Pressure-Induced Myogenic Responses in Isolated Bovine Retinal Arteries

Abstract

purpose. To investigate whether a pressure-induced myogenic vasoconstriction can be demonstrated in isolated bovine retinal arteries and to determine the cellular mechanisms involved.

methods. Isolated bovine retinal arteries were mounted on a pressure myograph without flow and exposed to stepwise increases in intraluminal pressure. Changes in internal diameter were monitored continuously using an inverted microscope video system.

results. Bovine retinal arteries showed myogenic tone at pressures higher than 10 mm Hg. This pressure-induced contraction was absent in calcium-free Krebs–Ringer bicarbonate solution. Inhibition of L-type voltage-operated calcium channels with nifedipine (1 μM) suppressed the myogenic contraction. After depolarization of the vascular smooth muscle cells with a K⁺ 120 mM solution, a pressure-induced contraction was still observed, indicating that besides stimulation of voltage-operated calcium channels, depolarization-independent mechanisms contribute to the pressure-induced myogenic vasoconstriction.

conclusions. Isolated bovine retinal arteries spontaneously contract when exposed to raised intraluminal pressure. This response depends on extracellular calcium and is blocked by nifedipine. In addition, depolarization-independent mechanisms seem to be involved.

Many investigators have demonstrated in vivo that pressure-induced adaptations of the arterial diameter maintain a constant retinal blood flow. However, as yet, no study has systematically investigated the pressure-induced autoregulatory responses on isolated retinal arteries. Nevertheless, indications for myogenic responses can be derived from some in vitro studies. In a study on the effects of adrenergic drugs on isolated bovine retinal arteries, Hoste et al. ¹ observed a myogenic contraction after rapid circumferential stretch. Similarly, Nyborg et al. ² observed a small contraction after a prompt increase in internal circumference and demonstrated that this contraction was dependent on extracellular calcium. Yu et al. ³ used a microperfusion system to compare the effects of intraluminal and extraluminal drug delivery on isolated porcine retinal arteries. They observed a myogenic constriction when flow through the arterial segment was stopped. 

These indications for myogenic contraction in isolated retinal arteries were not further investigated. The purposes of the present study were therefore to examine more systematically the myogenic responses of isolated bovine retinal arteries using a pressure myograph, to determine the pressure range in which these arteries show a myogenic response and to investigate the mechanisms involved. 

Materials and Methods

Bovine eyes, obtained from the local abattoir, were incised at the equator. The anterior segment and vitreous were removed, the eyecup was placed under a dissection microscope, and a segment of the most prominent retinal artery was isolated. This segment was transferred to the vessel chamber of the pressure myograph (model P100; JP Trading, Aarhus, Denmark). The vessel chamber was filled with oxygenated Krebs–Ringer bicarbonate (KRB) buffer kept at 37°C. 

The arterial segment was mounted on two glass cannulas and was secured with nylon 10-0 sutures. Each cannula was connected with a reservoir filled with oxygenated KRB solution. The height of the reservoirs determined the intraluminal pressure. The inflow and outflow pressures were measured by two pressure transducers. The internal diameter was monitored continuously by an inverted microscope digital video system. The video images were analyzed with a commercial software package (Vesselview; PhysioLogic, Aarhus, Denmark). 

The response of the vessel segments to changes in transmural pressure was studied without flow. No flow conditions were obtained by positioning the inflow and outflow reservoirs at the same height. 

After mounting, the retinal vessels were equilibrated for at least 30 minutes at a pressure of 35 mm Hg. Thereafter, the intraluminal pressure was set to 0 mm Hg and was increased with 20 mm Hg every 10 minutes until the intraluminal pressure reached 100 mm Hg. Vessels were discarded when no myogenic contraction was seen during this procedure (approximately 8% of the vessels) and subsequent application of prostaglandin F_2α (30 μM) failed to induce a stable and pronounced contraction. 

Drugs and Solutions

The experiments were performed using a KRB solution with the following composition (mM): 135 NaCl, 5 KCl, 20 NaHCO₃, 10 glucose, 2.5 CaCl₂, 1.3 MgSO₄, 1.2 KH₂PO₄, and 0.026 EDTA. A Ca²⁺-free KRB solution was prepared by equimolar replacement of CaCl₂ with MgCl₂ and by replacement of EDTA with 2 mM EGTA. A KRB solution containing 120 mM K⁺ was prepared by equimolar replacement of NaCl with KCl. Papaverine HCl (40 mg) was purchased from Federa (Brussels, Belgium), prostaglandin F_2α (dinoprostum trometamolum [Dinolytic]) from Upjohn (Puurs, Belgium), and nifedipine from Sigma (St. Louis, MO). Stock solutions were freshly prepared in distilled water, except for nifedipine, which was dissolved in dimethyl sulfoxide. 

Data Analysis

Preliminary experiments have shown that the myogenic contraction reaches a steady state within 10 minutes after a pressure increment and remains stable for at least 60 minutes. Internal diameters were therefore measured 10 minutes after a pressure increment (unless otherwise stated). Internal diameters are expressed in percentages (mean ± SEM) of the diameter at 100 mm Hg (or at 40 mm Hg for the experiments with nifedipine) measured in a Ca²⁺-free KRB solution. 

The effectiveness of myogenic autoregulation to maintain flow was quantified by a gain factor. ⁴ A gain value of 1 implies perfect autoregulation, and values less than unity indicate an insufficient myogenicity to maintain a constant blood flow. 

Statistical significance was evaluated using either a Student’s t-test for paired observations or a repeated measures analysis of variance (ANOVA). A Bonferroni test was performed when the repeated measures ANOVA revealed a significant difference (overall P < 0.05). The number of experiments is indicated by n. 

Results

Figure 1 shows the response of an isolated bovine retinal artery to stepwise increases in intraluminal pressure. The top tracing shows the response in a normal KRB solution. Changing the pressure from 0 to 10 mm Hg increased vessel diameter. When the pressure was further increased to 20 mm Hg, the artery showed an initial increase in diameter, followed by a constriction. The onset of the constriction was almost immediate, but it took more than 1 minute for the vessel response to attain 50% of the final response. Increasing the pressure to 30 or 40 mm Hg reduced the diameter even further. At pressures higher than 60 mm Hg, arterial diameter gradually increased. A sudden decrease in pressure to 0 mm Hg at the end of the pressure–response curve resulted in an initial decrease of the vascular diameter followed by a vasodilatation. 

When the pressure–response curve was repeated in a calcium-free solution, the diameter increased with every increment in pressure (middle tracing). This increase in diameter was most pronounced at low pressures and was almost absent at high pressures. Returning the pressure to 0 mm Hg at the end of the pressure–response curve resulted in a decrease of the arterial diameter, however, without the subsequent dilatation that was seen in a normal KRB solution. 

Papaverine (0.1 mM), which was added to the calcium-free solution at the end of the experiments (bottom tracing), did not enhance the pressure-induced increases in diameter. 

Figure 2 shows the normalized data for the responses of five retinal arteries to stepwise increases in intraluminal pressure. At all pressures higher than 10 mm Hg, the diameter was significantly larger in a calcium-free solution with papaverine than in a normal KRB solution. This indicates that retinal arteries show an active tone at pressures higher than 10 mm Hg. This pressure-induced tone was most pronounced at pressures between 10 and 60 mm Hg and resulted in positive gain values within this range (average gain value for this pressure range was 0.36 ± 0.07, n = 5). At pressures below 10 mm Hg, vascular diameter tended to increase with increasing pressure, whereas the ability to maintain a high degree of myogenic tone was reduced at pressures greater than 60 mm Hg, as shown by the gradual increases in diameter with further increments. This is also reflected in negative gain values for pressures lower than 10 mm Hg (−0.28 ± 0.090, n = 5) and higher than 60 mm Hg (−0.76 ± 0.357 for the pressure step from 60 to 80 mm Hg and −1.05 ± 0.289 for the pressure increment from 80 to 100 mm Hg; n = 5). 

The pressure–response curve performed in a calcium-free solution with papaverine (0.1 mM), did not significantly differ from the pressure–response curve in a calcium-free solution without papaverine. 

In a separate series of experiments, the response of retinal arteries to stepwise reductions in pressure was studied. The retinal arteries were first subjected to stepwise increases in intraluminal pressure and thereafter to stepwise reductions in intraluminal pressure. There was no significant difference between the two pressure–response curves (n = 4, paired Student’s t-test). The only difference noted was that it took approximately 20 minutes for the retinal arterial diameter to reach a steady state after a decrease in intraluminal pressure compared with less than 10 minutes after an increase in intraluminal pressure. 

Effect of Nifedipine on Myogenic Response

To investigate whether activation of voltage-operated Ca²⁺ channels (VOCs) was involved in the myogenic response of isolated bovine retinal arteries, the effect of nifedipine on the myogenic response was studied. 

Cumulative addition of increasing concentrations of nifedipine (0.1 nM to 1 μM) resulted in a concentration-dependent reduction of the myogenic tone (at 40 mm Hg). The highest concentration of nifedipine (1μ M) reduced the myogenic contraction 72.4% ± 17.7% (n = 4). 

At pressures higher than 10 mm Hg, the pressure–response curve (0–40 mm Hg) performed in the presence of nifedipine (1 μM) differed significantly from the pressure–response curve obtained with a normal KRB solution. There was no significant difference between the pressure–response curve obtained in the presence of nifedipine and that obtained in a Ca²⁺-free solution. 

Influence of K⁺ on Myogenic Response

In this series of experiments retinal arteries were depolarized with 120 mM K⁺ containing KRB solution and a pressure–response curve was determined. In the presence of 120 mM K⁺, consecutive pressure increments resulted in large increases in diameter, followed by moderate contractions (Fig. 3) . The latter was not sufficient, however, to completely reverse the initial increase in diameter. 

Table 1 shows the diameter of the retinal artery 1 and 10 minutes after an increase in pressure in the presence of 120 mM K⁺. When the pressure was increased from 0 to 10 mm Hg, the retinal artery showed no constriction after the initial diameter change. At higher intraluminal pressures, however, the diameter was systematically determined to be smaller 10 minutes after the pressure change than immediately after the pressure change. 

The initial dilatation and the contraction that follows the increase in intraluminal pressure were both more pronounced when the pressure increment was larger. Changing the intraluminal pressure from 10 to 40 mm Hg increased the diameter of the depolarized retinal artery from 159.4 ± 12.22 μm (n = 11) to 192.5 ± 14.18μ m (n = 11). This initial dilatation was quickly followed by a constriction, which, after 10 minutes, reduced the diameter to 171.0 ± 12.48 μm (P < 0.001, paired Student’s t-test, n = 11). However, this constriction was insufficient to completely abolish the increase in diameter. 

Discussion

The present study provides convincing evidence that pressure-induced myogenic tone develops in isolated bovine retinal arteries. This myogenic tone is elicited when intraluminal pressure exceeds 10 mm Hg and is most pronounced at pressures between 10 and 60 mm Hg. At pressures lower than 10 mm Hg and at pressures exceeding 60 mm Hg, vascular diameter tends to increase with increasing pressure. The changes in diameter and corresponding gain values therefore suggest a myogenic regulatory pressure range from 10 to 60 mm Hg. 

This autoregulatory range compares relatively well with the range reported in several in vivo studies. In cats, retinal blood flow showed autoregulation until the perfusion pressure was reduced to 25 mm Hg. ⁵ The lowest perfusion pressure at which the retina of 17 normal subjects was able to maintain normal blood flow corresponded to an average perfusion pressure of 27 mm Hg. ⁶ In healthy volunteers there was no detectable change in retinal blood flow until mean brachial blood pressure (i.e., mean arterial pressure [MAP]) was increased to 115 mm Hg ⁷ (representing an increase of MAP of 41% and a retinal perfusion pressure of approximately 60 mm Hg). Similarly, Rassam et al. ⁸ found that autoregulation began to breakdown during a 40% increase in MAP in normal subjects. Based on all these studies the autoregulatory range of the retinal circulation can be estimated between perfusion pressures of 25 and 60 mm Hg. 

Although these in vivo studies provide results that are generally of high physiological relevance, they cannot distinguish between the various mechanisms that may participate in the regulation of retinal blood flow (such as myogenic tone, local metabolic factors, circulating hormones, and neurotransmitters). The gain calculations in the present in vitro study suggest that myogenic mechanisms are only in part responsible for flow autoregulation and that they are supplemented by other mechanisms (positive-gain values <1). The influence of circulating hormones and neurotransmitters on retinal arterial resistance is, however, generally assumed to be negligible due to the blood–retinal barrier and the absence of retinal blood flow responses to electrical stimulation of the ocular sympathetic and parasympathetic nerves. By contrast, there is strong evidence for metabolic autoregulation. Our data therefore support the view that both metabolic and myogenic autoregulatory mechanisms may operate in vivo. 

Most contractile stimuli induce arterial smooth muscle contraction by increasing the concentration of cytosolic calcium ([Ca²⁺]_i).[ Ca²⁺]_i may increase by an influx of extracellular Ca²⁺ or by the release of Ca²⁺ from intracellular stores. In our experiments, a pressure elevation failed to induce a myogenic contraction of the retinal artery in a calcium-free solution. The presence of extracellular calcium therefore seems to be a prerequisite for the development of myogenic response. The experiments performed in the presence of nifedipine (1 μM) suggest that extracellular calcium enters the cell through L-type voltage-operated Ca²⁺-channels (VOCs). Besides activation of VOCs, a depolarization-independent mechanism also seems to contribute, albeit to a much smaller extent, to the pressure-induced contraction in bovine retinal arteries. This is suggested by the pressure–response curves performed in 120-mM K⁺ medium. K⁺ at 120 mM depolarized the vascular smooth muscle cell, which resulted in a maximal stimulation of the VOCs and contraction. Nevertheless, a small pressure-induced contraction could still be observed in a 120-mM K⁺ solution, indicating the involvement of additional mechanisms besides stimulation of VOCs. This depolarization-independent contraction could be due simply to a rearrangement of the active contractile filaments in the smooth muscle cells in response to a rapid pressure increase, but it may also be due to an increase in myofilament Ca²⁺ sensitivity. In rat cerebral arteries pressurization has been shown to activate phospholipase C ⁹ resulting in activation of protein kinase C (PKC). Pharmacologic activation of PKC produces constriction at otherwise subthreshold[ Ca²⁺]_i. ¹⁰ This suggests that PKC activation can increase the sensitivity of the contractile machinery to calcium. Pressure-induced activation of PKC could therefore be responsible for the small component of the myogenic response observed in 120 mM K⁺ solution. 

In summary, isolated bovine retinal arteries show a myogenic response in vitro. This response depends on extracellular calcium, which enters the vascular smooth muscle cell mainly through VOCs. In addition, a small depolarization-independent component seems to contribute to the pressure-induced myogenic contraction. 

 Supported by a Grant 011D0196 from Ghent University, and Grant 31.5056.96 from the Fund for Scientific Research Flanders (FWO Vlaanderen).

 Submitted for publication July 27, 1999; revised November 10 and December 20, 1999; accepted January 5, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

Representative tracings of the changes in internal diameter of an isolated bovine retinal artery during stepwise increases (0–100 mm Hg) and a sudden decrease (0 mm Hg) in intraluminal pressure. The responses of the retinal artery in a normal KRB buffer is represented in the top tracing. The middle tracing represents the changes in diameter in a calcium-free medium (Ca²⁺-free). The bottom tracing represents the changes in diameter in a calcium-free medium with papaverine (0.1 mM).

Figure 2.

 

The changes in diameter of isolated bovine retinal arteries (n= 5) in response to changes in intraluminal pressure in a normal KRB solution (▪), a calcium-free solution (▴), and a calcium-free solution with 0.1 mM papaverine (▾). The responses are expressed as a percentage of the diameter of each artery in a calcium-free solution with papaverine (0.1 mM) at 100 mm Hg.

Figure 3.

 

Representative tracings of the changes in internal diameter of an isolated bovine retinal artery during stepwise increases in intraluminal pressure in a solution containing 120 mM K⁺.

Table 1.

 

Diameter of the Retinal Artery with Increasing Pressure in the Presence of 120 mM K⁺

Arterial Pressure	After 1 Minute			After 10 Minutes
		Diameter	% of Initial Diameter	Diameter	% of Initial Diameter
0 to 10 mm Hg	161.3 ± 15.1	117.2 ± 2.94	161.9 ± 15.49	117.7 ± 3.09
10 to 20 mm Hg	175.5 ± 15.66	128.3 ± 3.85	169.9 ± 16.60	123.4 ± 3.87*
20 to 30 mm Hg	177.4 ± 16.48	129.5 ± 4.37	173.4 ± 16.47	126.5 ± 4.58*
30 to 40 mm Hg	179.0 ± 17.20	130.8 ± 5.02	175.4 ± 15.86	128.5 ± 5.03

 Mean diameter of isolated bovine retinal arteries in a K⁺ 120 mM containing solution measured 1 minute and 10 minutes after an increase in intraluminal pressure. The diameters are expressed as μm and as a percentage of the initial diameter (i.e., the diameter of the depolarized retinal artery at 0 mm Hg). Statistical significance was evaluated using a paired Student’s-t test. Asterisk indicates that the arterial diameter after 10 minutes was significantly different (P < 0.05) from the diameter after 1 minute.

 Data are means ± SEM. Diameters are in micrometers.