and facilitate calcium influx (14). Also, it is accepted that calcium and PKC play critical interactive roles in the mediation of vasoconstrictor responses to neurohormonal and myogenic stimuli (12).

The present study was undertaken to test the hypothesis that the development of calcium-dependent basal tone in the aorta of rats with aortic coarctation-induced hypertension is linked to activation by mechanical stimuli, stretch or increased transmural pressure, of a constrictor mechanism involving augmentation of calcium influx via a PKC-regulated entry pathway. First, we compared the intraluminal pressure-internal diameter relationship in segments of thoracic aorta from normotensive and hypertensive rats and evaluated the effect of removal of extracellular calcium on the internal diameter of pressurized aortas. Second, we examined the relationship between calcium influx and calcium-dependent basal tone in stretched and unstretched rings of thoracic aorta taken from normotensive and hypertensive rats. Third, we studied the effect of PKC inhibitors on calcium influx and basal tone in aortic rings of normotensive and hypertensive rats.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Drugs and solutions. Drugs and chemicals were obtained from Sigma Chemical (St. Louis, MO) unless otherwise noted. Pyrogen-free, deionized water was used in preparation of buffers and solutions. Stock solutions of a myristoylated pseudosubstrate-(19-27) PKC inhibitor (MPI, Biomol, Plymouth Meeting, PA; Ref. 31) were prepared in deionized water. Stock solutions of verapamil, nifedipine, staurosporine, thapsigargin, and ryanodine were prepared in dimethyl sulfoxide. Stock solutions were further diluted with Krebs buffer at the time of experiments.

Krebs buffer consisted of (mmol/l) 118.5 NaCl, 4.7 KCl, 2.8 CaCl2, 1.2 KH2PO4, 1.1 MgSO4, 25.0 NaHCO3, and 11.1 dextrose. The composition of calcium-free buffer was that of Krebs buffer with omission of CaCl2 and inclusion of 1.0 mmol/l EGTA. The composition of high potassium-Krebs buffer (mmol/l) was 18.5 NaCl, 100 KCl, 2.8 CaCl2, 1.2 KH2PO4, 1.1 MgSO4, 25.0 NaHCO3, and 11.1 dextrose.

Animals. Experiments were performed on male Crl:CD (SD)BR rats (Charles River, Wilmington, MA), either sham operated or made hypertensive by complete ligation of the abdominal aorta at a point between the renal arteries (6). Studies were conducted 7-14 days or 2-3 mo after surgery according to protocols approved by the Institutional Animal Care and Use Committee.

On the day of the experiment, the right carotid artery of rats anesthetized with methoxyflurane (Pitman-Moore, Mundelein, IL) was cannulated with polyethylene tubing (PE-50) connected to a pressure transducer (Statham, Division, Gould, Oxnard, CA) for recording of mean arterial pressure on a polygraph (Grass Instruments, Quincy, MA). Blood pressure was measured in awake rats 2 h after cannulation. Mean arterial pressure averages were 95 ± 3 mmHg in sham-operated rats, 184 ± 9 mmHg in rats 7-14 days after aortic coarctation, and 161 ± 15 mmHg in rats 2-3 mo after coarctation. After blood pressure measurement, animals were anesthetized with pentobarbital sodium (60 mg/kg ip). The thoracic and abdominal cavities were exposed, and the descending thoracic aortas, along in some experiments, with the abdominal aorta below the site of ligation, were excised and placed on a dish filled with ice-cold Krebs buffer.

Measurement of internal diameter in isolated segments of pressurized aorta. Segments (3.0-4.0 mm in length) of descending thoracic aorta were transferred to a water-jacketed vessel chamber (18 ml in vol) filled with Krebs buffer, and all visible branches were ligated. One end of the aorta was mounted on a cannula connected to a pressure servocontroller (model CH/200/Q, Living System Instrumentation, Burlington, VT). Subsequently, the vessel was flushed to remove residual blood and the other end of the vessel was mounted on a cannula connected to a stopcock. The vessel chamber was placed on the stage of a microscope fitted with a video camera (Javelin, Newburgh, NY) leading to a video caliper (Texas, A & M, College Station), monitor (Javelin), and recorder. Unless indicated otherwise, the vascular preparation was superfused (5 ml/min at 37°C) with Krebs buffer gassed with 95% O2-5% CO2. After the stopcock was closed, the intraluminal pressure was increased slowly to 120 mmHg, a level of pressure that was maintained during a 30-min equilibration period. Only vessels that did not leak were utilized. The internal diameter of the aorta was monitored throughout the experiment.

Measurement of isometric tension and calcium influx in aortic rings. Aortas were cleared of periadventitial tissue and cut transversely into ring segments 3.0-5.0 mm in length. Each aortic ring was placed in a water-jacketed tissue chamber filled with Krebs buffer bubbled with 95% O2-5% CO2 and was attached to a force-displacememt transducer (Grass Instruments) coupled to a polygraph (Grass Instruments). Aortic rings were subjected to 2.0 g of passive stretch unless otherwise noted. Upon stabilization of isometric tension at 23°C for 20-30 min, the temperature of the tissue chambers was increased to 37°C unless otherwise indicated. Then 30-60 min later, the Krebs buffer was replaced with calcium-free buffer; 15 min later, calcium-containing buffer was returned to the bath. Any increase in tension caused by the re-addition of calcium-containing buffer is taken to indicate the calcium-dependent basal tone of aortic rings.

After assessment of calcium-dependent basal tone, calcium influx was determined by exposing the aortic rings to Krebs buffer containing 45Ca2+ (1 µCi/ml) (Du Pont-NEN Research Products, Boston, MA) for 2 min (16). Rings were then removed from the transducers and immersed for 45 min in 250 ml of ice-cold (4°C) calcium-free buffer containing 2 mmol/l EGTA to remove extracellular 45Ca2+. Each ring was blotted, weighed, and placed in 1 ml of 5 mmol/l EDTA at room temperature for 18-20 h, followed by addition of 4 ml of scintillation fluid and measurement of radioactivity in a liquid scintillation counter. The appropriateness of selecting a 2-min period of exposure of the tissues to 45Ca2+ was established in preliminary experiments showing that calcium uptake is directly proportional to the time of exposure (0.5-2.5 min) of the tissue to 45Ca2+. Calcium influx is expressed as nanomoles of Ca2+ per gram of aortic tissue per minute.

Experimental protocols. Protocol 1 examined the pressure-diameter relationship in pressurized segments of descending thoracic aorta taken from rats with aortic coarctation-induced hypertension of 7-14 days duration and from corresponding normotensive controls. The intraluminal pressure-internal diameter relationship was studied as described previously (29). After equilibration for 30 min at an intraluminal pressure of 120 mmHg, the pressure was decreased to ~0 mmHg and after 10 min it was increased in 20-mmHg steps until it reached 180 mmHg. The pressure was maintained for ~10 min at each pressure step so that the vessel could reach a steady-state diameter. Before an experiment was concluded, the vascular preparation was superfused with calcium-free Krebs buffer and the pressure-diameter relationship was examined again to obtain the passive diameter of the aorta at each level of intraluminal pressure. The internal diameter of the aorta during superfusion with calcium-containing buffer (absolute diameter) and with calcium-free buffer (passive diameter) is expressed in micrometers (µm). The normalized diameter refers to the absolute diameter expressed as the percentage of the passive diameter.

Protocol 2 investigated the effect of changes in extracellular calcium on the diameter of pressurized segments of thoracic aortas from normotensive rats and hypertensive rats 7-14 days after coarctation. The intraluminal pressure of the vessels was set at 120 mmHg, and, after equilibration, the internal diameter was monitored during three 30-min periods during which the vascular preparations were superfused consecutively with calcium-containing buffer, calcium-free buffer, and again with calcium-containing buffer.

Protocol 3 examined the effect of temperature and extracellular calcium on the basal tone of aortic rings. Aortic rings from normotensive and hypertensive rats were placed in tissue chambers and subjected to 2 g of passive stretch. The temperature of the buffer was increased in some experiments from 23 to 37°C, whereas in other experiments it remained at 23°C throughout. Calcium-dependent tone and calcium influx were assessed as described.

Protocol 4 examined the effect of stretch on calcium influx in thoracic aortic rings of sham-operated rats and rats 7-14 days after coarctation. Aortic rings placed in tissue chambers were subjected or not subjected to 2 g of passive stretch. The temperature of the bathing buffer was increased to 37°C, and the aortic rings were sequentially exposed to calcium-free and calcium-containing Krebs buffer as described in protocol 1. Calcium influx measurements were then taken. In additional experiments, in hypertensive rats 7-14 days after coarctation, calcium influx and calcium-dependent tone were assessed in rings of thoracic aorta subjected to varying degrees of passive stretch (0.5-2.0 g).

Protocol 5 examined the effect of calcium channel blockers and PKC inhibitors on basal tone and calcium influx in rings of thoracic aorta from normotensive rats and hypertensive rats 7-14 days after coarctation. Calcium-dependent tone was measured in aortic rings bathed in buffer at 37°C. Once isometric tension was stable, the aortic rings were exposed to a blocker of voltage gated calcium channels (verapamil, 10 µmol/l, or nifedipine, 100 nmol/l), an inhibitor of PKC (staurosporine, 10 nmol/l, or MPI, 10 µmol/l), a blocker of stretch-activated cation channels, gadolinium (10 µmol/l; Ref. 8), or the appropriate vehicle. Changes in isometric tension produced by drugs were monitored, and calcium influx was assessed once isometric tension was stable. In some experiments, stretched aortic rings from hypertensive rats were treated with MPI (10 µmol/l). Once the MPI-induced change in isometric tension had reached a plateau, the rings were exposed to high-potassium buffer containing MPI. Calcium influx was measured once isometric tension was stable.

Protocol 6 examined the effects of thapsigargin and ryanodine on calcium-dependent tone and calcium influx in aortic rings from rats 7-14 days after aortic coarctation. Experiments were conducted, as described in protocol 3, in rings of thoracic aorta bathed from the outset (before stretching) in buffer containing and not containing thapsigargin (1 µmol/l) and ryanodine (30 µmol/l), agents known to produce depletion of intracellular calcium stores (27).

Statistical analysis. Results are means ± SE; n in figure legends and text denotes the number of animals from which vascular rings were obtained. A paired or unpaired Student's t-test was used for single variable comparisons. Multiple comparisons were analyzed by analysis of variance. Newman-Kuels modified t-test was used to make specific comparisons. The null hypothesis was rejected when the P value was <0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Comparison of the effects of intraluminal pressure and extracellular calcium on the internal diameter of the descending thoracic aorta taken from normotensive rats and hypertensive rats 7-14 days after aortic coarctation. As shown in Fig. 1, stepwise elevation of intraluminal pressure elicited comparable increases of passive diameter in segments of thoracic aorta taken from normotensive and hypertensive rats. Increments of pressure over the range 0-100 mmHg in the presence of normal bath calcium also produced comparable increases of absolute diameter in the thoracic aorta of normotensive and hypertensive rats. However, further increments in pressure to 180 mmHg brought about reduction (P < 0.05) of absolute diameter in the aorta of hypertensive rats while continuing to increase that of the aorta from normotensive rats. Over the range 100-180 mmHg, increments of intraluminal pressure had little effect on the normalized diameter of aortas from normotensive rats but decreased (P < 0.05) the normalized diameter of aortas from hypertensive rats. Neither the absolute diameter nor the normalized diameter of thoracic aortas from normotensive rats differed from the corresponding diameters in aortas from hypertensive rats over the range 0-100 mmHg. In contrast, at pressures >100 mmHg both the absolute diameter and the normalized diameter of aortas from hypertensive rats were smaller (P < 0.05) than the corresponding diameter in aortas of normotensive rats.


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  		Fig. 1.   Effect of stepwise increments in intraluminal pressure on passive, absolute, and normalized internal diameter (ID) of segments of descending thoracic aorta taken from sham-operated normotensive rats and rats with aortic coarctation-induced hypertension 7-14 days after coarctation. Results are means ± SE. *P < 0.05 relative to corresponding data in aorta of normotensive rats.

Figure 2 shows the effect of sequential exposure to calcium-free and calcium-containing buffer on the internal diameter of segments of descending thoracic aortas equilibrated at an intraluminal pressure of 120 mmHg. The internal diameter of aortas from sham-operated normotensive rats was not affected by the exposure to calcium-free buffer nor by the subsequent reintroduction of calcium into the buffer. In contrast, the internal diameter of aortas from hypertensive rats 7-14 days after aortic coarctation increased (P < 0.05) during exposure to calcium-free buffer, an effect that was reversed by the subsequent reintroduction of calcium into the buffer.


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  		Fig. 2.   Internal diameter of segments of pressurized (120 mmHg) descending thoracic aorta before, during, and after superfusion with calcium-free buffer. Results means ± SE. *P < 0.05 relative to control.

Comparison of isometric tension development in rings of descending thoracic aorta from normotensive rats and rats with aortic coarctation-induced hypertension: effect of temperature and extracellular calcium and relationship to stretch and calcium influx. Figure 3 illustrates the effect of increases in buffer temperature from 23 to 37°C, and of sequential exposure to calcium-free and calcium-containing buffer, on isometric tension development in rings of descending thoracic aorta taken from sham-operated normotensive rats (n = 22) and hypertensive rats 7-14 days (n = 29) and 2-3 mo (n = 6) after coarctation. Progressive elevation of buffer temperature increased isometric tension in rings of thoracic aorta from hypertensive rats 7-14 days after coarctation (1.2 ± 0.1 g) but not in aortic rings from normotensive rats (0 ± 0 g) or hypertensive rats 2-3 mo after coarctation (0 ± 0 g). Exposure of aortic rings taken from hypertensive rats 7-14 days after coarctation to calcium-free buffer caused isometric tension to fall (1.4 ± 0.1 g), whereas reexposure to calcium-containing buffer prompted isometric tension to increase (1.6 ± 0.1 g), a response that is taken to reflect the calcium-dependent tone of the rings. In contrast, aortic rings taken from normotensive controls, or from hypertensive rats 2-3 mo after coarctation, displayed no change in isometric tension when exposed sequentially to calcium-free and calcium-containing buffer, indicating absence of calcium-dependent tone. As shown in Fig. 3, the expression of calcium-dependent tone at 37°C in rings of thoracic aorta taken from hypertensive rats 7-14 days after coarctation is accompanied by increased calcium influx (P < 0.05) relative to the calcium influx in aortic rings from normotensive rats and hypertensive rats 2-3 mo after coarctation. Rings of thoracic aorta from normotensive rats and hypertensive rats 7-14 days after coarctation had comparable calcium influx values (25 ± 2 vs. 31 ± 5 nmol Ca2+ · min-1 · g-1) when bathed in buffer at 23°C to prevent development of tone.


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  		Fig. 3.   Representative tracings illustrating temperature- and calcium-dependent changes in isometric tension in rings of thoracic aorta taken from normotensive rats (A), rats 7-14 days after (B), and 2-3 mo after aortic coarctation (C). Aortic rings subjected to 2 g of passive stretch were assessed for temperature-dependent tone, calcium-dependent tone, and calcium influx.

In experiments using rings of abdominal aorta from sham-operated normotensive rats (n = 3) and rats with the aortic coarctation-induced hypertension of 7-14 days duration (n = 4), neither elevation of buffer temperature to 37°C nor sequential exposure of the preparations to calcium-free and calcium-containing buffers had any effect on isometric tension. Also, calcium influx was comparable in rings of abdominal aorta from normotensive (22 ± 6 nmol Ca2+ · min-1 · g-1) and hypertensive rats (28 ± 8 nmol Ca2+ · min-1 · g-1).

Figure 4 shows the effect of passive stretch (2 g) on calcium influx in rings of descending thoracic aorta taken from normotensive (n = 4) and hypertensive rats (n = 5). Calcium influx was similar in stretched and unstretched aortic rings taken from normotensive rats. In contrast, calcium influx measured in stretched aortic rings from hypertensive rats 7-14 rats after coarctation surpassed that measured in unstretched preparations from the same animals. As shown in Fig. 5, in rings of thoracic aorta from hypertensive rats 7-14 days after coarctation, measurements of calcium influx correlated well (r = 0.968, P < 0.01) with estimates of calcium-dependent tone in preparations subjected to passive stretch in varying degrees (n = 9). In these experiments, calcium-dependent tone in aortic rings subjected to either 1.0 or 2.0 g of passive stretch was greater than the calcium-dependent tone in rings subjected to 0.5 g of passive stretch.


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  		Fig. 4.   Calcium influx in stretched and unstretched rings of thoracic aorta taken from normotensive (n = 4) and hypertensive rats (n = 5). Aortic rings were subjected to 0 or 2 g of passive stretch. Calcium influx was measured at 37°C. Results are means ± SE. *P < 0.05 relative to data in unstretched rings.



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  		Fig. 5.   Relationship between calcium influx and calcium-dependent tone in rings of thoracic aorta taken from hypertensive rats 7-14 days after aortic coarctation. Aortic rings were subjected to 0.5 g (open circle ), 1.0 g (), and 2.0 g (triangle ) of passive stretch before assessment of calcium-dependent tone and calcium influx. +, Individual observations.

Comparison of the effects of calcium channel blockers, gadolinium, and inhibitors of PKC on isometric tension and calcium influx in rings of the thoracic aorta taken from normotensive rats and hypertensive rats 7-14 days after coarctation. Figure 6 illustrates the effects of blockers of voltage-gated calcium channels, verapamil and nifedipine, and of stretch-activated cation channels, gadolinium, on isometric tension and calcium influx in rings of thoracic aorta taken from normotensive rats and hypertensive rats 7-14 days after coarctation. Neither verapamil (n = 4) nor nifedipine (n = 4) affected isometric tension or calcium influx in aortic rings from normotensive rats. In contrast, both verapamil (n = 5) and nifedipine (n = 5) elicited reductions of isometric tension in aortic rings of hypertensive rats corresponding to 108 ± 4% and 106 ± 5% of the prevailing calcium-dependent tone, respectively. Verapamil and nifedipine also decreased calcium influx in aortic rings of hypertensive rats to a level comparable to that in normotensive rats. Gadolinium was without effect on isometric tension and calcium influx in aortic rings from normotensive (n = 3) and hypertensive rats (n = 4).


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  		Fig. 6.   Effect of vehicle, verapamil, nifedipine, and gadolinium on calcium influx in stretched rings of thoracic aorta taken from normotensive (n = 4) and hypertensive rats (n = 5). Accompanying decreases of isometric tension in response to treatment are indicated. Results are means ± SE. *P < 0.05 relative to data in vehicle-treated rings.

Figure 7 depicts the effects of PKC inhibitors staurosporine and MPI on isometric tension and calcium influx in rings of thoracic aorta taken from normotensive rats and hypertensive rats 7-14 days after coarctation. Neither staurosporine (n = 3) nor MPI (n = 4) affected isometric tension or calcium influx in aortic rings from normotensive rats. In contrast, both staurosporine (n = 6) and MPI (n = 4) elicited reductions of isometric tension in aortic rings of hypertensive rats to ~3 and 13% of the prevailing calcium-dependent tone, respectively. Staurosporine and MPI also decreased calcium influx in aortic rings of hypertensive rats to a level comparable to that in normotensive rats. However, MPI at a concentration that blunts the expression of calcium-dependent tone and normalizes calcium influx in aortic rings from hypertensive rats could not prevent the increase in isometric tension (2.7 ± 0.3 g with MPI vs. 2.8 ± 0.2 g without MPI) and calcium influx (55 ± 1 nmol Ca2+ · min-1 · g-1 with MPI vs. 53 ± 3 nmol Ca2+ · min-1 · g-1 without MPI) produced by exposure of the rings to high potassium buffer (n = 4). Likewise, in rings of thoracic aorta taken from normotensive rats, the tone (2.1 ± 0.1 g) and calcium influx (45 ± 2 nmol Ca2+ · min-1 · g-1 ) imposed by exposure to high potassium buffer were not affected by staurosporine (2.1 ± 0.1 g; 46 ± 4 nmol Ca2+ · min-1 · g-1; n = 5) or MPI (2.1 ± 0.1 g; 53 ± 4 nmol Ca2+ · min-1 · g-1; n = 6).


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  		Fig. 7.   Effects of vehicle, staurosporine, and myristoylated, cell-permeable PKC inhibitor (MPI) on calcium influx in stretched rings of thoracic aorta taken from normotensive rats (staurosporine, n = 4; MPI, n = 4) and hypertensive rats (staurosporine, n = 6; MPI, n = 4). Accompanying decreases in isometric tension in response to treatment are indicated. Results are means ± SE. *P < 0.05 relative to data in vehicle-treated aortic rings.

The calcium-dependent tone in vehicle-pretreated rings of thoracic aorta from hypertensive rats 7-14 days after coarctation (1.1 ± 0.2 g; n = 5) did not differ from the calcium-dependent tone in rings pretreated with thapsigargin and ryanodine (n = 4) to deplete intracellular calcium stores (1.3 ± 0.2 g). Likewise, calcium influx values in aortic rings pretreated with vehicle (50 ± 7 nmol Ca2+ · min-1 · g-1) were comparable to calcium influx values in aortic rings pretreated with thapsigargin plus ryanodine (57 ± 9 nmol Ca2+ · min-1 · g-1).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows that pressurized segments of descending thoracic aorta taken from hypertensive rats 7-14 days after aortic coarctation constrict in response to stepwise increments of intraluminal pressure >100 mmHg. This is in contrast to observations in the thoracic aorta of sham-operated normotensive rats, which does not constrict when intraluminal pressure is increased in the range 0-180 mmHg. The pressure-induced constrictor response in the aorta of hypertensive rats depends on extracellular calcium, as elevation of intraluminal pressure >100 mmHg does not decrease the internal diameter of aortas superfused with calcium-free buffer. Bearing on this point, the internal diameter of pressurized segments of thoracic aorta from hypertensive rats, but not from normotensive rats, was found to increase during superfusion with calcium-free buffer and, subsequently, to decrease when calcium was reintroduced into the buffer. These observations are taken as evidence that the pressurized aorta of rats with aortic coarctation-induced hypertension expresses a calcium-dependent constrictor tone that is absent from the aorta of normotensive rats.

The present study also demonstrates that rings of descending thoracic aorta taken from hypertensive rats 7-14 days after aortic coarctation exhibit a calcium-dependent basal tone that is absent from aortic rings taken from normotensive rats or hypertensive rats 2-3 mo after coarctation, the late phase of aortic coarctation-induced hypertension. The basal tone is expressed at 37°C but not at 23°C, is accompanied by elevated calcium influx, and varies in relation to the degree of passive stretch imposed on the preparation. Based on the aforementioned findings it would appear that mechanical stimuli, either an increase of intraluminal pressure or passive stretch, activates a calcium-dependent constrictor mechanism in the thoracic aorta of rats in the early phase of aortic coarctation-induced hypertension but not in the aorta of normotensive rats. Previous studies documented extensively that distention of certain blood vessels, due to stretch or increased transmural pressure, triggers a myogenic constrictor response that contributes to the vascular tone (13, 21, 30). Hence, the calcium-dependent tone displayed by the pressurized or stretched thoracic aorta of hypertensive rats, 7-14 days after coarctation, may be a manifestation of myogenic behavior.

According to our study, only in preparations subjected to passive stretch did calcium influx in aortic rings of hypertensive rats 7-14 days after coarctation surpass the calcium influx values in aortic rings of normotensive controls. One interpretation of this finding is that the increased calcium influx found in aortic rings of hypertensive rats is also a manifestation of the myogenic response to stretch. This notion is in agreement with reports that in myogenically active vascular preparations, constrictor responses elicited by stretch or elevation of transmural pressure rely on the presence of extracellular calcium and on augmentation of calcium influx (4, 13, 15, 16, 21). Indeed, depending on the type of blood vessel and experimental conditions, vascular contraction induced by distension of the vessel wall has been linked to calcium entry via dihydropyridine-sensitive, voltage-gated calcium channels (9, 18, 24), stretch-activated cation channels (3), or a combination thereof (2).

In our study, in aortic rings of hypertensive rats 7-14 days after coarctation, blockade of L-type voltage-gated calcium channels with verapamil or nifedipine resulted in virtual elimination of basal tone and in marked reduction of calcium influx. In contrast, neither basal tone nor calcium influx were decreased in aortic rings treated with gadolinium, a trivalent cation that blocks stretch-activated cation channels (8). These findings suggest that the calcium-dependent tone displayed by stretched aortic rings of rats in the early phase of aortic coarctation-induced hypertension relies on increased calcium influx through L-type channels. However, it would be premature to exclude the possibility that calcium entry through stretch-activated cation channels also contributes to the development of basal tone in such preparations. For example, the calcium-dependent basal tone exhibited by aortic rings of hypertensive rats, 7-14 days after coarctation, may rely on activation of stretch-activated channels insensitive to gadolinium (32), leading to increased influx of cations, membrane depolarization, and activation of voltage-gated calcium channels (2). That treatment with ryanodine and thapsigargin did not attenuate calcium-dependent tone and calcium influx in aortic rings suggests that the underlying stretch-responsive mechanism(s) is not linked to release of calcium from intracellular stores (27).

Previous studies have documented augmentation of PKC activity and redistribution of PKC isoforms in the thoracic aorta of rats with aortic coarctation-induced hypertension (20, 26). Moreover, treatment with PKC inhibitors was shown to decrease the basal tone displayed by aortic rings of rats 7-14 days after aortic coarctation (26). The present study demonstrates that the reduction of basal tone elicited by PKC inhibitors, staurosporine or MPI, in aortic rings of hypertensive rats is accompanied by reduction of calcium influx to values not different from those found in aortic rings of normotensive rats. These findings suggest that the stretch-responsive mechanism that promotes calcium influx and basal tone in aortic rings of hypertensive rats 7-14 days after coarctation is subject to stimulatory regulation by PKC. A role for PKC supporting expression of myogenic tone in blood vessels is well documented (11, 15, 25). However, the mechanisms underlying such a role are less well known. There are reports that PKC activation increases the sensitivity of contractile proteins to intracellular calcium (19, 28), decreases potassium channel activity (1), and increases dihydropyridine-sensitive calcium conductance in vascular smooth muscle cells (7). However, our study offers no indication of a direct regulatory influence of PKC on the function of L-type calcium channels, because treatment with PKC inhibitors did not interfere with the contractile response or the increased calcium influx induced by high potassium buffer in aortic rings of normotensive or hypertensive rats. Another possibility to consider is that PKC inhibitors decrease calcium influx and basal tone in aortic rings of hypertensive rats, 7-14 days after coarctation, by blunting an action of PKC on upstream events that would otherwise lead to activation of calcium entry via channels sensitive to verapamil and nifedipine.

That the thoracic aorta from normotensive rats does not exhibit increased calcium influx and calcium-dependent tone when stretched or pressurized fits well with the notion that conduit vessels display little or no myogenic behavior (30). That pressurization or the application of passive stretch to the thoracic aorta of rats 7-14 days after coarctation promotes constrictor tone development implies that the aortic smooth muscle of these hypertensive rats has acquired myogenic properties. Our findings that stretched rings of abdominal aorta from below the site of coarctation (a segment of the vessel that is not exposed to high blood pressure) exhibit neither calcium-dependent basal tone nor increased calcium influx may signify that the expression of myogenic behavior in aortic smooth muscle is conditioned by exposure to high blood pressure. However, it is unlikely that increased blood pressure alone promotes expression of myogenic behavior, because no evidence of such was apparent in rings of thoracic aorta from hypertensive rats 2-3 mo after coarctation. One possibility to consider is that unknown factors, occurring in the early phase but not the late phase of aortic coarctation-induced hypertension, act in concert with the increased blood pressure to foster myogenic behavior in aortic smooth muscle of rats in the early phase of the hypertension.

In summary, this study demonstrates that the descending thoracic aorta of rats in the early phase of aortic coarctation-induced hypertension, 7-14 days after coarctation, develops a calcium-dependent constrictor tone in response to either increments of intraluminal pressure or the application of passive stretch. The expression of calcium-dependent tone in the aorta of these animals relies on increased calcium influx via a PKC-regulated entry pathway that utilizes L-type calcium channels. This pressure- and stretch-activated constrictor mechanism is not apparent in the thoracic aorta of normotensive rats. We propose that its expression in the aorta of hypertensive rats 7-14 days after coarctation reflects newly acquired myogenic properties. In this regard, several previous studies have documented enhancement of pressure-induced myogenic vasoconstriction in hypertensive animal models (2, 5, 22, 25).

Perspectives

In hypertension, augmentation of myogenic behavior in small arteries and arterioles and the acquisition of myogenic behavior by large arterial vessels are expected to impact on systemic hemodynamics (2). Augmented activity of a pressure-activated, myogenic, mechanism of vasoconstriction may reinforce the elevation of blood pressure brought about by neurohormonal mechanisms that mediate arteriolar constriction (22). Along this line, there is evidence that as blood pressure rises driven by neurohormonal mechanisms in animal models of renal hypertension it brings about activation of a myogenic vasoconstrictor mechanism that produces further elevation of blood pressure (2). Also, in hypertension the acquisition of myogenic behavior by conduit arteries may favor elevation of systolic blood pressure by generating a force that opposes passive distention of the wall of large arteries during systole.


    ACKNOWLEDGEMENTS

We thank Jennifer Brown for secretarial assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-18579 and HL-34300 and by the American Heart Association New York Affiliate Grant 99-30291T.

Address for reprint requests and other correspondence: A. Nasjletti, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595.

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

Received 17 October 2000; accepted in final form 8 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 280(4):R968-R975
0363-6119/01 $5.00 Copyright © 2001 the American Physiological Society


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