Materials and Methods

Sample population—Laminar arteries and veins obtained from 8 adult horses that ranged from 5 to 14 years of age (mean, 9 years) were included in the study. Inclusion criteria included that each horse did not have clinical evidence of lameness and that results of examination of survey radiographs of the forelimb digits were within acceptable limits. All protocols were approved by the University of Georgia Institutional Animal Care and Use Committee.

Isolation of laminar vessels—Laminar arteries and veins were isolated as described elsewhere.^9-11 Briefly, the horses were euthanized by use of a penetrating captive bolt.¹⁴ The distal portions of each forelimb were disarticulated at the metacarpophalangeal joint, and 2 full-thickness segments of the dorsal hoof were isolated via sectioning with a band saw. The segments were placed in ice-cold PSS containing 118mM NaCl, 24mM NaHCO₃, 1mM MgSO₄, 0.435mM NaH₂PO₄, 5.56mM glucose, 1.8mM CaCl₂, and 4mM KCl. Segments were exposed to gases (21% oxygen and 5% carbon dioxide [mean ± SEM pH, 7.40 ± 0.01]). On the stage of a high-powered 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) were isolated by use of microfine surgical instruments and mounted on small vessel myographs.^a Vessels were bathed in PSS, and the bath temperature was increased to and then maintained at 37°C while the vessels equilibrated for 1 hour. Laminar arteries and veins were then stretched to generate equivalent transmural pressures of 3.1 and 1.9 kPA, respectively, which reportedly¹⁰ is optimal for these vessels. Mean ± SEM internal diameters for arteries and veins were 339 ± 11 μm (range, 201 to 503 μm) and 377 ± 21 μm (range, 205 to 712 μm), respectively, and each vessel had a length of 1 to 2 mm. Data were collected from 1 or 2 arteries and veins from a minimum of 3 horses for each agonist. The numbers of vessels and horses used to obtain data for the study reported here were determined on the basis of other studies^9-11 conducted by our laboratory group on isolated laminar arteries and veins.

Experimental protocols—All vessels were exposed 3 times (2 min/exposure) to 80mM KCl-PSS (ie, isotonic replacement of NaCl with KCl). Exposures were at intervals of 15 minutes to establish the maximal contractile response to a depolarizing stimulus. Concentration response curves were then obtained to various concentrations of PE (1nM to 10μM), 5-HT (1nM to 10μM), PGF_2α (1nM to 100μM), or ET-1 (1pM to 1μM) by cumulative addition of each agonist. Before generation of response curves by addition of the agonists in some experiments, vessels were incubated with the PKC inhibitor, Ro-31-8220, at a concentration (3μM) that inhibits PKC-dependent contractile responses in isolated resistance arteries.¹⁵

Data and statistical analysis—Contractile responses were calculated as a percentage of the maximal contractile response to KCl-PSS for each vessel. Data were reported as mean ± SEM. Data were subjected to logistic regression analyses to determine threshold concentrations for contraction and EC₅₀ values.¹⁶ The Max:EC₅₀, a determinant of the total response curve (ie, approximation of area under the curve), was also determined.¹⁶ Data were also subjected to a repeated-measures ANOVA to allow for the determination of differences between means, which were determined with a Student modified t test by use of the Bonferroni correction for multiple comparisons between means and the error mean square term from the ANOVA.¹⁷ Values of P < 0.05 were considered significant.

Results

Agonist-induced vasoconstrictor responses in equine laminar arteries and veins—The agonists 5-HT, PE, and ET-1 each elicited concentration-dependent contractions in laminar arteries and veins. However, these agonists were considerably more potent constrictors in laminar veins than in laminar arteries (Figure 1; Table 1). Mean ± SEM threshold concentrations for 5-HT (ie, calculated lowest concentration that elicited significant contraction as derived from logistic regression analyses) differed significantly between laminar veins and arteries (2.8 ± 0.4nM and 52.4 ± 8.2nM, respectively). The maximal contractions elicited by 5-HT were similar in veins and arteries, although the EC₅₀ values were lower and Max:EC₅₀ values were higher in laminar veins than in laminar arteries. Mean threshold concentrations for PE in laminar veins (6.8 ± 0.8nM) and arteries (72.6 ± 9.4nM) differed significantly. The EC₅₀ values for PE were lower, whereas maximal contractions and Max:EC₅₀ values were higher, in laminar veins than in laminar arteries. Mean threshold concentrations for ET-1 differed significantly between laminar veins (0.04 ± 0.02nM) and arteries (8 ± 2nM). The EC₅₀ values for ET-1 were lower, whereas the maximal contractions and Max:EC₅₀ values were higher, in laminar veins than in laminar arteries. Laminar veins responded robustly to PGF_2α, with a mean threshold concentration of 64 ± 10nM (Figure 2). In contrast, laminar arteries were unresponsive to PGF_2α (data not shown).

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Mean ± SEM responses of laminar veins (A, C, and E) and arteries (B, D, and F) to various concentrations of PE (1nM to 10μM; A and B), 5-HT (1nM to 10μM; C and D), or ET-1 (1pM to 1μM; E and F) after incubation with Ro-31-8220 (3μM [circles]) or without Ro-31-8220 (control treatment [squares]). Contractile responses were significantly (P < 0.05) reduced in laminar veins after incubation with Ro-31-8220, whereas responses in laminar arteries were unaffected by incubation with Ro-31-8220. Notice that values on the x- and y-axes differ among portions of the figure. %T_K = Maximal response expressed as a percentage of the maximal contractile response to KCl-PSS.

Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.664

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Effects of Ro-31-8220 on agonist-induced contractile responses of equine laminar veins—Prior incubation of laminar veins with the PKC inhibitor, Ro-31-8220, significantly reduced contractile responses to 5-HT, PE, and ET-1 (Figure 1; Table 1). Although incubation with Ro-31-8220 did not significantly affect the mean ± SEM threshold concentration for 5-HT (5-HT without Ro-31-8220 incubation, 2.8 ± 0.4nM; 5-HT with Ro-31-8220 incubation, 3.1 ± 0.5nM), the PKC inhibitor significantly increased EC₅₀ values and decreased the maximal response and Max:EC₅₀ value. Incubation with Ro-31-8220 significantly increased the mean threshold concentration for PE (PE without Ro-31-8220 incubation, 6.8 ± 0.8nM; PE with Ro-31-8220 incubation, 49.7 ± 7.3nM), decreased the maximal response to PE, increased the EC₅₀ value, and decreased the Max:EC₅₀ value. Although incubation with Ro-31-8220 did not significantly affect the mean threshold concentration for ET-1 (ET-1 without Ro-31-8220 incubation, 0.04 ± 0.02nM; ET-1 with Ro-31-8220 incubation, 0.05 ± 0.02nM), it significantly increased the EC₅₀ value, decreased the maximal response, and decreased the Max:EC₅₀ value.

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Mean ± SEM responses of laminar veins to various concentrations of PGF_2α (1nM to 100μM) after incubation with or without Ro-31-8220. Notice that contractile responses of laminar veins were significantly (P < 0.05) reduced after incubation with Ro-31-8220. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 68, 6; 10.2460/ajvr.68.6.664

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Similar to its effects on the other vasoconstrictor agonists, incubation with Ro-31-8220 significantly reduced the contractile responses of laminar veins to PGF_2α. Although incubation with Ro-31-8220 did not significantly affect the mean ± SEM threshold concentration for PGF_2α (PGF_2α without Ro-31-8220 incubation, 64 ± 10nM; PGF_2α with Ro-31-8220 incubation, 73 ± 12nM), it significantly increased the EC₅₀ value, decreased the maximal response, and decreased the Max:EC₅₀ value (Figure 2; Table 1).

Effects of Ro-31-8220 on agonist-induced contractile responses of equine laminar arteries—In contrast to its effects on laminar veins, incubation with Ro-31-8220 did not affect the contractile properties of 5-HT, PE, and ET-1 in laminar arteries (Figure 1; Table 1). Similarly, it did not affect the threshold concentrations of any of the constrictor agents (data not shown). Analysis revealed that incubation with Ro-31-8220 did not affect the maximal response, EC₅₀ value, or Max:EC₅₀ value for 5-HT, PE, or ET-1 in equine laminar arteries.

Discussion

Analysis of results of other studies^{3–5,7,10,18–20} on the pathogenesis of acute laminitis in horses indicates that there is an increase in postcapillary resistance within the digits, which most likely is the result of selective venoconstriction. Evidence for venoconstriction in the digits of horses was first provided in a study⁴ in which investigators used an extracorporeal digital preparation obtained from control horses and horses with experimental laminitis induced by carbohydrate overload. Those investigators determined that there was an increase in postcapillary resistance during the early stages of experimentally induced laminitis, a finding that was subsequently confirmed in another study⁵ in which black walnut heartwood extract was used to induce laminitis. These findings revealed that, at least during the developmental stages of laminitis, there is an increase in postcapillary resistance that is likely attributable to venoconstriction within the affected digit.^4,5 An increase in postcapillary resistance would lead to enhanced capillary filtration, accumulation of interstitial fluid, and an increase in tissue pressure that could eventually lead to collapse of the capillary bed and ischemia.^4,5 These findings are consistent with the hypothesis that venoconstriction within affected digits during the development of laminitis could be an important contributing factor, in addition to local inflammation and ischemia, in the pathogenesis of laminitis. However, it is also possible that venoconstriction may not contribute to the development of laminitis and is merely a coincidental finding or a compensatory mechanism. To determine whether venoconstriction is indeed an important contributory factor in the pathogenesis of this condition, it is necessary to determine the mechanisms that are responsible for venoconstriction during laminitis.

To identify the mechanisms responsible for alterations in vascular function during the onset of laminitis, it is first necessary to elucidate pathways responsible for the physiologic responses of laminar arteries and veins. By use of this approach, our laboratory group has determined that laminar veins are far more sensitive to physiologically relevant vasoconstrictor agonists, compared with the response of laminar arteries. This finding is consistent with the fact that the digits of horses may be predisposed to venoconstriction as well as that this predisposition to venoconstriction could explain the reason various dissimilar systemic conditions may lead to the same end result (ie, acute laminitis) and, in turn, predispose horses to laminitis.¹¹ Because this increased sensitivity of laminar veins to vasoconstrictor agonists may be a result of specific vasoconstrictor effector pathways in laminar veins that may not be found in laminar arteries, the study reported here was performed to provide insights into the role of PKC activation in these vessels and whether PKC contributes differentially to agonist-induced constriction in laminar veins and arteries.

Protein kinase C was among the first protein kinases to be identified.¹² It has been found to be a key player in a diverse array of cellular responses, including muscle contraction and cell permeability, proliferation, and secretion of cells.¹³ Smooth muscle contractility is determined by the phosphorylation state of myosin light chain, which is in turn regulated by myosin lightchain kinase and myosin light-chain phosphatase. Increases in intracellular calcium concentrations, either via influx or through release from intracellular stores, result in activation of myosin light-chain kinase, phosphorylation of myosin light chain, and contraction. However, increased myosin light-chain phosphorylation can also result from a decrease in activity of myosin light-chain phosphatase. Activation of specific kinases, such as PKC, decreases activity of myosin lightchain phosphatase, an effect that is purportedly mediated by a C kinase–potentiated phosphatase inhibitor, which phosphorylates and inhibits myosin light-chain phosphatase.

Protein kinase exists in at least 12 isoforms, each of which has a different function, depending on the cell type. Because pharmacologic efforts to differentiate PKC isoforms are controversial,²¹ in the study reported here, we used the broad-spectrum PKC inhibitor, Ro-31-8220, which inhibits PKC-associated constriction of isolated blood vessels, independent of calcium-mediated constriction.¹⁵ We determined that the contractile responses of laminar veins to PE, 5-HT, ET-1, and PGF_2α were significantly diminished after prior incubation with Ro-31-8220. In contrast, contractile responses of laminar arteries to PE, 5-HT, and ET-1 were unaffected. In addition, the laminar arteries were unresponsive to PGF_2α, which thereby eliminated the need to test the effects of Ro-31-8220. Collectively, analysis of these findings suggests that PKC plays an important role in agonist-induced constriction of laminar veins but not in laminar arteries. Because of similar results for other pharmacologic tools and nonspecific effects of enzyme inhibitors, such as Ro-31-8220, it is unwise to attribute their actions solely to specific targets.

Although it would have been preferable to provide direct evidence for PKC activation in laminar veins, the smooth muscle content of these vessels is extremely small.¹⁰ Thus, it is highly unlikely that sufficient amounts of protein could have been isolated from these small veins to permit performance of isolated kinase assays. Moreover, there appears to be as much endothelial cell mass as smooth cell mass in these vessels,¹⁰ which would make it impossible to confidently ascribe the results of such assays to the kinase resident in the smooth muscle and not to that in the endothelium. Although it would be preferable to delineate the specific role or roles of each PKC isoform in the vasoconstrictor responses of laminar veins, the lack of isoform-specific pharmacologic inhibitors renders such studies problematic. The development of peptide inhibitors of PKC, which are based on the pseudosubstrate regions of PKC groups, individual isoforms, or both, may offer novel insights into the specific functions of PKC isoforms. Unfortunately, the efficacy of these peptide inhibitors has yet to be established in equine tissues.

One potentially confounding factor that was not addressed in the study reported here was the possible modulation of PKC activity by locally produced factors, such as endothelium-derived nitric oxide and other vasoactive factors. Indeed, although the information reported here provided an initial insight into the activation of PKC by vasoconstrictor agonists, additional studies will be needed to determine how PKC per se and individual PKC isoforms are affected by locally produced vasoactive modulators and whether these pathways are altered during the development of laminitis in horses.

Results of this initial investigation were consistent with PKC activation being important in, and exclusive to, agonist-induced venoconstriction within the equine laminar dermis. The fact that PKC activation appeared to be evident in laminar veins, regardless of the agonist, may indicate that PKC activation is a universal facet of the constrictor responses of laminar veins. Consequently, the possible involvement of PKC in venoconstriction detected during the development of laminitis is worthy of further investigation.