Effect of voltage-gated and capacitative calcium entry blockade on agonist-induced constriction of equine laminar blood vessels

John F. Peroni Department of Physiology and Pharmacology, Institute of Comparative Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7389
Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7389

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James N. Moore Department of Physiology and Pharmacology, Institute of Comparative Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7389
Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7389

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Erik Noschka Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7389

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Tristan H. Lewis Department of Physiology and Pharmacology, Institute of Comparative Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7389

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Stephen J. Lewis Department of Physiology and Pharmacology, Institute of Comparative Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7389

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Tom P. Robertson Department of Physiology and Pharmacology, Institute of Comparative Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7389
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Abstract

Objective—To characterize the relative contributions of voltage-gated and capacitative Ca2+ entry to agonist-induced contractions of equine laminar arteries and veins.

Animals—16 adult mixed-breed horses.

Procedures—Laminar arteries and veins were isolated and mounted on small vessel myographs for the measurement of isometric tension. Concentration-response curves were obtained for the vasoconstrictor agonists phenylephrine, 5-hydroxytryptamine (5-HT), prostaglandin F (PGF), and endothelin-1 (ET-1) either in the absence of extracellular Ca2+ or in the presence of the voltage-gated Ca2+ channel inhibitor diltiazem or the putative inhibitor of capacitative Ca2+ entry, trifluoromethylphenylimidazole.

Results—In the absence of extracellular Ca2+, maximal responses of veins to 5-HT, phenylephrine, ET-1 and PGF were reduced by 80%, 50%, 50%, and 45%, respectively; responses of arteries to 5-HT, phenylephrine, and ET-1 were reduced by 95%, 90%, and 20%, respectively. Although diltiazem did not affect the maximal responses of veins to any agonist, responses of arteries to 5-HT, phenylephrine, and ET-1 were reduced by 40%, 50%, and 27%, respectively. Trifluoromethylphenylimidazole did not affect maximal responses of veins, but did reduce their contractile responses to low concentrations of ET-1 and PGF.

Conclusions and Clinical Relevance—Results suggested that the contribution of extracellular Ca2+ to laminar vessel contractile responses differs between arteries and veins and also between contractile agonists, voltage-gated Ca2+ entry is more predominant in laminar arteries than in veins, and capacitative Ca2+ entry has a minor role in agonist-induced contractile responses of laminar veins.

Abstract

Objective—To characterize the relative contributions of voltage-gated and capacitative Ca2+ entry to agonist-induced contractions of equine laminar arteries and veins.

Animals—16 adult mixed-breed horses.

Procedures—Laminar arteries and veins were isolated and mounted on small vessel myographs for the measurement of isometric tension. Concentration-response curves were obtained for the vasoconstrictor agonists phenylephrine, 5-hydroxytryptamine (5-HT), prostaglandin F (PGF), and endothelin-1 (ET-1) either in the absence of extracellular Ca2+ or in the presence of the voltage-gated Ca2+ channel inhibitor diltiazem or the putative inhibitor of capacitative Ca2+ entry, trifluoromethylphenylimidazole.

Results—In the absence of extracellular Ca2+, maximal responses of veins to 5-HT, phenylephrine, ET-1 and PGF were reduced by 80%, 50%, 50%, and 45%, respectively; responses of arteries to 5-HT, phenylephrine, and ET-1 were reduced by 95%, 90%, and 20%, respectively. Although diltiazem did not affect the maximal responses of veins to any agonist, responses of arteries to 5-HT, phenylephrine, and ET-1 were reduced by 40%, 50%, and 27%, respectively. Trifluoromethylphenylimidazole did not affect maximal responses of veins, but did reduce their contractile responses to low concentrations of ET-1 and PGF.

Conclusions and Clinical Relevance—Results suggested that the contribution of extracellular Ca2+ to laminar vessel contractile responses differs between arteries and veins and also between contractile agonists, voltage-gated Ca2+ entry is more predominant in laminar arteries than in veins, and capacitative Ca2+ entry has a minor role in agonist-induced contractile responses of laminar veins.

Laminitis is a crippling musculoskeletal condition in horses, the etiology of which remains obscure.1,2 It is apparent that the prodromal stages of equine laminitis are associated with vascular dysfunction at the level of the laminar dermis3–7 and, specifically, an increase in postcapillary resistance.8,9 Thus, studies detailing the regulation of laminar arterial and venous tone would likely prove helpful in the search for therapeutic strategies that effectively target the vascular dysfunction that occurs in acute laminitis. However, no information presently exists pertaining to the signal transduction pathways that regulate vascular tone in laminar micro-vessels. The authors have, therefore, recently developed techniques that allow for the routine isolation and functional examination of small laminar arteries and veins of the laminar dermis of horses.

Vascular tone is primarily determined by the [Ca2+]i within smooth muscle and by the sensitivity of the smooth muscle contractile apparatus to [Ca2+]i. Increases in [Ca2+]i can be the result of either Ca2+ entry (eg, via voltage-gated, capacitative, or receptor-operated Ca2+ entry) or via the release of Ca2+ from intracellular stores. Voltage-gated Ca2+ entry is an important pathway through which agonists elicit increases in [Ca2+]i via depolarization of the smooth muscle plasma membrane and via the subsequent opening of voltage-gated Ca2+ channels. The influx of Ca2+ through these channels causes vascular smooth muscle contraction by increasing actin-myosin interactions, and consequently, voltage-gated Ca2+ entry is a primary determinant of tone in vascular tissues.10 Moreover, emerging lines of evidence indicate that voltage-gated Ca2+ entry is also an important regulator of the genes expressed in response to agonist stimulation.11

In contrast to voltage-gated Ca2+ entry, capacitative Ca2+ entry is a voltage-independent event that is activated upon depletion of intracellular Ca2+ stores.12 This process is also referred to as store-operated Ca2+ entry. Whereas voltage-gated Ca2+ entry is a ubiquitous mechanism for increasing tone in vascular smooth muscle, capacitative Ca2+ entry is present in certain artery types, such as pulmonary arteries, and absent in other arteries, such as renal, femoral, and mesenteric arteries.13 Recently, the authors' laboratory has determined that the induction of capacitative Ca2+ entry results in constriction of isolated laminar veins, but not laminar arteries, of horses.14 This latter observation is consistent with laminar veins possessing discrete pathways leading to vasoconstriction that are not present in laminar arteries.

The aim of the study reported here was to characterize the relative contributions of voltage-gated and capacitative Ca2+ entry to agonist-induced contractions of equine laminar arteries and veins. Agonists included the α-adrenergic receptor agonists phenylephrine, 5-HT, ET-1, and PGF. The possible roles of voltage-gated or capacitative Ca2+ entry was assessed by determining the effects of inhibitors of these Ca2+ entry pathways, namely diltiazem and TRIM, respectively.

Materials and Methods

Animals—This study involved 16 adult horses ranging in age from 6 to 14 years (mean, 11 years). To be included in the study, each horse lacked clinical evidence of lameness, and survey radiographs of the forelimb digits revealed no abnormalities. All protocols were approved by the University of Georgia Institutional Animal Care and Use Committee. The horses were euthanized by use of a penetrating captive bolt, as approved by the Report of the AVMA Panel on Euthanasia.15

Isolation of laminar vessels—Laminar arteries and veins were isolated as previously described in detail.16 Briefly, the distal portion of each forelimb was disarticulated at the level of the metacarpophalangeal joint, and 2 full-thickness segments of the dorsal portion of the hoof were isolated via sectioning with a band saw. The segments were placed in ice-cold PSS containing NaCl, 118mM; NaHCO3, 24mM; MgSO4, 1mM; NaH2PO4, 0.435mM; glucose, 5.56mM; CaCl2, 1.8mM; and KCl, 4mM and were gassed with 21% O2 and 5% CO2 (mean ± SEM pH, 7.40 ± 0.01). On the stage of a high-powered light microscope, the lamellar portion of the dermis was shaved until only a thin layer covered the laminar vascular bed. Laminar arteries and veins (2 to 3 cm distal to the coronary band, 200 to 800 μm in internal diameter, and 1 to 2 mm in length) were isolated with microfine surgical instruments and mounted on small vessel myographs.a The vessels were bathed in PSS, and the bath temperature was raised to and maintained at 37°C while the vessels equilibrated for 1 hour. Laminar arteries and veins were then stretched to produce equivalent transmural pressures of 3.1 and 1.9 kPa, respectively.16 Data were collected for each agonist from 1 to 4 arteries and veins from a minimum of 3 horses. The numbers of vessels and horses used to obtain the data in this study were based on previous experience with isolated laminar arteries and veins.14,16,17

Experimental protocols—All vessels were given three 2-minute exposures to 80mM KCl-PSS (isotonic replacement of NaCl with KCl) 15 minutes apart to establish the maximal contractile response to a depolarizing stimulus. Concentration-response curves for either phenylephrine (1nM to 10μM), 5-HT (1nM to 10μM), or ET-1 (1pM to 1μM) were obtained by cumulative addition of each agonist. As previously reported,17 laminar arteries do not contract upon exposure to PGF; therefore, concentration-response curves to this agonist (1nM to 100μM) were established only in laminar veins. In some experiments, agonist-induced contractile responses were recorded in the absence of extracellular Ca2+ (0 Ca2+ PSS, by omission of Ca2+ from PSS) or in the presence of the voltage-gated Ca2+ blocker, diltiazem (10μM) or the putative inhibitor of capacitative Ca2+ entry, TRIM (300μM).

Data and statistical analysis—Contractile responses were calculated as a percentage of the maximal contractile response to KCl-PSS for each vessel. Data are presented as mean ± SEM. Data were analyzed via logistic regression analyses to determine EC50 values.18 The ratio of maximal contractions to the EC50, a determinant of the total response curve (ie, approx area under the curve),18 was also determined. The EC50 and the ratio of maximal contractions to the EC50 values are an indication of the potency and efficacy of an agonist in eliciting a given response, such as vasoconstriction, and can be used to compare the relative sensitivities of different vessel types. Data were also analyzed via nested repeated-measures ANOVA to allow for identification of differences between individual means, which were determined by use of the modified Student t test with the Bonferroni correction for multiple comparisons between means by use of the error mean square term from the ANOVA (taking into account that some vessels were from the same horse).19 A value of P < 0.05 was considered significant.

Results

Contractile responses of laminar arteries and veins to phenylephrine, 5-HT, and ET-1 in the presence and absence of extracellular Ca2+—In the presence of extracellular Ca2+, the contractile responses of laminar arteries and veins to phenylephrine, 5-HT, or ET-1 were similar to those previously reported,17 with laminar veins being more sensitive and contracting to a greater relative degree than laminar arteries (Tables 1 and 2). In the absence of extracellular Ca2+, responses to phenylephrine and 5-HT were virtually abolished in laminar arteries (Figure 1). Although responses to phenylephrine and 5-HT were also significantly reduced in laminar veins, this was to a lesser extent than those observed in laminar arteries. In contrast, responses of laminar veins and arteries to ET-1 in the absence of extracellular Ca2+ were still robust.

Table 1—

Variables (mean ± SEM) of contractility in laminar veins of horses in response to various agonists.

AgentTreatmentNo.Max (%TK)EC50 (nM)Max:EC50
5-HTControl16168 ± 1252 ± 53.5 ± 0.4
0 Ca2+638 ± 6*35 ± 4*1.2 ± 0.2*
Diltiazem7159 ± 1647 ± 63.2 ± 0.4
TRIM6148 ± 2444 ± 53.6 ± 0.4
PEControl16213 ± 2194 ± 82.6 ± 0.3
0 Ca2+6112 ± 13*146 ± 14*0.8 ± 0.1*
Diltiazem8247 ± 2189 ± 83.0 ± 0.4
TRIM8235 ± 3690 ± 112.8 ± 0.4
ET-1Control12244 ± 180.29 ± 0.03874 ± 174
0 Ca2+9160 ± 15*6.7 ± 0.5*23 ± 4*
Diltiazem8267 ± 152.8 ± 0.4*,90 ± 12*
TRIM7236 ± 134.3 ± 0.5*52 ± 7*
PGF2xControl12174 ± 14493 ± 490.38 ± 0.05
0 Ca2+6100 ± 9*1025 ± 81*0.09 ± 0.01*
Diltiazem7173 ± 121532 ± 169*,0.15 ± 0.02*,
TRIM6185 ± 71079 ± 91*0.19 ± 0.02*

No. = Number of veins. Max (%TK) = Maximal response expressed as percentage of the maximal contractile response to isotonic replacement of NaCl with KCl. Max:EC50 = The ratio of maximal contractions to the EC50. PE = Phenylephrine.

Significant (P < 0.05) difference versus control value.

Significant (P < 0.05) difference versus 0 Ca2+.

Table 2—

Variables (mean ± SEM) of contractility in laminar arteries of horses in response to various agonists.

AgentTreatmentNo.Max (%TK)EC50 (nM)Max:EC50
5-HTControl24154 ± 7268 ± 350.68 ± 0.11
0 Ca2+811 ± 2*334 ± 400.04 ± 0.01*
Diltiazem1490 ± 9*,760 ± 87*,0.15 ± 0.03*,
PEControl39127 ± 9699 ± 460.21 ± 0.03
0 Ca2+811 ± 2*726 ± 850.02 ± 0.01*
Diltiazem1062 ± 12*,398 ± 48*,0.14 ± 0.03*,
ET-1Control18124 ± 1565 ± 61.93 ± 0.14
0 Ca2+8101 ± 8154 ± 17*0.61 ± 0.09*
Diltiazem895 ± 1296 ± 8*,0.98 ± 0.13*,

No. = Number of arteries.

See Table 1 for remainder of key.

Figure 1—
Figure 1—

Mean responses (%TK [percentage of the maximal contractile response to KCl-PSS]) of equine laminar veins and arteries to 5-HT (1nM to 10μM), phenylephrine (PE; 1nM to 10μM), or ET-1 (1pM to 1μM) in the presence (control) or absence (0 calcium) of extracellular calcium. *Significant (P < 0.05) difference between groups.

Citation: American Journal of Veterinary Research 68, 7; 10.2460/ajvr.68.7.722

Effects of diltiazem on contractile responses of laminar arteries and veins to phenylephrine, 5-HT, and ET-1—Preincubation of laminar arteries with diltiazem (10μM) significantly reduced contractile responses to phenylephrine, 5-HT, and ET-1 (Table 2; Figure 2). In contrast, diltiazem had no effect on the responses of laminar veins to either phenylephrine or 5-HT (Table 1). Contractile responses of laminar veins to concentrations of ET-1 of ≤ 1nM were significantly attenuated by diltiazem, whereas responses to higher concentrations of ET-1 were not affected.

Figure 2—
Figure 2—

Mean responses (%TK) of equine laminar veins and arteries to 5-HT (1nM to 10μM), PE (1nM to 10μM), or ET-1 (1pM to 1μM) in the absence (control) or presence of the voltage-gated Ca2+ channel blocker diltiazem (10μM). See Figure 1 for key.

Citation: American Journal of Veterinary Research 68, 7; 10.2460/ajvr.68.7.722

Effects of TRIM on the contractile responses of laminar veins to phenylephrine, 5-HT, and ET-1—Although TRIM had no effect on either phenylephrine- or 5-HT–induced contractile responses (Table 1; Figure 3), it significantly reduced responses to all, except the highest, concentrations of ET-1.

Figure 3—
Figure 3—

Mean responses (%TK) of equine laminar veins to 5-HT (1nM to 10μM), PE (1nM to 10μM), or ET-1 (1pM to 1μM) in the absence (control) or presence of TRIM (300μM). See Figure 1 for key.

Citation: American Journal of Veterinary Research 68, 7; 10.2460/ajvr.68.7.722

Effects of diltiazem, TRIM, and removal of extracellular Ca2+ on contractile responses of laminar veins to PGF—In the absence of extracellular Ca2+, responses of laminar veins to PGF were significantly reduced, yet substantial contractile responses were still observed (Table 1; Figure 4). Preincubation with either diltiazem or TRIM reduced contractile responses to low concentrations (≤ 1μM) of PGF, but was without effect at higher concentrations.

Figure 4—
Figure 4—

Effects of removal of extracellular Ca2+ (panel A), diltiazem (10μM; panel B), orTRIM (300μM; panel C) on the mean responses of laminar veins to PGF (1nM to 100μM). See Figure 1 for key.

Citation: American Journal of Veterinary Research 68, 7; 10.2460/ajvr.68.7.722

Discussion

Vascular smooth muscle tone is governed by the concentration of cytosolic Ca2+ and the sensitivity of the contractile apparatus to Ca2+. The aim of the present study was to provide information regarding the role of Ca2+ entry in agonist-induced contractile responses of laminar vessels and, in particular, voltage-gated and capacitative Ca2+ entry. Because no previous data existed regarding contractile mechanisms in laminar vessels, the experiments performed in this study represent the first steps taken towards elucidating the regulation of tone in these functionally important vessels. In this initial study, we did not examine the possible roles of either receptor-operated Ca2+ entry or the possible contribution of the release of Ca2+ from intracellular stores to agonist-induced contraction in laminar vessels, although these important pathways certainly warrant examination in future studies. The agonists used in the present study were selected for several reasons. Phenylephrine is an α-adrenergic receptor agonist, and stimulation of these receptors elicits contraction in isolated digital arteries of horses.20–22 Moreover, α-adrenergic receptor antagonists induce vasodilation of the digital circulation in healthy horses, a finding that is consistent with basal activation of these receptors in this vascular bed.1 Serum concentration of 5-HT increases during experimentally induced laminitis, and 5-HT constricts isolated digital arteries but not other peripheral arteries of horses.23 Prostaglandin F is a representative vasoconstrictor produced by the enzyme cyclooxygenase 2, for which expression is upregulated in the laminae of horses during the early stages of laminitis.24 Endothelin-1 is a potent vasoconstrictor, and concentrations of ET-1 are increased in laminar tissues during acute laminitis.25 Moreover, administration of an ET-1 receptor antagonist reduces vascular resistance in the isolated perfused digit of anesthetized horses after carbohydrate overload.26

The obvious way to obtain an indication of the contribution of Ca2+ influx to agonist-induced vascular contractility is to remove Ca2+ from the solution bathing the vessels. It should be noted, however, that the 0 Ca2+ PSS concentration used was only nominally Ca2+ free because no Ca2+ chelating agents, such as ethylene glycol-bis(B-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, were included. However, in preliminary experiments, it was determined that constrictor responses of the vessels to KCl-PSS were abolished in nominally Ca2+-free PSS (ie, where Ca2+ was simply omitted from the PSS), which indicated that any residual Ca2+ in this solution was not sufficient to sustain extracellular Ca2+-dependent contractile responses. In the absence of extracellular Ca2+, the responses of laminar arteries to phenylephrine and 5-HT were virtually abolished, which is consistent with the generally held belief that voltage-gated Ca2+ entry is a primary pathway through which agonists increase [Ca2+]i and elicit vascular contraction.27 Although the responses of laminar veins to phenylephrine and 5-HT were significantly reduced in the absence of extracellular Ca2+, these vessels retained some of their ability to constrict. This finding is consistent with constriction of laminar veins in response to phenylephrine and 5-HT being less dependent on Ca2+ influx than laminar arteries.

In direct contrast, responses of laminar arteries and veins to ET-1 and of laminar veins to PGF remained robust in the absence of extracellular Ca2+. Collectively, these results are consistent with Ca2+ influx being important in agonist-induced constriction of laminar arteries and veins. However, it should be noted that the effects of removing extracellular Ca2+ may not be limited to ablating Ca2+ influx because this manipulation may also reduce basal concentrations of cytosolic Ca2+ in the vascular smooth muscle cells, as has been noted in other vascular preparations.13 It is possible that the contractions observed in the absence of extracellular Ca2+ were attributable to the release of Ca2+ from intracellular stores. However, because contractions obtained in response to intracellular Ca2+ release are most often transient in nature, in the absence of extracellular Ca2+, it is unlikely that the sustained agonist-induced responses observed in laminar vessels in 0 Ca2+ PSS were attributable to Ca2+ store release.

One of the main pathways for Ca2+ entry in vascular smooth muscle are via voltage-gated Ca2+ entry, which is activated by depolarization of the vascular smooth muscle and the resultant opening, and Ca2+ influx through voltage-gated Ca2+ channels. It has been determined that the voltage-gated Ca2+ channel blocker diltiazem, at the concentration used in this study (10μM), ablates the contractile responses of laminar arteries and veins to a depolarizing stimulus.14 In the present study, diltiazem substantially reduced the responses of laminar arteries to phenylephrine or 5-HT. However, diltiazem did not alter the contractile responses of laminar veins to either of these agonists. These findings are consistent with voltage-gated Ca2+ entry playing a prominent role in the contractile responses of laminar arteries, but not laminar veins, to phenylephrine or 5-HT. This apparent dependence of laminar artery vasoconstriction on voltage-gated Ca2+ entry was also observed when ET-1 was used as the contractile agonist, although the magnitude of the effect of diltiazem was less than that observed for phenylephrine or 5-HT in these vessels. In laminar veins, diltiazem reduced contractile responses to low concentrations of ET-1 or PGF, whereas no effect of diltiazem was observed at higher concentrations of these agonists. It should be noted that the concentration of diltiazem used in these studies does not inhibit capacitative Ca2+ entry in laminar vessels.14

In recent years, it has become apparent that capacitative Ca2+ entry is an important pathway for the influx of Ca2+ in a diverse array of cell-signalling events.12 It has been reported that induction of capacitative Ca2+ entry elicits contractile responses in laminar veins, but not in laminar arteries.14 Because one of the early vascular events in equine laminitis appears to be selective venoconstriction in the digit, capacitative Ca2+ entry would appear to be a viable candidate as a pathway that, upon activation, could initiate and maintain laminar venoconstriction. However, one problem that arises when attempting to elucidate the role of capacitative Ca2+ entry in agonist-induced responses is the present lack of specific inhibitors. Recently, TRIM was reported as a selective inhibitor of this pathway in smooth muscle,28 and TRIM reduces capacitative Ca2+ entry by approximately 50% in laminar veins while not affecting voltage-gated Ca2+ entry in these vessels.14 In the present study, TRIM had no effect on either phenylephrine- or 5-HT–induced contractions and responses to PGF were affected at only low concentrations of PGF. Trifluoromethylphenylimidazole had a greater effect on responses to ET-1, but this effect was overcome by the highest concentration of ET-1 used. These results are consistent with a heterogeneous contribution of capacitative Ca2+ entry to laminar vein contractile responses, depending on the identity and concentration of the agonist used. However, it should be noted that TRIM does not completely inhibit capacitative Ca2+ entry in laminar veins,14 and as such, the possible contribution of this pathway to laminar vein constriction may have been underestimated in the present study. Further delineation of the importance of this pathway in agonist-induced constriction of laminar veins would be greatly aided by the development of more specific and efficacious inhibitors of capacitative Ca2+ entry.

It is interesting to note that responses of laminar veins to low concentrations of ET-1 and PGF were inhibited markedly by diltiazem or TRIM or in the absence of extracellular Ca2+, whereas responses to the highest concentrations of these agonists were inhibited to a much lesser extent. This finding is consistent with Ca2+-dependent mechanisms being more prevalent at lower concentrations of these agonists and Ca2+-independent mechanisms being recruited at higher concentrations. It is, however, questionable as to whether the highest concentrations of these agonists used in the present study would ever be achieved in vivo, rendering these concentrations supraphysiologic.

The data from this initial study are consistent with a generalized contribution of voltage-gated Ca2+ entry in agonist-induced contractile responses of laminar arteries. In contrast, voltage-gated events appear to be comparatively less important to the responses of laminar veins. The present study also provides support for a role of capacitative Ca2+ entry in laminar veins, but because of the lack of availability of specific and effective inhibitors, these data remain equivocal. Interestingly, the fact that substantial constrictor responses could be observed for all 4 agonists used in laminar veins in the absence of extracellular Ca2+ is supportive of the possibility that Ca2+-independent mechanisms (ie, Ca2+ sensitization) may be more prominent in laminar veins than in laminar arteries. Because the mechanisms underlying vasoconstriction appear to differ between laminar arteries and veins, it is possible that therapeutic agents could be developed that may selectively alleviate the venoconstriction that occurs in laminitis, yet do not adversely affect the arterial side of the digital circulation.

ABBREVIATIONS

[Ca2+]i

Concentration of intracellular Ca2+

5-HT

5-hydroxytryptamine

ET-1

Endothelin-1

PGF

Prostaglandin F

TRIM

Trifluoromethylphenylimidazole

PSS

Physiologic saline solution

EC50

Effective concentration that induces 50% of the maximum response

a.

Model 500A, Danish Myo Technology, Aarhus, Denmark.

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