(Hypertension. 1995;26:1051-1055.)
© 1995 American Heart Association, Inc.
Articles |
Intracellular Ca2+ Release in Flow-Induced Contraction of Venous Smooth Muscle
From the Department of Pharmacology, College of Medicine, The University of Vermont, Burlington.
Correspondence to John A. Bevan, Department of Pharmacology, College of Medicine, The University of Vermont, Burlington, VT 05405-0068.
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Abstract |
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Abstract We designed the present study to determine whether Ca2+ release from intracellular stores contributes to flow-induced contraction. We carried out experiments on segments of rabbit facial vein under isometric conditions. Intraluminal flow by perfusion of physiological salt solution (10 to 80 µL/min) caused contraction in this vessel, which was significantly inhibited by (1) 30-minute pretreatment with 10 µmol/L ryanodine, the sarcoplasmic reticulum Ca2+ channel opener, and (2) 30-minute pretreatment with concomitant application of 20 mmol/L caffeine and 1 µmol/L cyclopiazonic acid in Ca2+-free medium to deplete the sarcoplasmic reticulum. In comparison, contraction initiated by 300 nmol/L histamine was significantly attenuated by the same interventions. K+ (25 mmol/L)–induced contraction was unaffected by ryanodine but was reduced after depletion of the sarcoplasmic reticulum. The phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (10 µmol/L) inhibited contractions induced by flow and histamine but not by K+. These findings indicate that Ca2+ release from intracellular stores, presumably via the phosphatidylinositol pathway, contributes to flow- and histamine- but not raised K+–induced contractions in this vessel.
Key Words: calcium • potassium • muscle, smooth, vascular • muscle contraction
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Introduction |
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In essential hypertension, development and maintenance of increased peripheral resistance results in elevated blood pressure. Increased vascular smooth muscle contractility is one of the factors leading to increased peripheral resistance. The sympathetic nervous system, humoral vasoactive substances, and mechanical forces are all stimuli that can initiate contraction of peripheral resistance vessels. The present study is concerned with the contraction induced by flow, a shear force, and its cellular mechanism or mechanisms. Shear stress plays an important role in the regulation of vascular tone,1 and changes in response to flow have been seen in hypertension.2
It is well accepted that a rise in intracellular Ca2+ concentration is the key requirement for vascular smooth muscle contraction. This rise results from Ca2+ influx from the extracellular space, Ca2+ release from intracellular stores, or both. The contribution from each site varies greatly among different blood vessels and with the contractile influence.3 4 5
Ca2+ release from intracellular stores is an important component for contractions induced by a variety of agonists.6 7 8 9 At least two mechanisms have been proposed for Ca2+ release from the SR. The major trigger for this release is IP3, one of the products of the hydrolysis of membrane phosphatidylinositides.5 10 The other mechanism is known as Ca2+-induced Ca2+ release. The latter has been observed in both nonvascular and vascular smooth muscle,11 12 13 although it is favored to function in the excitation-contraction coupling of skeletal and cardiac muscle.14 15
Shear stress can cause Ca2+ release in endothelial cells.16 Shear stress by intraluminal flow has been shown to produce vascular contraction in several isolated vessels.17 18 19 20 21 Flow contraction is endothelium-independent1 and associated with depolarization22 and an increase in 45Ca2+ influx in part through voltage-gated Ca2+ channels.20 23 However, many aspects of the mechanism of flow contraction have not been elucidated.
In the present study we investigated whether Ca2+ release from the SR contributes to flow-induced contraction in vascular smooth muscle. Our results indicate that in the RFV Ca2+ release from the SR, presumably via the phosphatidylinositol pathway, contributes to flow- and histamine- but not raised K+-induced contractions.
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Methods |
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General Preparations
Male New Zealand White rabbits weighing 2 to 3 kg were anesthetized with sodium pentobarbital (40 mg/kg IV) and heparinized (1000 U/kg heparin IV). They were killed by exsanguination, and RFVs were removed immediately. The experiments were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee, The University of Vermont.
Mandibular segments of the RFV were isolated. The standard myograph technique described previously by Bevan and Joyce17 18 was used. Changes in isometric tension were recorded with a force displacement transducer (UL-2GR) and displayed on a chart recorder (SOLTEC 1242, model 43 TD).
Flow-dependent responses were achieved as described previously.17 18 In brief, PSS was infused into the lumen of the mounted segment by a syringe infusion pump (model 22, Harvard Apparatus) through a glass micropipette whose tip was positioned within about 0.3 mm of one open end of the segment. Veins were always infused in the direction of normal blood flow.
All experiments were carried out on endothelium-denuded ring preparations. Endothelial cells were removed by gentle rubbing of the intimal surface with rough plastic tubing. Endothelium removal was assumed to be successful if the relaxation to acetylcholine (3 µmol/L) was lost. It was verified in some experiments by scanning electron microscopy.
Experimental Protocols
Effects of Ryanodine
Flow (10 to 80 µL/min)–induced contractions were examined
before and after pretreatment with ryanodine (1 to 10 µmol/L for 30
minutes), the SR Ca2+ release channel
opener.24 25 Effects of ryanodine on responses to 25
mmol/L K+ and 300 nmol/L histamine were also compared.
Effects of Depleting the SR
To empty the SR, we exposed tissues to 20 mmol/L caffeine in
Ca2+-free PSS for 30 minutes and washed them every
15 minutes, during which we added CPA (1 µmol/L), the
Ca2+-ATPase inhibitor,25 to
block the uptake of Ca2+ by the SR
Ca2+ pump. The loss of caffeine-induced
contraction was considered an indication that the SR was empty.
Responses to flow (40 µL/min), K+ (25 mmol/L), and
histamine (300 nmol/L) were examined in the presence of
Ca2+ (1.6 mmol/L) before and after depletion of the
SR.
Effects
of 2-Nitro-4-Carboxyphenyl-N,N-Diphenylcarbamate
Contractions induced by flow, 25 mmol/L K+,
and 300 nmol/L histamine were investigated before and after a 30-minute
preincubation with NCDC (10 µmol/L), the putative PLC
inhibitor.26
Drugs
Acetylcholine, caffeine, cimetidine, CPA, histamine, NCDC, and
ryanodine were purchased from Sigma Chemical Co. CPA and ryanodine were
dissolved in dimethyl sulfoxide, NCDC in ethanol, and the rest of the
drugs in deionized water. The volume of solvent used in the present
study had no effect on the tissues. The composition of the PSS was
(mmol/L) NaCl 135, KCl 4.7, CaCl2 1.6,
MgSO4 1.2, KH2PO4 1.2,
NaHCO3 15, dextrose 11.1, and EDTA 0.026, pH 7.4. KCl (25
mmol/L) was prepared by substitution of an equimolar amount of NaCl
with KCl. Nominal Ca2+-free PSS was prepared by
simple omission of calcium from normal PSS, whereas
Ca2+-free (plus EGTA) PSS was used in previous
studies.20 23 PSS always contained cimetidine (1 µmol/L)
to block H2 receptors.
Data Analysis
Data points plotted in the figures represent the
mean±SEM; n values indicate the number of animals examined. Paired
data were analyzed with Student's t test.
Differences were considered significant at a value of
P<.05.
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Results |
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Effects of Ryanodine on Responses to Flow, K+, and Histamine
Ryanodine (10 µmol/L) elevated the basal tone of the RFV. At 10 µmol/L ryanodine, caffeine (20 mmol/L)-induced contraction was reversed to relaxation (Fig 1a), indicating that the ryanodine concentration was effective in emptying the SR Ca2+ contents. Contractions induced by flow (40 µL/min, n=8) and histamine (300 nmol/L, n=6) were sensitive to pretreatment with ryanodine (10 µmol/L). However, the response to 25 mmol/L K+ (n=6) was unaffected (Fig 1b and 1c). Ryanodine (1 to 10 µmol/L) inhibited flow contraction (10 to 80 µL/min) in a concentration-dependent manner (n=5, Fig 2).
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.0001 compared with control), 25 mmol/L K+
(K, n=6, P>.05), and 300 nmol/L histamine (H, n=6,
**P
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Effects of Depleting the SR on Responses to Flow,
K+, and Histamine
Caffeine (20 mmol/L) caused a transient contraction in both normal
and Ca2+-free PSS. After two washes with
Ca2+-free PSS plus 1 µmol/L CPA, caffeine did not
elicit contraction (Fig 3a), indicating
that the SR Ca2+ contents were emptied. Responses to
flow (40 µL/min, n=8), histamine (300 nmol/L, n=5), and
K+ (25 mmol/L, n=5) were significantly reduced after
depletion of the SR, although the extent of reduction was different
(Fig 3b and 3c).
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Effects of NCDC on Responses to Flow, K+,
and Histamine
Contractions induced by flow and histamine (300 nmol/L) were
significantly inhibited by 30 minutes of pretreatment with 10 µmol/L
NCDC, the PLC inhibitor. However, NCDC had no effect on the
response induced by 25 mmol/L K+ (Fig 4,
n=4 to 5).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Our findings indicate that Ca2+ release from the SR contributes to flow-induced contraction in RFV. Previous studies showed that flow contraction is Ca2+ dependent and associated with an increase in 45Ca2+ uptake.20 The pathway for Ca2+ entry partly involves voltage-gated Ca2+ channels.23 However, the existence of a nifedipine-resistant component of flow contraction was evident.23 Although the nifedipine-resistant component of flow contraction was eliminated by removal of extracellular Ca2+ with Ca2+-free medium plus EGTA (2 mmol/L), this observation could reflect either an involvement of dihydropyridine-insensitive Ca2+ channels or a loss of a component mediated by Ca2+ release from intracellular stores.27
We investigated the possible involvement of SR Ca2+ release in flow contraction using two approaches. First, the experiments presented here show that ryanodine (1 to 10 µmol/L) significantly inhibited flow-induced contraction. Ryanodine can open SR Ca2+ channels and result in Ca2+ release from the SR.24 25 Our data confirmed that ryanodine antagonized caffeine-induced Ca2+ release, as indicated by the loss of caffeine-induced transient contraction. The reversion of caffeine-induced contraction to relaxation after pretreatment with ryanodine could be due to an increase in cytosolic cAMP by the inhibition of phosphodiesterase,28 an increase in Ca2+ extrusion,29 or both. The contractile response to 300 nmol/L histamine was also inhibited by ryanodine, but K+ (25 mmol/L)–induced contraction was unaffected. Thus, the effect of ryanodine in these experiments is selective.
Second, our results indicate that flow contraction was greatly attenuated after depletion of the SR by pretreatment of tissues with 20 mmol/L caffeine plus 1 µmol/L CPA in Ca2+-free medium. Caffeine releases Ca2+ from the SR in a variety of preparations,8 30 31 and CPA specifically inhibits the SR Ca2+-ATPase.25 Thus, our procedure ensured that the SR was empty because concomitant application of CPA can prevent the possible uptake of Ca2+ by the SR. However, responses to histamine (300 nmol/L) and K+ (25 mmol/L) were also affected when the SR was emptied by the same intervention.
Our results with histamine are in agreement with other reports that this amine can induce contraction via Ca2+ release from the SR in vascular smooth muscle.7 32 As for the decrease in response to 25 mmol/L K+ after depletion of the SR, two possible explanations can be provided. One is related to the superficial buffer barrier function of the SR.33 When the SR is loaded with Ca2+, extracellular Ca2+ enters cells and causes contraction by acting on the contractile apparatus; but when the SR is emptied, a larger portion of Ca2+ entering the cells would then be taken up by the SR, resulting in a reduction in Ca2+ availability for contraction. However, it is unlikely that the attenuated contraction by K+ observed here is due to Ca2+ uptake by the SR because this pathway was blocked by pretreatment with CPA. Alternatively, it is possible that depletion of the SR affected the response to K+ by interfering with the Ca2+-induced Ca2+ release mechanism. This is consistent with observations in other vascular smooth muscle.34 After depletion of the SR, depolarization with K+ causes Ca2+ entry, which will no longer release additional Ca2+ for contraction. Thus, Ca2+ entering the cells would act only on contractile proteins and cause a delayed and smaller response.
We also explored the possible trigger for Ca2+ release in the present study. NCDC is claimed to be the inhibitor of PLC.26 In agonist- or hormone-initiated signal-transduction cascades, the activation of PLC through at least one closely coupled G protein results in the generation of two second messengers, IP3 and diacylglycerol, which are involved in intracellular Ca2+ release and protein kinase C activation, respectively.4 5 Since NCDC (10 µmol/L) had inhibitory effects on flow- and histamine-induced contractions but did not alter the response to K+, a nonspecific action of NCDC on the contractile process is unlikely. Thus, our data suggest that IP3-induced Ca2+ release might be involved in flow- and histamine-induced contractions. Interestingly, it has been noted that shear stress can increase phosphatidylinositol turnover.35 On the other hand, we are aware that our data are indirect, and the possibility that the inhibition of flow contraction by NCDC might be related to a decrease in diacylglycerol formation and protein kinase C activation cannot be excluded.
On the basis of our findings and those of others,16 we propose that flow can act on the cell membrane and cause conformational changes within membrane proteins via an unidentified G protein coupling event. One of these changes involves activation of the phosphatidylinositol-PLC pathway. Consequently, IP3 and Ca2+, as second messengers, are responsible for flow contraction.
Finally, the characterization of the mechanism or mechanisms responsible for flow contraction would help advance our understanding of the physiology of blood vessels. One of the fundamental features of hypertension is increased contractility of vascular smooth muscle. It has been reported that cytosolic free Ca2+ and phosphatidylinositol turnover increase in response to agonists in spontaneously hypertensive rats.36 37 Furthermore, endothelium-dependent flow dilation is impaired during the development of hypertension.2 However, whether changes in flow contraction in hypertension are primary or secondary and whether Ca2+ handling associated with flow contraction is altered await future study.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by US Public Health Service grant HL-32985. We wish to thank John Dodge for help in scanning electron microscopy.
Received June 19, 1995; first decision August 1, 1995; accepted August 18, 1995.
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References |
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