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Am J Physiol Heart Circ Physiol 284: H1808-H1817, 2003. First published February 13, 2003; doi:10.1152/ajpheart.00637.2002
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Vol. 284, Issue 5, H1808-H1817, May 2003

Phasic contractions of the rat portal vein depend on intracellular Ca2+ release stimulated by depolarization

Richard P. Burt

Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The phasic contraction to phenylephrine of the rat isolated portal vein was investigated using functional studies. Phasic contractions to phenylephrine and caffeine could be produced after several minutes in Ca2+-free Krebs solution, which were inhibited by cyclopiazonic acid or ryanodine. The phenylephrine and caffeine contractions were abolished, however, within 10 min in Ca2+-free Krebs solution and by nifedipine. This indicated the Ca2+ stores were depleted in the absence of Ca2+ influx through voltage-gated channels. The phasic contraction to phenylephrine was also abolished by niflumic acid even in Ca2+-free Krebs solution. This showed that the response depended on intracellular Ca2+ release stimulated directly by depolarization, resulting from opening of Ca2+-activated Cl- channels, but did not require Ca2+ influx. In support of this, K+-induced phasic contractions were also produced in Ca2+-free Krebs solution. The phenylephrine but not K+-induced phasic contractions in Ca2+-free Krebs solution were inhibited by ryanodine or cyclopiazonic acid. This would be consistent with Ca2+ release from more superficial intracellular stores (affected most by these agents), probably by inositol 1,4,5-trisphospate, being required to stimulate the phenylephrine depolarization.

voltage-sensitive Ca2+ release; Ca2+-activated Cl- channels; niflumic acid


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CONTRACTION OF THE RAT PORTAL VEIN to phenylephrine is mediated by alpha 1-adrenoceptors (38). These are G protein-coupled receptors that are often linked to activation of phospholipase C, resulting in a rise of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol produced from phosphatidylinositol 4,5-bisphosphate (30). Ins(1,4,5)P3 releases Ca2+ from intracellular stores via specific Ins(1,4,5)P3 receptors (4), whereas diacylglycerol can activate PKC (25). Another Ca2+ release channel of the sarcoplasmic reticulum is the ryanodine receptor (RYR) (45). Ryanodine either opens or blocks these channels, depending on concentration. They are Ca2+-activated channels involved in Ca2+-induced Ca2+ release (CICR) from intracellular stores. This has been shown to be stimulated in rat portal vein myocytes by Ca2+ influx through voltage-gated channels (16) but not by Ca2+ released through Ins(1,4,5)P3 receptors (33). In portal vein cells, Ins(1,4,5)P3-sensitive and ryanodine-sensitive Ca2+ pools may be functionally linked (27).

Spontaneous transient inward currents stimulated by opening of Ca2+ activated Cl- channels have been shown in rabbit (8) and rat (35) portal vein cells. Rat portal vein tissues display spontaneous phasic contractions, an ability shared by small resistance vessels (10). These spontaneous contractions are thought to result from depolarizations stimulated by spontaneous transient inward currents. Stimulation of alpha 1-adrenoceptors can also activate Ca2+-activated Cl- current [ICl(Ca)] in rat portal vein cells (34, 36), which results from release of intracellular Ca2+ (5). ICl(Ca) can be selectively inhibited by niflumic acid in rabbit (17) and rat portal vein cells (20, 35).

In isolated tissues, evidence indicates that spontaneous contractions of the portal vein are completely dependent on influx of extracellular Ca2+ (13, 29). The phasic contraction to phenylephrine, however, was reported to be still present in the absence of extracellular Ca2+ (39). The aim of the present study, therefore, was to investigate the phasic contraction to phenylephrine of the rat isolated portal vein and to compare this with K+-induced and spontaneous contractions. This was done using drugs that may show how cellular mechanisms described in portal vein myocytes might relate to the function of whole tissues.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Male Sprague-Dawley rats between 350 and 450 g were stunned and killed by cervical dislocation. The portal vein was removed into Krebs solution, and associated connective tissue was dissected away. The tissues (10-15 mm) were suspended longitudinally in 5-ml tissue baths containing Krebs solution of the following composition (in mM): 143 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg2+, 128 Cl-, 25 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 1.2 HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 1.2 SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, and 11 glucose, at 25°C and bubbled with 95% O2-5% CO2. The portal veins were placed under 0.5 g of resting tension and equilibrated for 1 h. A modified high-K+ Krebs solution was sometimes used of the same composition except for an increase in K+ to 50 mM and an equivalent decrease in Na+ to 98.9 mM. Changes in isometric tension were measured using Grass FT.03 transducers and recorded by Biopac Systems MP100WS for Windows.

When Ca2+-free Krebs solution was used, it always contained EGTA (1 mM). Tissues were washed for 30 s with 0.5 liter of Ca2+-free Krebs solution to remove extracellular Ca2+. Although some responses in this study were measured after only 45 s in Ca2+-free Krebs solution, the spontaneous contractions were completely abolished by this. Previous studies have shown that Ca2+ is depleted quickly from intracellular stores in the rat portal vein when placed in a Ca2+-free medium (13, 39), and in the rat uterus and portal vein this effect occurs more slowly at lower temperatures (13, 23). Preliminary investigations supported this, and so all the present experiments were performed at 25°C so that contractions in Ca2+-free Krebs solution could be recorded.

A single dose of phenylephrine (10-4 M) was added to the tissues, which were then left to recover for 45 min until the spontaneous phasic contractions had become reproducible again before further tests were carried out. The concentration of phenylephrine used throughout the study was 10-4 M (which produced a maximal response) unless otherwise stated. When drugs were tested on responses to phenylephrine (10-6 M), reproducible control responses were recorded at 45-min intervals before drug addition. Drugs were incubated for 30 min with tissues unless otherwise stated.

Experiments in normal and Ca2+-free Krebs solution. The effect of nifedipine (3 × 10-7 M), niflumic acid (3 × 10-5 M), and calphostin C (10-6 M, 1-h incubation) was recorded on the spontaneous contractions and response to phenylephrine. The effect of Ca2+-free Krebs solution on the spontaneous contractions and response to phenylephrine after 45 s, 2 min, 4 min, and 10 min in Ca2+-free Krebs solution was recorded, and the effect of ryanodine (10-4 M), cyclopiazonic acid (10-5 M), nifedipine (3 × 10-7 M), or niflumic acid (3 × 10-5 M) was measured on the response to phenylephrine after 2 min in Ca2+-free Krebs solution. Contractions to caffeine (25 mM) were also recorded, and the effects of nifedipine (3 × 10-7 M) and niflumic acid (3 × 10-5 M) were measured on this response. The effect of either 45 s, 2 min, or 5 min in Ca2+-free Krebs solution on the response to caffeine was measured.

Responses to K+ (20, 50, or 100 mM) were measured in normal Krebs solution, after 2 min in Ca2+-free Krebs solution, and after 2 min in Ca2+-free Krebs solution in the presence of ryanodine (10-4 M). Responses to K+ (20 or 50 mM) were also measured after 45 s in Ca2+-free Krebs solution and after 45 s in Ca2+-free Krebs solution in the presence of ryanodine (10-4 M). The effect of a combination of prazosin (10-7 M) and alpha ,beta -methylene ATP (10-5 M) on responses to K+ (50 mM) was measured in normal Krebs solution.

Experiments in high-K+ Krebs solution. For some experiments, after the initial response to phenylephrine, the Krebs solution was changed to a modified high-K+ (50 mM) Krebs solution. Tissues were left until this contraction had returned to baseline or close to it (after ~40 min), and the response to phenylephrine was then measured. The effect of nifedipine (3 × 10-7 M) or niflumic acid (3 × 10-5 M) on the response to phenylephrine in high K+-Krebs solution was measured. The effect of equilibrating tissues in high-K+ Krebs solution and then changing to high-K+, Ca2+-free Krebs solution for 2 min on the response to phenylephrine was then recorded, and the effect of ryanodine (10-4 M) or cyclopiazonic acid (10-5 M) on this response was measured. The effect of changing from normal to Ca2+-free Krebs solution for 1 min followed by 1 min in high-K+, Ca2+-free Krebs solution on the response to phenylephrine was also measured. The response to caffeine (25 mM) in high-K+ Krebs solution was also recorded, and the effect of niflumic acid (3 × 10-5 M) was measured on this response.

Data analysis. All contractions were measured as a percentage of the maximum tonic response to phenylephrine (10-4 M) and calculated as the mean from four separate experiments (n = 4). Statistical significance of differences between control and test means was tested for on raw data using a paired t-test where a control and test value are given together and a nonpaired t-test if given separately. A P value of <0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using Prism (GraphPad Software; San Diego, CA).

Drugs and solutions. Nifedipine, niflumic acid, caffeine, and phenylephrine were obtained from Sigma. Ryanodine, cyclopiazonic acid, and calphostin C were obtained from Calbiochem. Prazosin and alpha ,beta -methylene ATP were obtained from RBI. All stock solutions were made in distilled water except for niflumic acid, cyclopiazonic acid, and calphostin C, which were dissolved along with further dilutions in DMSO, and nifedipine, which was dissolved in ethanol and then diluted in distilled water. Prazosin was dissolved in DMSO with further dilutions in distilled water. Stock solutions were stored frozen except for phenylephrine and caffeine, which were made fresh each day in distilled water.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All phasic contractions were measured at their maximum and given as a percentage of the maximum tonic response to phenylephrine (10-4 M) for each tissue. The phasic contraction to phenylephrine always preceeds the tonic response and is not a component of the latter (see Fig. 1). Values for phasic contractions, therefore, are smaller than 100% but are the maximum contractile response of the tissue at the point of measurement. The duration of time tissues were in Ca2+-free Krebs solution was measured from the point of Ca2+ removal to the point of drug addition.


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  		Fig. 1.   A: spontaneous contractions and contraction to phenylephrine (10-4 M) of the rat portal vein. B: phasic and tonic contractions to phenylephrine (10-4 M). C: several phasic contractions to phenylephrine (10-4 M) preceeding the tonic response.

Spontaneous and phenylephrine-induced phasic contractions of the portal vein. Spontaneous contractions of the rat portal vein occurred with a frequency of 3.4 ± 0.6 contractions/min, a duration of 8.2 ± 0.5 s, and a maximum tension of 0.59 ± 0.05 g (Fig. 1A). Phenylephrine (10-4 M) produced a contraction consisting of an initial phasic response (55 ± 4%) or, in some cases, several phasic responses merged together, which developed into a larger tonic response (Fig. 1; maximum response 1.72 ± 0.05 g). All subsequent responses to phenylephrine are for 10-4 M unless otherwise stated.

Dependence of phasic contractions on extracellular Ca2+. The spontaneous contractions were abolished within 45 s in Ca2+ free-Krebs solution (Fig. 2). The contraction to phenylephrine after 45 s in Ca2+-free Krebs solution consisted of an initial phasic response (Fig. 2A; 39 ± 4%), followed by a more slowly developing second phasic response. After 2 min in Ca2+-free Krebs solution, the initial phasic response was 29 ± 4% (Fig. 2B) and after 4 min was 21 ± 2% (Fig. 2C). After 10 min in Ca2+-free Krebs solution, the contractions to phenylephrine were abolished (results not shown). There was no maintained tonic component to the phenylephrine contractions at any time in Ca2+-free Krebs solution. Cyclopiazonic acid (10-5 M) reduced the initial phasic response to phenylephrine after 2 min in Ca2+-free Krebs solution to 8 ± 3% (P < 0.05; Fig. 2D), and ryanodine (10-4 M) abolished it (Fig. 2E). These results indicated the initial phasic response to phenylephrine involved release of intracellular Ca2+ from stores that were depleted quickly in the absence of extracellular Ca2+.


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  		Fig. 2.   Effect of Ca2+-free Krebs solution on the spontaneous contractions and on the contraction to phenylephrine (10-4 M) after 45 s (A), 2 min (B), and 4 min (C). D and E: effect of cyclopiazonic acid (10-5 M; D) and ryanodine (10-4 M; E) on the contraction to phenylephrine (10-4 M) after 2 min in Ca2+-free Krebs solution.

Effect of high-K+ Krebs solution. Equilibration of tissues in high-K+ Krebs solution has been shown previously to potentiate contractions in the absence of extracellular Ca2+ (7, 28). When the Krebs solution was changed to a modified high-K+ (50 mM) Krebs solution, this produced an initial phasic contraction (75 ± 5%) of the portal vein, followed by a smaller tonic response (47 ± 5%), which returned to the baseline, or close to it (within 5%), after ~40 min (Fig. 3A). The high-K+ Krebs solution completely abolished the spontaneous activity in all tissues. Phenylephrine in high-K+ Krebs solution produced a contraction consisting of an initial phasic response (47 ± 2%), followed by a tonic response (82 ± 1%) (Fig. 3B). After equilibration in high-K+ Krebs solution, some tissues were placed in high-K+, Ca2+-free Krebs solution for 2 min before a response to phenylephrine was recorded. The initial phasic response to phenylephrine that occurred after 2 min in high-K+, Ca2+-free Krebs solution was potentiated (49 ± 2%, P < 0.05; Fig. 3C) compared with after 2 min in Ca2+-free Krebs solution with no high-K+ Krebs solution equilibration (Fig. 2B). When tissues were placed in Ca2+-free Krebs solution for 1 min followed by high-K+, Ca2+-free Krebs solution for 1 min, the initial phasic response to phenylephrine was not potentiated (31 ± 2%, results not shown). This showed that the potentiating effect of high-K+ Krebs solution involved influx of extracellular Ca2+. Cyclopiazonic acid (10-5 M) reduced the initial phasic response to phenylephrine after 2 min in high-K+ Ca2+-free Krebs solution after high-K+ Krebs solution to 24 ± 2% (P < 0.05; Fig. 3D), and ryanodine (10-4 M) abolished it (Fig. 3E). These results indicate that equilibration of the portal vein in high-K+ Krebs solution inhibits the depletion of Ca2+ from intracellular stores in the absence of extracellular Ca2+.


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  		Fig. 3.   A: contraction of the rat portal vein to high-K+ (50 mM) Krebs solution. B and C: contraction to phenylephrine (10-4 M) in high-K+ Krebs solution (B) and after 2 min in high-K+, Ca2+-free Krebs solution after high-K+ Krebs solution (C). D and E: contraction to phenylephrine (10-4 M) after 2 min in high-K+, Ca2+-free Krebs solution after high-K+ Krebs solution and in the presence of cyclopiazonic acid (10-5 M; D) and ryanodine (10-4 M; E).

Effect of caffeine. Caffeine activates the ryanodine channel and can release Ca2+ from ryanodine-sensitive stores (37). A series of consecutive phasic contractions of the portal vein was produced by caffeine (25 mM) in normal Krebs solution (maximum reponse 48 ± 2%), which became progressively smaller, lasting ~1 min (Fig. 4A). In Ca2+-free Krebs solution, the response to caffeine (25 mM) again consisted of a series of consecutive phasic contractions, but after 45 s in Ca2+-free Krebs solution the maximum response was reduced to 33 ± 4% (Fig. 4B), and after 2 min it was reduced to 16 ± 1% and was abolished after 5 min (results not shown). After the response to caffeine (25 mM) in normal Krebs solution, spontaneous activity was completely abolished but returned after washout of caffeine. Caffeine (25 mM) produced a single phasic contraction in high-K+ Krebs solution (maximum response 27 ± 2%; Fig. 4C). These results indicate that release of intracellular Ca2+ from ryanodine-sensitive stores can produce a phasic contraction of the portal vein.


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  		Fig. 4.   Contraction of the rat portal vein to caffeine (25 mM) in normal Krebs solution (A), after 45 s in Ca2+-free Krebs solution (B), and in high-K+ Krebs solution (C).

Effect of nifedipine and niflumic acid. These experiments were performed to see whether depolarization stimulated by opening of Ca2+-activated Cl- channels and influx of Ca2+ through voltage-gated channels is involved in the phasic contractions. Niflumic acid was used to block the former, and nifedipine was used to block the latter. Nifedipine (3 × 10-7 M) or niflumic acid (3 × 10-5 M) abolished the spontaneous contractions of the portal vein (Fig. 5, A and B). Nifedipine (3 × 10-7 M) abolished the phasic contraction to phenylephrine (and inhibited the tonic response, 15 ± 2%) in normal Krebs solution (Fig. 5C). Niflumic acid (3 × 10-5 M) abolished the phasic and tonic contractions to phenylephrine in normal Krebs solution (Fig. 5D; some phasic activity returned in the presence of phenylephrine, but this was at least 30 s after the initial phasic response should have occurred). Nifedipine (3 × 10-7 M) also inhibited the phasic response to phenylephrine after 2 min in Ca2+-free Krebs solution (4 ± 1%), and niflumic acid (3 × 10-5 M) abolished this response (results not shown). This indicated that influx of Ca2+ through voltage-gated channels is required to prevent intracellular Ca2+ stores from being depleted. In high-K+ Krebs solution, nifedipine (3 × 10-7 M) inhibited the phasic and tonic contractions to phenylephrine (24 ± 2%, P < 0.05, and 16 ± 2%, respectively; Fig. 5E), whereas niflumic acid (3 × 10-5 M) abolished the phasic contraction to phenylephrine and inhibited the tonic response (14 ± 4%; Fig. 5F). The response to caffeine (25 mM) was abolished by nifedipine (3 × 10-7 M) and niflumic acid (3 × 10-5 M) in normal Krebs solution (results not shown). In high-K+ Krebs solution, the phasic contraction to caffeine (25 mM) was not inhibited by 3 × 10-5 M niflumic acid (27 ± 2%, results not shown). This showed that in high-K+ Krebs solution, niflumic acid does not inhibit phasic contractions by depleting Ca2+ from intracellular stores. The abolition of the phasic contraction to phenylephrine by niflumic acid in high-K+ Krebs solution suggests, therefore, that this response depends directly on depolarization.


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  		Fig. 5.   A and B: inhibition of spontaneous contractions of the rat portal vein by cumulative additions of nifedipine (A) and niflumic acid (3 × 10-5 M; B). C and D: inhibition of phasic and tonic contractions to phenylephrine (10-4 M) of the rat portal vein by nifedipine (3 × 10-7 M; C) and niflumic acid (3 × 10-5 M; D). E and F: inhibition of phasic and tonic contractions to phenylephrine (10-4 M) of the rat portal vein in high-K+ Krebs solution by nifedipine (3 × 10-7 M; E) and niflumic acid (3 × 10-5 M; F).

Effect of PKC inhibition on phasic contractions. The PKC inhibitor calphostin C (10-6 M) did not significantly affect spontaneous contractions (control 35 ± 3%, with calphostin C 34 ± 3%) or the phasic response to 10-4 M phenylephrine (control 53 ± 2%, with calphostin C 55 ± 3%) (results not shown). The highest concentration of DMSO (0.5%) resulting from the addition of any drugs in this study did not affect the spontaneous contractions or contraction to phenylephrine.

Second phasic contraction to phenylephrine in Ca2+-free Krebs solution. The more slowly developing second phasic contraction to phenylephrine (10-4 M), which followed the initial phasic response in Ca2+-free Krebs solution, was not significantly inhibited by cyclopiazonic acid (10-5 M) or ryanodine (10-4 M). The control response after 2 min in Ca2+-free Krebs solution was 37 ± 3% (Fig. 2B), with cyclopiazonic acid was 35 ± 6% (Fig. 2D), and with ryanodine was 34 ± 5% (Fig. 2E), but was abolished by nifedipine (3 × 10-7 M, results not shown). The second phasic response was reduced to 21 ± 2% after 4 min in Ca2+-free Krebs solution (Fig. 2C) and abolished after 5 min in Ca2+-free Krebs solution (results not shown). The second phasic contraction to phenylephrine (10-4 M) was potentiated after 2 min in high-K+, Ca2+-free Krebs solution after equilibration in high-K+ Krebs solution (45 ± 3%, P < 0.05; Fig. 3C) compared with after 2 min in Ca2+-free Krebs solution. These results indicate that the second phasic contraction to phenylephrine in Ca2+-free Krebs solution involved release of intracellular Ca2+ from a different pool which produces the initial phasic response.

K+-induced contractions. Contractions to K+ of the portal vein consisted of an initial phasic contraction followed by a smaller tonic response. Maximum contractions to K+ (20, 50, and 100 mM) were 61 ± 2%, 75 ± 2%, and 77 ± 5%, respectively (see Fig. 7, A-C, for typical traces). The response to K+ (50 mM) was not affected by a combination of 10-7 M prazosin and 10-5 M alpha ,beta -methylene ATP (control maximum response 41 ± 3%, with prazosin and alpha ,beta -methylene ATP 39 ± 3%, results not shown).

After 45 s in Ca2+-free Krebs solution (which abolished the spontaneous contractions), K+ (20 and 50 mM) produced phasic contractions of 28 ± 2% and 42 ± 5%, respectively (Fig. 6, A and C). After 45 s in Ca2+-free Krebs solution and in the presence of ryanodine (10-4 M), the responses to K+ (20 and 50 mM) were 35 ± 5% and 52 ± 6%, respectively (Fig. 6, B and D). After 2 min in Ca2+-free Krebs solution, phasic contractions to K+ (20, 50, and 100 mM) were 13 ± 2%, 18 ± 2%, and 39 ± 5%, respectively (Fig. 7, D-F). Contractions to K+ (20, 50, and 100 mM) after 2 min in Ca2+-free Krebs solution and in the presence of ryanodine (10-4 M) were 15 ± 2%, 22 ± 3%, and 47 ± 7%, respectively (Fig. 7, G-I). These results indicate that depolarization can directly stimulate release of intracellular Ca2+.


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  		Fig. 6.   A and C: contractions after 45 s in Ca2+-free Krebs solution to 20 mM K+ (A) and 50 mM K+ (B). B and D: contractions after 45 s in Ca2+-free Krebs solution and in the presence of ryanodine (10-4 M) to 20 mM K+ (B) and 50 mM K+ (D).



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  		Fig. 7.   A-C: contractions in normal Krebs solution to 20 mM (A), 50 mM (B), and 100 mM K+ (C). D-F: contractions after 2 min in Ca2+-free Krebs solution to 20 mM (D), 50 mM (E), and 100 mM K+ (F). G-I: contractions after 2 min in Ca2+-free Krebs solution and in the presence of ryanodine (10-4 M) to 20 mM (G), 50 mM (H), and 100 mM K+ (I).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The rat isolated portal vein displayed regular spontaneous phasic contractions, as described in previous studies on this tissue (13, 39, 41). Phenylephrine produced a maximum contraction at 10-4 M, which is mediated by alpha 1-adrenoceptors (38), consisting of an initial phasic contraction that then formed a larger sustained tonic response. Sometimes the initial response to phenylephrine consisted of several phasic contractions, merged together with increasing magnitude. The phasic responses to phenylephrine were similar in appearance to the spontaneous contractions. The frequency of the spontaneous activity for each tissue, however, showed the phasic response to phenylephrine was stimulated directly by the agonist and was not a spontaneous contraction. The aim of this study, therefore, was to compare the mechanism of contraction involved in the phasic response with phenylephrine (10-4 M unless otherwise stated) and the spontaneous contractions. The results suggest the phasic contraction to phenylephrine depends on release of intracellular Ca2+ from ryanodine-sensitive stores. This is stimulated directly by depolarization resulting from opening of Ca2+-activated Cl- channels. The spontaneous activity may involve a similar mechanism, but requires Ca2+ influx to stimulate Ca2+ release from the ryanodine-sensitive stores.

Phasic contraction to phenylephrine. The dependence of the phasic contraction to phenylephrine on intracellular or extracellular Ca2+ was first investigated. The phasic response was still present up to 4 min in Ca2+-free Krebs solution but became smaller with time, being completely abolished within 10 min. This suggested the contraction involved release of Ca2+ from intracellular stores but that these stores were depleted in the absence of extracellular Ca2+ (in agreement with Refs. 13 and 39). The initial phasic contraction in Ca2+-free Krebs solution was followed by another slower phasic response (discussed later), with no tonic contraction.

The phasic phenylephrine contraction was quickly reduced in Ca2+-free Krebs solution, but equilibration in high-K+ Krebs solution has been shown to potentiate contractions dependent on intracellular Ca2+ release (7, 28). This effect is consistent with the Ca2+ content of the intracellular stores being increased during sustained influx of extracellular Ca2+. In high-K+ Krebs solution (which abolished the spontaneous activity), once contraction of the portal vein to this had returned to baseline, phenylephrine still produced a phasic and tonic contraction. The phasic response to phenylephrine in high-K+ Krebs solution was not potentiated when compared with that in normal Krebs solution. If tissues were equilibrated in high-K+ Krebs solution followed by 2 min in high-K+ Ca2+-free Krebs solution, however, the phasic contraction was potentiated compared with that in Ca2+-free Krebs solution. High-K+ Krebs solution therefore reduced the depletion of Ca2+ from intracellular stores of the portal vein when extracellular Ca2+ was removed. When tissues were placed in Ca2+-free Krebs solution followed by high-K+, Ca2+-free Krebs solution, the phasic response was not potentiated, showing that this effect was not due simply to high K+. Furthermore, in the DISCUSSION, when responses in high-K+, Ca2+-free Krebs solution are mentioned, tissues will always have been first equilibrated in high-K+ Krebs solution.

Cyclopiazonic acid is a Ca2+-ATPase inhibitor of the sarcoplasmic reticulum that can deplete Ca2+ from Ins(1,4,5)P3-sensitive intracellular stores in smooth muscle (40). Cyclopiazonic acid and ryanodine both inhibited the phasic contraction to phenylephrine in Ca2+-free Krebs and high-K+ Ca2+-free Krebs solution This showed that the response involved release of Ca2+ from ryanodine-sensitive intracellular stores in both cases. Caffeine, which activates the ryanodine channel (37), produced a phasic contraction of the portal vein in normal and Ca2+-free Krebs solution, indicating that the release of Ca2+ from intracellular stores could be responsible for the phasic contraction to phenylephrine. The contraction to caffeine was abolished after 5 min in Ca2+-free Krebs solution, due to depletion of Ca2+ from intracellular stores. The phasic phenylephrine contraction, however, took longer to be abolished in Ca2+-free Krebs solution. This may be because the caffeine contraction is more dependent on release of the most superficially stored Ca2+, which would be depleted first in Ca2+-free Krebs solution. Ca2+ released initially could then release more Ca2+ by CICR. Caffeine produced a series of phasic contractions in normal and Ca2+-free Krebs solution, indicating that Ca2+ is released in waves during the response.

The phasic phenylephrine contraction was abolished by niflumic acid and nifedipine despite being dependent on release of intracellular Ca2+. These drugs block Ca2+-activated Cl- channels and L-type Ca2+ channels, respectively. Opening of Ca2+-activated Cl- channels can result in depolarization. Nifedipine and niflumic acid, however, also inhibited this response in Ca2+-free Krebs solution. This suggested that inhibiting Ca2+ influx with these drugs depleted intracellular Ca2+ stores and thus responses that are dependent on this in the same way that removing extracellular Ca2+ does. In support of this, nifedipine inhibited the phasic phenylephrine response in Ca2+-free Krebs solution to a greater extent than that in high-K+ Krebs solution. Influx of extracellular Ca2+ to refill intracellular stores could occur during the spontaneous contractions, which were also abolished by nifedipine and niflumic acid. Contractions to caffeine were abolished by nifedipine and niflumic acid in normal Krebs solution but were not affected by niflumic acid in high-K+ Krebs solution. This also showed that niflumic acid inhibited responses dependent on intracellular Ca2+ release by inhibiting Ca2+ influx in normal Krebs solution. In high-K+ Krebs solution, however, some Ca2+ influx through voltage-gated channels occurs, which is not dependent on opening of Ca2+-activated Cl- channels. The intracellular Ca2+ stores were therefore not depleted by niflumic acid in high-K+ Krebs solution.

The mechanism by which release of Ca2+ from ryanodine-sensitive stores is initiated in the phasic phenylephrine contraction was then investigated. The phasic response was still abolished by niflumic acid in high-K+ Krebs solution, showing that its effect was not due to depletion of intracellular Ca2+ stores, as in normal Krebs solution (discussed above). Influx of Ca2+ through voltage-gated channels has been shown to release Ca2+ from ryanodine-sensitive stores by CICR in portal vein cells (3, 16). The phasic contraction to phenylephrine was not abolished in Ca2+-free Krebs solution or high-K+, Ca2+-free Krebs solution, however. This showed that depolarization alone rather than Ca2+ influx can stimulate Ca2+ release from intracellular stores for the phasic phenylephrine contraction, sometimes called voltage-sensitive Ca2+ release. In support of this, niflumic acid was more effective at inhibiting the phasic rather than tonic response to phenylephrine in high-K+ Krebs solution, whereas the opposite was true for nifedipine. In normal Krebs solution, the phasic response may additionally involve some CICR, however. RYR1 has been shown to be associated with voltage-sensitive Ca2+ release, and RYR2 has been shown to be associated with CICR (42). All three RYR subtypes have been shown to be expressed in the rat portal vein, although voltage-sensitive Ca2+ release was not detected in isolated cells (11).

High-K+ Krebs solution should depolarize tissues to the extent that opening of Ca2+-activated Cl- channels would not produce further depolarization. The phasic and tonic phenylephrine contractions were not abolished in high-K+ Krebs solution, however, as this implies, and both were still inhibited by niflumic acid and nifedipine. This suggests that when the contraction to high-K+ Krebs solution has returned to baseline, the tissues have repolarized to the extent that opening Ca2+-activated Cl- channels can still produce depolarization. Niflumic acid would otherwise have to inhibit these responses by another mechanism. It did not, however, inhibit the contraction to caffeine in high-K+ Krebs solution, showing that it did not directly block Ca2+ release or have nonspecific effects on contraction. In other studies, niflumic acid inhibited norepinephrine but not K+-induced contractions of the rat aorta (12) and did not interact with alpha -adrenoceptors or voltage-dependent Ca2+ channels or evoke a K+ current in rabbit portal vein cells (17), further supporting its selectivity.

Stimulation of alpha 1-adrenoceptors produces a rise in Ins(1,4,5)P3 levels of rat portal vein cells (26). Intracellular Ca2+ released by Ins(1,4,5)P3 has also been shown to open Ca2+-activated Cl- channels in portal vein cells, resulting in depolarization (5, 31). A rise in Ins(1,4,5)P3 stimulated by phenylephrine should preceed any depolarization, and so contraction due directly to Ca2+ released by Ins(1,4,5)P3 should not be inhibited by niflumic acid in high-K+ Krebs solution. The phasic contraction to phenylephrine in the present study was abolished by niflumic acid, however, indicating that any Ins(1,4,5)P3-mediated Ca2+ release did not directly contribute to the phasic response. A "noncontractile" cellular Ca2+ compartment has been reported to be present in the ferret portal vein between the superficial sarcoplasmic reticulum and cell membrane (1, 2). A rise in Ins(1,4,5)P3 stimulated by phenylephrine may therefore release Ca2+ into this noncontractile compartment where it can stimulate opening of Ca2+-activated Cl- channels, resulting in depolarization and Ca2+ release from ryanodine-sensitive stores. The phenylephrine contraction was not affected by the PKC inhibitor calphostin C (21), which has been previously shown to inhibit alpha 1-adrenoceptor mediated contractions of the rat epididymal vas deferens (6).

The rise in intracellular Ca2+ concentration due to intracellular Ca2+ release, and which opens Ca2+-activated Cl- channels, would be transient. Influx of Ca2+ through voltage-gated channels resulting from this, however, also stimulates Ca2+-activated Cl- channels of the portal vein and may prolong ICl(Ca) (15). The time course of this appears to be regulated by Ca2+ uptake into mitochondria rather than by sarcoplasmic reticulum Ca2+-ATPase (14). Removal of Ca2+ from portal vein cells by Na+/Ca2+ exchange may also regulate the time course of ICl(Ca), as inhibition of this process can stimulate ICl(Ca) (24). Changes in intracellular pH could also affect the intracellular Ca2+ concentration and time course of ICl(Ca), although the effects of pH on phasic contractions of the portal vein are complex. Increasing pH has been shown to increase Ca2+ influx through voltage-gated channels but reduce peak intracellular Ca2+ concentration and tension in response to K+ (18). For spontaneous activity, increasing pH produced a transient decrease, followed by potentiation of the contractions (43).

After the initial phasic response in Ca2+-free Krebs solution, there was a slow phasic contraction to phenylephrine. The latter was potentiated by high-K+ Krebs solution and abolished by nifedipine but was not significantly affected by ryanodine or cyclopiazonic acid. This indicated that the response depended on a superficial source of Ca2+ different from the one responsible for the initial phasic response. The slow phasic contraction might therefore result from Ca2+ within the noncontractile cellular compartment. In normal Krebs solution, this response simply becomes part of the tonic contraction.

K+-induced phasic contractions. K+-induced phasic contractions were also produced in Ca2+-free Krebs solution at 25°C but reduced with time, showing that depolarization could directly stimulate intracellular Ca2+ release in the portal vein. The tonic contraction was always abolished in Ca2+-free Krebs solution. Involvement of intracellular Ca2+ release in K+-induced phasic contractions of the rat portal vein has previously been suggested (39). Contractions to K+ in the absence of extracellular Ca2+ have also been reported in the rat bladder (32) and aortic cells (22). K+-induced contractions were not affected by prazosin or alpha ,beta -methylene ATP, indicating that neuronal release of norepinephrine or ATP did not contribute to the response. There was no response to K+ (50 mM) at 37°C in Ca2+-free Krebs solution, consistent with the effect of temperature on the phasic contraction to phenylephrine (unpublished observation).

Work on portal vein cells has shown depolarization also stimulates CICR from ryanodine-sensitive stores in the presence of extracellular Ca2+ (3, 16). Although all the concentrations of K+ used in normal Krebs solution produced phasic responses of similar magnitude, lower concentrations of K+ had their responses reduced to a much greater extent in Ca2+-free Krebs solution. K+ (20 mM)-induced contractions in normal Krebs solution may therefore depend mostly on CICR, whereas the phasic response to high concentrations of phenylephrine or K+ depend more on voltage-sensitive Ca2+ release.

The presence of a superficial buffer barrier in portal vein cells (1, 2) may also prevent Ca2+ influx from directly stimulating phasic contractions during K+- or phenylephrine-induced depolarizations. Such a mechanism was proposed in the rabbit vena cava (9). The sarcoplasmic reticulum close to the cell membrane acts as a superficial buffer barrier for Ca2+ entering the cell, which first passes through a noncontractile Ca2+ compartment (44). It has been reported that virtually all Ca2+ influx on depolarization is taken up by the superficial sarcoplasmic reticulum in rat portal vein cells (19).

The phasic contraction to phenylephrine was abolished by ryanodine in Ca2+-free Krebs solution but contractions to K+ were not inhibited by ryanodine. The inhibitory effect of ryanodine and cyclopiazonic acid on the phenylephrine contraction in Ca2+-free Krebs solution may therefore result from these drugs inhibiting Ca2+ release more from the superficial part of the sarcoplasmic reticulum. Ca2+ release from this part of the intracellular stores by Ins(1,4,5)P3 may be required to stimulate niflumic acid-sensitive depolarization for the phenylephrine contraction. This would not be so for K+-induced depolarizations. Ryanodine and cyclopiazonic acid may therefore not affect Ca2+ release into the contractile compartment, at least in whole portal vein tissues.

Spontaneous contractions. The spontaneous activity was abolished within 45 s in Ca2+-free Krebs solution, before intracellular Ca2+ stores would have been depleted, and by nifedipine and niflumic acid (in agreement with Refs. 13, 20, and 29). This indicated the spontaneous contractions were dependent directly on Ca2+ influx through voltage-gated channels stimulated by opening of Ca2+-activated Cl- channels, resulting in depolarization. The presence of a superficial buffer barrier in this tissue may, however, prevent extracellular Ca2+ influx from directly stimulating contraction (discussed above). The spontaneous contractions were also similar in appearance compared with the phasic response to phenylephrine. If the spontaneous contractions involve Ca2+ release from ryanodine-sensitive stores, this must result from CICR, stimulated by Ca2+ influx through voltage-gated channels (3, 16).

In conclusion, the results of this study were consistent with the phasic contraction to phenylephrine of the rat portal vein being directly dependent on depolarization due to opening of Ca2+-activated Cl- channels. This may be stimulated by Ins(1,4,5)P3-mediated release of intracellular Ca2+, which does not directly result in contraction. Depolarization resulted in release of Ca2+ from intracellular stores (voltage-sensitive Ca2+ release), which does produce contraction. Influx of extracellular Ca2+ through voltage-gated channels was not directly necessary for the phasic contraction but was required to refill the intracellular Ca2+ stores. The spontaneous contractions were also stimulated by depolarization via opening of Ca2+-activated Cl- channels. They may also involve intracellular Ca2+ release but in this case resulting from CICR, stimulated by Ca2+ influx through voltage-gated channels.


    FOOTNOTES

Address for reprint requests and other correspondence: R. Burt, Dept. of Pharmacology, Univ. College London, Gower St., London WC1E 6BT, UK (E-mail: r.burt{at}ucl.ac.uk).

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.

10.1152/ajpheart.00637.2002

Received 24 July 2002; accepted in final form 7 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 284(5):H1808-H1817
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