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Background Hyperinsulinemia is associated with equine laminitis, and digital lamellar inflammation in equine metabolic syndrome‐associated laminitis (EMSAL) is modest when compared with sepsis‐associated laminitis. Objectives To characterize digital lamellar inflammation in horses in a euglycemic‐hyperinsulinemic clamp (EHC) model of laminitis. Animals Sixteen healthy adult Standardbred horses. Methods Prospective experimental study. Horses underwent EHC or saline infusion (CON) for 48 hours or until the onset of Obel grade 1 laminitis. Horses were euthanized, and digital lamellar tissue was collected and analyzed via polymerase chain reaction (pro‐inflammatory cytokine and chemokine genes—CXCL1, CXCL6, CXCL8, IL‐6, MCP‐1, MCP‐2, IL‐1β, IL11, cyclooxygenases 1 and 2, tumor necrosis factor alpha [TNF‐α], E‐selectin, and ICAM‐1), immunoblotting (phosphorylated and total signal transducer and activator of transcription 1 [STAT1], STAT3, and p38MAPK), and immunohistochemistry (markers of leukocyte infiltration: CD163, MAC387). Results Lamellar mRNA concentrations of IL‐1β, IL‐6, IL‐11, COX‐2, and E‐selectin were increased; the concentration of COX‐1 was decreased; and concentrations of CXCL1, CXCL6, MCP‐1, MCP‐2, IL‐8, TNF‐α and ICAM‐1 were not significantly different in the EHC group compared to the CON group (P ≤ .003). Lamellar concentrations of phosphorylated STAT proteins (P‐STAT1 [S727], P‐STAT1 [Y701], P‐STAT3 [S727], and P‐STAT3 [Y705]) were increased in the EHC group compared to the CON group, with phosphorylated STAT3 localizing to nuclei of lamellar basal epithelial cells. There was no change in the lamellar concentration of P‐p38 MAPK (T180/Y182), but the concentration of total p38 MAPK was decreased in the EHC samples. There was no evidence of notable lamellar leukocyte emigration. Conclusions and Clinical Importance These results establish a role for lamellar inflammatory signaling under conditions associated with EMSAL.

Summary An 8-year-old Arabian stallion weighing 361 kg presented to Louisiana State University Veterinary Teaching Hospital with a 3-month history of weight loss, exercise intolerance, long hair coat and recent history of seizures and aimless wandering in the pasture. An initial presumptive diagnosis of pituitary pars intermedia dysfunction (PPID) was made based on clinical signs. The initial examination revealed weight loss and loss of body condition (BCS 3/9), hypertrichosis, muscle wasting and reluctance to move when prompted. A neurological examination revealed dull mentation with no evidence of proprioceptive deficits in the limbs. Mild hyperglycaemia and a stress leucogram were noted on initial biochemical panel and haematology, respectively. Plasma adrenocorticotrophic hormone (ACTH) concentrations before and after thyrotropin releasing hormone (TRH) stimulation were markedly increased. Rapid slice computed tomography (CT) scan of the head before and after contrast revealed a large mass in the region of the pituitary gland suggestive of macroadenoma causing PPID. Prior to imaging, treatment consisted of supportive nursing care. Due to size of the pituitary gland (measuring 4.6 × 4.6 × 3.8 cm) and the presence of seizure-like activity and dull mentation, the stallion was subjected to euthanasia. A necropsy was not performed. Pituitary macroadenomas in horses affected with PPID, who show neurological signs such as seizure-like activity, dull mentation and aimless wandering, might have a poor prognosis and treatment with pergolide mesylate might not reduce pituitary gland size or relieve clinical signs. A CT scan is indicated in horses with neurological signs suspected of PPID to further evaluate pituitary gland size and surrounding structures and rule out other causes to better assess prognosis.

Hyperinsulinemia leads to insulin resistance of blood vessels and interferes with circulation of laminae of the equine foot. The objective of the study was to compare the digital vessel responses of clinically healthy and laminitic horses with and without experimental induction of insulin resistance. Vessel segments (artery and vein) collected after euthanasia were cut into 3-mm wide rings and were prepared in tissue baths containing Tyrode solution for response studies. Two rings of each vessel type from each horse were used as nonresistant, and two were made insulin resistant. Insulin resistance was induced by incubating the rings with 10-μM bath concentration of insulin. Then, all rings were contracted with phenylephrine (5 μM). When the contraction reached a plateau, insulin (10 μM) was added, and the response monitored for 30 minutes. The response to insulin was calculated as a percentage of the phenylephrine response. The results showed that the responses of the nonresistant rings differed significantly between the groups. However, when all rings were made insulin resistant, the significance between the groups disappeared. In the laminitic group, responses of resistant and nonresistant rings did not differ, whereas in the healthy group, the vessel responses differed significantly. In all experiments, arterial and venous rings followed the same pattern, but the magnitudes were greater in arterial rings. The findings suggest that the vessel responses to insulin are altered in laminitis which could be due to insulin resistance.

Summary Reasons for performing study: One of the causes of equine laminitis is hyperinsulinaemia, which may be associated with endothelial dysfunction and insulin resistance of vessels. Hypothesis and objectives: Insulin resistance can be induced in palmar digital vessels by continued exposure to insulin in vitro. The objective was to evaluate this in vitro model for future studies. Methods: Palmar digital vessel segments were collected immediately after euthanasia from horses with normal insulin/glucose blood values. Four arterial and 4 venous rings (3 mm wide) were prepared and each ring mounted in a tissue bath, containing Tyrode's solution at 37°C, 2 g tension was applied and the rings allowed to equilibrate for 45 min. Of the 4 rings of each vessel type, one was used as a control. One each of the remaining 3 rings was used for incubation with insulin (to induce resistance), wortmannin (to block PI3-kinase) and PD-098059 (to block MAP-kinase), respectively, for 30 min. After the incubation period, the rings were contracted with phenylephrine. When the response reached a plateau, a single dose of insulin was added to the baths and the response of each ring monitored for 30 min. Results: Insulin relaxed the control rings and those treated with PD 098059 but contracted those pretreated with insulin and wortmannin. Normal relaxation responses of the rings were converted to contractions by insulin resistance. Insulin resistance was confirmed by the qualitative response of insulin-incubated and wortmannin-incubated rings. Conclusions: This study demonstrated successful induction of insulin resistance in both arterial and venous rings. It also suggested that the MAP-kinase pathway plays a minor role in controlling vasomotor tone under normal physiological conditions. Potential relevance: The study suggests that the induction of insulin resistance in equine palmar digital vessel rings is reliable and provides a good in vitro model for studying the vascular insulin resistance which may occur in equine laminitis.

To date, there is a substantial amount of data to support the hypotheses that vascular and enzymatic changes are ongoing in experimental laminitis. Furthermore, there is substantial in vitro evidence that the enzymatic changes weaken the dermo-epidermal attachments leading to mechanical failure of the hoof-bone interface of the equine digit. However, investigators of both the vascular and enzymatic theories have, to date, been unable to substantiate the effects of these pathophysiological changes in vivo on laminar tissues of horses afflicted with experimentally induced or naturally acquired laminitis. In addition, the effects of laminitis-inducing treatment have not been prevented or reversed by treatment with an MMP inhibitor or a vasoactive antagonist. It is possible that there is simultaneous activation of the vascular and enzymatic pathways and/or other inflammatory processes. Moreover, the third theory involving mechanical factors cannot be discounted simply because strong evidence for vascular and enzymatic changes exists. It is common for horses with severe musculoskeletal disease affecting weightbearing on a limb to develop laminitis in the contralateral limb. It remains to be determined what factors are responsible for initiation of laminitis in these individuals. Evidence has not been presented that precludes the possibility of coincident occurrence of vascular and enzymatic changes. In fact, many of the inflammatory mediators (e.g. interleukin-1beta) found in laminitic tissues can concurrently stimulate synthesis of vasoactive substances and activate MMPs. Because enzymatic action on proteins is largely dependent on the concentrations of proteins and enzyme, the enzymatic theory is not dependent upon increased delivery of enzymes via increased capillary flow. Likewise, because vascular changes can alter tissue function via increased capillary flow and oedema formation, the vascular theory is not dependent upon decreased capillary flow. It is true that naturally acquired laminitis is widely variable in severity and predisposing diseases. Therefore, most probably there are multiple mechanisms involved in the initiation and propagation of the pathophysiologic cascade(s) and, therefore, successful intervention will necessitate multiple treatment modalities.

Objective—To quantify changes in endothelium-derived factors and relate those changes to various aspects of digital hemodynamics during the prodromal stages of carbohydrate overload (CHO)-induced laminitis in horses. Animals—20 adult horses without abnormalities of the digit. Procedures—Digital and jugular venous blood samples were collected at 1-hour intervals (for assessment of endothelin-1 [ET-1] immunoreactivity and measurement of glucose, insulin, and nitric oxide [NO] concentrations) or 4-hour intervals (CBC and platelet-neutrophil aggregate assessment) for 8 hours or 16 hours after induction of CHO-associated laminitis in horses treated with an ET-1 antagonist. Effects of treatment, collection site, and time and the random effects of horse on each variable were analyzed by use of a repeated-measures model. Where treatment and collection site had no significant effect, data were combined. Results—Compared with baseline values, CHO resulted in changes in several variables, including a significant increase from baseline in digital blood ET-like immunoreactivity at 11 hours; digital blood ET-like immunoreactivity was significantly greater than that in jugular venous blood at 8, 9, 11, and 12 hours. Digital and jugular venous blood concentrations of glucose increased from baseline significantly at 3, 4, and 5 hours; insulin concentration increased significantly at 5 hours; and the number of platelet-neutrophil aggregates increased significantly at 12 hours. Conclusions and Clinical Relevance—In horses, concurrent increases in venous blood ET-1 immunoreactivity, insulin and glucose concentrations, and platelet-neutrophil aggregates support a role of endothelial dysfunction in the pathogenesis of CHO-induced laminitis.

Objective—To characterize the in vitro response of equine cecal longitudinal smooth muscle (CLSM) to endothelin (ET)-1 and assess the role of ETA and ETB receptors in those ET-1–induced responses. Animals—36 horses without gastrointestinal tract disease. Procedure—To determine cumulative concentrationresponse relationships, CLSM strips were suspended in tissue baths containing graded concentrations of ET-1 (10–9 to 10–6M) with or without BQ-123 (ETA receptor antagonist); with or without IRL-1038 (ETB receptor antagonist); or with both antagonists at concentrations of 10–9, 10–7, and 10–5M. To determine the percentage change in baseline tension of CLSM, the areas under the curve during the 3-minute periods before and after addition of each dose were compared . Also, the effects of ET-1 and a combination of selective ETA and ETB receptor antagonists on electrically evoked contractions were studied. Results—ET-1 caused sustained increases in CLSM tension in a concentration-dependent manner. Contractile responses to ET-1 were not significantly inhibited by either BQ-123 or IRL-1038 alone at any concentration; however, responses were significantly inhibited by exposure to the antagonists together at a concentration of 10–5M. Electrical field stimulation did not change the spontaneous contractile activity of CLSM and did not significantly alter the tissue response to ET-1, BQ-123, or IRL-1038. Conclusions and Clinical Relevance—Results indicated that ET-1 has a contractile effect on equine CLSM that is mediated via ETA and ETB receptors. In vitro spontaneous contractions of equine CLSM apparently originate in the smooth muscle and not the enteric nervous system. (Am J Vet Res 2005;66:1202–1208)

We describe the echocardiographic findings in a 4-day-old thoroughbred foal with an aortico-pulmonary septal defect. The foal had labored breathing, cyanotic mucous membranes and a continuous grade 5/6 heart murmur with point of maximal intensity over the base of the heart on the right side. Echocardiographically, there was a large communication between the aorta and the pulmonary artery just dorsal to the base of the heart. The cardiac anomaly seen during the echocardiographic exam was confirmed at necropsy where a large communication between the two great vessels was observed. These findings correlate with previous studies in humans, dogs, and cats. The possible failure in the embryologic development that led to this unusual cardiac anomaly is discussed.

Hyperinsulinemia causes laminitis experimentally and is a risk factor for naturally occurring laminitis. The aim of this study was to investigate the effects of insulin on laminar vascular relaxation and to induce insulin-associated vascular dysfunction in vitro. Relaxation responses of isolated laminar arterial and venous rings to acetylcholine and insulin were evaluated. To alter vascular function in response to insulin, all vessel rings were incubated with insulin or vehicle, submaximally contracted, administered insulin again and relaxation responses recorded. Laminar arteries were also incubated with the mitogen-activated protein kinase (MAPK) inhibitor, PD-98059. Relaxation in response to acetylcholine was not different between arteries and veins, but veins relaxed less in response to insulin than arteries. In arteries incubated with insulin, the subsequent relaxation response to insulin was blunted. Veins had minimal relaxation to insulin regardless of incubation. Arteries incubated with PD-98059 relaxed more in response to insulin than arteries not exposed to PD-98059, indicating that MAPK plays a role in maintenance of basal tone in laminar arteries. A differing response of laminar veins and arteries to insulin-induced relaxation may be important in understanding the link between hyperinsulinemia and laminitis. In vitro induction of vascular dysfunction in response to insulin in laminar arteries may be useful for testing therapeutic interventions and for understanding the pathophysiology of laminitis.

Tumor necrosis factor α (TNFα) occupies a pivotal role in the development of shock and tissue injury during endotoxemia and septicemia, and may be an important trigger in the pathogenesis of endotoxin-induced intestinal mucosal dysfunction. This study investigated the contribution of TNFα to endotoxin-induced mucosal dysfunction and the efficacy of polyclonal anti-TNFα antibody in preventing endotoxin-induced mucosal dysfunction. To evaluate mucosal dysfunction, jejunal blood-to-lumen clearances of chromium 51-labeled ethylenediaminetetraacetate ([51Cr]-EDTA) were measured in cats administered fetal calf serum (controls), endotoxin, TNFα, or polyclonal anti-TNFα antibody and endotoxin. Serum TNFα activity was determined using a modified in vitro cytotoxicity bioassay using the murine fibrosarcoma cell line, WEHI-164 clone 13. Endotoxin and TNFα induced jejunal mucosal dysfunction as indicated by increases in [51 Cr]-EDTA clearance. Mucosal dysfunction was accompanied by marked increases in serum TNFα activity. Furthermore, pretreatment with polyclonal anti-TNFα antibody prevented endotoxin-induced mucosal dysfunction and markedly reduced the associated increase in serum TNFα activity. The findings of this study suggest that TNFα is an important mediator of endotoxin-induced mucosal epithelial barrier dysfunction.

The effects of an intravenous infusion of endotoxin (750 μg/kg) or an equal volume of saline solution (control) on the proximal and distal jejunal permeability and blood flow were evaluated in cats. In 8 cats, proximal and distal jejunal segments were isolated and mucosal clearance of 51Cr-ethylenediaminetetraacetic acid (EDTA) was measured. The mucosal clearance in the distal loop was not altered by endotoxin infusion in 6 cats given endotoxin or 2 cats given saline solution. In 6 cats given endotoxin, the proximal jejunal segment exhibited a 10-fold increase in mucosal permeability. In contrast, mucosal clearance remained stable in the proximal jejunal loop in 2 cats infused with saline solution. In a second group of 13 cats (7 cats infused with endotoxin; 6 cats infused with saline solution), blood flow to the proximal and distal segments was measured. Either endotoxin (n = 7) or saline solution (n = 6) did not significantly alter blood flow to the proximal and distal jejunum in these experiments. Samples of proximal and distal jejunum were collected from 12 cats (6 cats infused with endotoxin and 6 cats infused with saline solution). There was significantly more epithelial necrosis in the endotoxin treated cats than in the saline treated cats. Neutrophil infiltration was greater in the jejunal segments of endotoxin treated cats than in the jejunal segments of saline treated cats. In the endotoxin treated cats, there was significantly greater necrosis in the proximal jejunal segment than in the distal jejunal segment. There were no significant differences in numbers of neutrophils in the proximal and distal jejunal segments. These results demonstrate that the proximal jejunum is more sensitive to endotoxin-induced increases in mucosal permeability than is the distal jejunum. The increases in mucosal permeability in the proximal jejunum were not accompanied by significant reductions in jejunal arterial blood flow. Endotoxemia induced neutrophil infiltration to the proximal and distal jejunum.

Objective—To determine the effects of clenbuterol, at a dosage of up to 3.2 μg/kg for 14 days, PO, on skeletal and cardiac muscle in healthy horses undergoing treadmill exercise. Animals—12 healthy horses from 3 to 10 years old. Procedures—Horses were randomly assigned to a control group (n = 6) or clenbuterol group (6) and received either saline (0.9% NaCl) solution or clenbuterol, PO, every 12 hours for 14 days. Horses were subjected to submaximal treadmill exercise daily during treatment. Muscle biopsy specimens were collected before and after treatment for determination of apoptosis. Echocardiographic measurements, serum clenbuterol and cardiac troponin I concentrations, and serum activities of creatine kinase and aspartate aminotransferase were measured before, during, and after treatment. Jugular venous blood samples were collected every 3 days during treatment. Echocardiography was repeated every 7 days after beginning treatment. Response variables were compared between treatment groups and across time periods. Results—No significant effect of clenbuterol or exercise on response variables was found between treatment and control groups at any time point or within groups over time. Conclusions and Clinical Relevance—Results did not reveal any adverse effects of treatment with an approved dose of clenbuterol on equine cardiac or skeletal muscle in the small number of horses tested.

The role of arachidonic acid metabolites in the forestomach stasis induced by Escherichia coli endotoxin was evaluated. Six adult Holstein cows received saline solution; endotoxin at 1, 10, and 100 ng/kg of body weight; flunixin meglumine at 1.1 mg/kg of body weight; and flunixin meglumine at 1.1 mg/kg plus endotoxin at 100 ng/kg. The frequency of reticulorumen contractions, mental attitude, body temperature, respiratory rate, heart rate, and plasma concentration of prostaglandin E2, prostacyclin, and thromboxane were evaluated. Administration of saline solution and endotoxin at 1 ng/kg had no significant effects. Administration of endotoxin at 10 ng/kg did not cause significant clinical effects or alter reticulorumen contractions but enhanced synthesis of thromboxane. Administration of endotoxin at 100 ng/kg caused mild clinical signs of stasis, reduced the frequency of reticulorumen contractions, and enhanced synthesis of thromboxane and prostacyclin. Reticulorumen stasis was not accompanied by an increase in the plasma concentration of prostaglandin E2. Flunixin meglumine abolished endotoxin-induced reticulorumen stasis, tachycardia, and synthesis of arachidonic acid metabolites. Reticulorumen stasis during bovine endotoxemia is caused either by enhanced synthesis of an arachidonic acid metabolite other than prostaglandin E2 or by local synthesis of prostaglandin E2.

Background: Summer pasture-associated recurrent airway obstruction (SPA-RAO), a seasonal airway obstructive disease of horses, is characterized by clinical exacerbation after exposure to pasture during warm months of the year. Endothelin (ET)-1, potent bronchoconstrictor, mitogen, secretagogue, and proinflammatory mediator, has been implicated in the pathogenesis of asthma and equine heaves. Hypothesis: Immunoreactive ET-1 concentrations increase during clinical exacerbation and return to basal values during periods of disease remission. Animals: Twelve horses, 6 affected with SPA-RAO and 6 nonaffected. Methods: Prospective, observational study. Bronchoalveolar lavage fluid (BALF), arterial and venous plasma samples, and clinical variables were obtained from affected horses during clinical exacerbation and remission. Samples and data of nonaffected horses were collected during the summer and winter on dates similar to affected horses. Immunoreactive ET-1 was determined using a commercial ELISA. Results: The median and range ET-1 concentrations (pg/ml) in arterial (1.3, 0.7–1.8) and venous (1.3, 1.2–1.7) plasma and in BALF (0.3, 0.2–0.4), and pulmonary epithelial lining fluid (PELF) (25.5, 21–50) were greater in affected horses during clinical exacerbation compared with remission (P < .01). The concentrations of immunoreactive ET-1 were greater in affected horses during clinical exacerbation compared with nonaffected horses (P < .05). Conclusions and Clinical Importance: During clinical exacerbation of SPA-RAO, ET-1 is increased in circulation and pulmonary secretions. Intervention with ET receptor antagonists should provide further information on the role of ET-1 in SPA-RAO.

Objective—To characterize the in vitro response of circular and longitudinal myometrial layers of the uterine horn (CMLH and LMLH, respectively) of horses to endothelin (ET)-1 by use of specific ETA (BQ-123) and ETB (IRL-1038) receptor antagonists. Sample Population—Uteruses from 10 nongravid mares in anestrus. Procedure—Muscle strips from the CMLH and LMLH were suspended in tissue baths and connected to force-displacement transducers interfaced with a polygraph. Strips were incubated for 45-minute intervals with no antagonist (control specimens), and 3 concentrations (10–9, 10–7, and 10–5M) of BQ-123, IRL- 1038, or BQ-123 and IRL-1038 before concentrationresponse curves to ET-1 were generated. Contractile response to cumulative concentrations of ET-1 (10–9 to 10–6M) was quantified by measuring change in the area under the curve (AUC) for the 3-minute period after each ET-1 dose. Results—ET-1 caused concentration-dependent contraction of the CMLH and LMLH specimens. Application of BQ-123 decreased AUC values for both layers. Application of IRL-1038 increased the AUC value for LMLH specimens but did not affect the CMLH value. The combination of BQ-123 and IRL-1038 decreased the AUC value for LMLH tissue and increased that for CMLH tissue. Conclusions and Clinical Relevance—ET-1 causes contraction of the CMLH and LMLH in nongravid horses. In both layers, ETA receptors mediate contraction but the role of ETB receptors remains unclear. In the LMLH, ETA receptors have a dominant role; the presence of another receptor or receptor subtype within this layer is suggested. These findings support a physiologic role for ET-1 in uterine contractility. (Am J Vet Res 2005;66:1094–1100)

Objective—To characterize the in vitro effects of oxytocin, acepromazine, xylazine, butorphanol, detomidine, dantrolene, isoproterenol, and terbutaline on skeletal and smooth muscle from the equine esophagus. Animals—14 adult horses without digestive tract disease. Procedure—Circular and longitudinal strips from the skeletal and smooth muscle of the esophagus were suspended in tissue baths, connected to force-displacement transducers interfaced with a physiograph, and electrical field stimulation was applied. Cumulative concentration-response curves were generated for oxytocin, acepromazine, xylazine, detomidine, butorphanol, isoproterenol, terbutaline, and dantrolene. Mean maximum twitch amplitude for 3 contractions/min was recorded and compared with predrug-vehicle values for the skeletal muscle segments, and area under the curve (AUC) for 3 contractions/min was compared with predrug-vehicle values for the smooth muscle segments. Results—No drugs caused a significant change in skeletal muscle response. In smooth muscle, isoproterenol, terbutaline, and oxytocin significantly reduced AUC in a concentration-dependent manner. Maximum reduction in AUC was 69% at 10–4M for isoproterenol, 63% at 10–5M for terbutaline, and 64% at 10–4M for oxytocin. Conclusions and Clinical Relevance—Isoproterenol, terbutaline, and oxytocin cause relaxation of the smooth muscle portion of the esophagus. The clinical relaxant effects on the proximal portion of the esophagus reported of drugs such as oxytocin, detomidine, and acepromazine may be the result of centrally mediated mechanisms. (Am J Vet Res 2002;63:1732–1737)

The present study investigated the possibility that nitric oxide is a nonadrenergic, noncholinergic neurotransmitter of nerves that are intrinsic to the forestomach. Tunica muscularis, myenteric plexus preparations of bovine reticulum and rumen were maintained in vitro in a physiological solution of buffer that contained scopolamine. Trains of electric field stimulation transiently reduced (relaxed) the tone induced by BaCl2. NG-Nitro-L-arginine methyl ester, a nitric oxide synthase competitive antagonist, inhibited relaxation of the rumen and reticulum preparations that had been induced by the electrical field. The actions of NG-nitro-L-arginine methyl ester were partially reversed by L-arginine. These data suggest that nitric oxide, or a related substance, is an inhibitory neurotransmitter of nerves that are intrinsic to tunica muscularis, myenteric plexus preparations of the bovine forestomach.

The objective of this study was to determine whether blockade of alpha(-2) adrenergic receptors would restore reticulorumen motility during toxaemia in cows. Reticulorumen contractions were measured via a water-filled balloon connected to a pressure transducer. Intravenous infusion of endotoxin (100 ng kg-1 over 30 min) significantly decreased the number of reticulorumen contractions. Intravenous infusion of yohimbine (125 micrograms kg-1 over 30 min) alone did not affect reticulorumen contractions. However, when yohimbine (125 micrograms kg-1 over 30 min) was infused concurrently with endotoxin (100 ng kg-1 over 30 min), the effects of endotoxin on reticulorumen contraction frequency decreased, suggesting that endotoxaemia causes reticulorumen stasis via a mechanism that involves alpha(-2) adrenergic receptors.