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From the Department of Physiology and Pathophysiology, Ghent University, Belgium.
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Abstract |
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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.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 F2
(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
F2 (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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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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 (1 µM) 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.
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Discussion |
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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 Hg7 (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 C9 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.
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Footnotes |
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Submitted for publication July 27, 1999; revised November 10 and December 20, 1999; accepted January 5, 2000.
Commercial relationships policy: N.
Corresponding author: Johan Van de Voorde, Department of Physiology and Pathophysiology, Ghent University, De Pintelaan 185, Blok B, B-9000 Gent, Belgium. johan.vandevoorde{at}rug.ac.be
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 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.