Yucatan Swine Research Articles

Multifunctional Photopolymerized Semiinterpenetrating Network (sIPN) System Containing Bupivacaine and Silver Sulfadiazine Is an Effective Donor Site Treatment in a Swine Model

Previously, we have shown in a cross-comparison study that multifunctional photopolymerized semiinterpenetrating network (sIPN) system is an effective donor site treatment in a swine model. The advantages of sIPN include spray-on application, in situ photopolymerization, and ability to cover large contoured areas. sIPN has also been shown to be an effective delivery vehicle for keratinocyte growth factor, dexamethasone, bupivacaine, and silver sulfadiazine in vitro. Our aim for this study was to show that these products delivered to the wound bed with sIPN would not change the wound healing characteristics compared with the control site through qualitative clinical evaluation and to compare the rate and quality of donor site healing through histologic evaluation. Eight Yucatan swine of 40 lbs each were randomly divided into four groups of two pigs before surgery. Each animal had 5.6% TBSA of skin harvested from two different dorsal regions, with one at 22/1000th-inch and the other at 30/1000th-inch setting on the dermatome. Each test site on each animal was then sequentially dressed with 50 cm(2) of Xeroform gauze, sIPN, sIPN loaded with 0.5% bupivacaine, or sIPN loaded with 1% silver sulfadiazine. sIPN with or without soluble drugs were applied as liquid, then photopolymerized in situ to form an elastic covering. Each of the test areas was separated by 50 cm(2) of autograft, which was used to divide the test areas. Wound assessment and killing occurred at days 7, 9, 14, and 21. A full-thickness biopsy was taken from each of the study areas for histological analysis. By 14 days, all areas showed complete epidermal coverage histologically. The 30/1000th-inch site revealed a thicker, more irregular dermis compared with the 22/1000th-site. Evaluation of the day-21 sites revealed equal thinning and flattening of the new epidermis. No site showed full restoration of the rete ridges. No signs of infection were seen in clinical or histological evaluations of any treatment. The addition of bupivacaine and silver sulfadiazine to sIPN does not show any alterations in wound healing of a donor site in a swine model when compared with sIPN without loaded drugs and a standard control dressing. This efficacy may be coupled with established localized sIPN drug delivery profiles and allow further studies to evaluate the efficacy of these drugs to promote healing, eradicate and prevent infection, and manage pain.

The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines

Solar lentigines (SLs) are hyperpigmentary lesions presented on sun-exposed areas of the skin and associated with ageing. The molecular mechanism of SL initiation is not completely understood. Ultraviolet B (UVB) stimulates keratinocytes to produce interlukin-1 alpha (IL-1α), which then induces keratinocyte growth factor (KGF) secretion; therefore, we examined their possible roles in the induction of SLs. We found that KGF increases pigment production in both pigmented epidermal equivalents and human skin explants. In addition, UVB exposure increases KGF expression, and KGF treatment induces tyrosinase (TYR) expression in primary melanocytes. The KGF-induced pigmentary changes were confirmed using pigmented Yucatan swine, and human skins grafted onto immuno-deficient mice. In both model systems, the topical treatment with KGF, alone or in combination with IL-1α, resulted in the in vivo formation of hyperpigmentary lesions with increased pigment deposition and elongated rete ridges, which resemble the histological features of human SLs. Preliminary immunohistochemical analysis of human skins showed a moderate increase in KGF, and a strong induction in KGF receptor (KGFR) in SL lesions. In summary, KGF increases pigment production and deposition in vitro and in vivo. Moreover, we show for the first time the in vivo generation of hyperpigmentary lesions with histological resemblance to human SLs and indicate the involvement of KGF/KGFR in the molecular pathology of human SLs.

Exercise training decreases the size and alters the composition of the neointima in a porcine model of percutaneous transluminal coronary angioplasty (PTCA)

Exercise training (EX) following percutaneous transluminal coronary angiography (PTCA) reduces progression to restenosis and increases event-free survival rates. Our aim was to determine whether EX inhibits lesion development and/or alters the extracellular matrix (ECM) composition of the neointima (NI) in a porcine PTCA model. Miniature Yucatan swine were assigned to cage confinement (SED) or EX for 20 wk. After 16 wk, all animals underwent a PTCA procedure of the left anterior descending artery (LAD) and left circumflex artery (LCX), with subsequent placement of an externalized jugular catheter. Animals recovered for 2 days and then resumed the previous protocol of SED or EX. Twelve days following PTCA, all animals received an intravenous bromodeoxyuridine (BrdU) injection to label proliferating cells. At 28 days following PTCA, the animals were euthanized, the LAD and LCX excised, and underwent standard histological processing for total collagen, type I collagen, fibronectin, BrdU, and Verhoeff-van Gieson stain. Our results demonstrate that EX significantly decreased lesion size and NI proliferation (-48%) in the LAD (P < 0.05) but not the LCX. Furthermore, EX attenuated type I collagen expression only in LAD, whereas total collagen was increased (5.9%) and fibronectin was decreased (-7.9%) in the NI of both vessels (P < 0.05). In conclusion, EX following PTCA may increase event-free survival rates following PTCA by decreasing lesion size and altering ECM composition.

Late Stent Expansion and Neointimal Proliferation of Oversized Nitinol Stents in Peripheral Arteries

For peripheral endovascular intervention, self-expanding (SE) stents are commonly oversized in relation to target arteries to assure optimal wall apposition and prevent migration. However, the consequences of oversizing have not been well studied. The purpose of this study was to examine the effects of SE stent oversizing (OS) with respect to the kinetics of late stent expansion and the long-term histological effects of OS. Pairs of overlapped 8 x 28-mm Nitinol SE stents were implanted into the iliofemoral arteries of 14 Yucatan swine. Due to variations in target artery size, the stent-to-artery ratio ranged from 1.2:1 to 1.9:1. Lumen and stent diameters were assessed by quantitative angiography at the time of implantation. Following angiographic assessment at 6 months, stented arteries were perfusion-fixed, sectioned, and stained for histological analysis. Immediately following implantation, the stents were found to be expanded to a range of 4.7-7.1 mm, largely conforming to the diameter of the recipient target artery. The stents continued to expand over time, however, and all stents had enlarged to nearly their 8-mm nominal diameter by 6 months. The histological effects of OS were profound, with marked increases in injury and luminal area stenosis, including a statistically significant linear correlation between stent-to-artery ratio and area stenosis. In this experimental model of peripheral endovascular intervention, oversized Nitinol SE stents are constrained by their target artery diameter upon implantation but expand to their nominal diameter within 6 months. Severe OS (stent-to-artery ratio >1.4:1) results in a profound long-term histological response including exuberant neointimal proliferation and luminal stenosis.

Atorvastatin Increases Myocardial Indices of Oxidative Stress in a Porcine Model of Hypercholesterolemia and Chronic Ischemia

Atorvastatin has previously been shown to reduce the endogenous angiogenic response to chronic ischemia in a porcine model. One possible mechanism for this effect is reduced bioavailability of nitric oxide, a key mediator of angiogenesis, secondary to increased oxygen free radicals. We sought to determine if atorvastatin modulates oxidative stress in myocardial tissue. Dietary induction of hypercholesterolemia was performed over 20 weeks in Yucatan swine with treated animals receiving atorvastatin 3 mg/kg/day. Chronic myocardial ischemia was induced via surgical placement of an ameroid constrictor ring around the proximal circumflex artery at age 20 weeks, followed by tissue harvest at age 27 weeks. Myocardial levels of protein, lipid, and DNA biomarkers of oxidative stress, serum levels of 8-isoprostane, nitric oxide (NO) dependent, and independent coronary microvascular reactivity, as well as isotope-labeled microsphere myocardial perfusion analysis and histologic analysis for endothelial cell density was performed. Atorvastatin treatment was associated with elevated levels of myocardial protein oxidation and lipid peroxidation. Conversely, serum oxidant stress biomarkers were not elevated. Atorvastatin treatment improved nitric oxide dependent and independent microvascular reactivity, and was associated with decreased perfusion in the ischemic myocardial territory. Treatment with atorvastatin was associated with increased levels of myocardial tissue protein and lipid oxidative stress biomarkers and a reduced functional endogenous angiogenic response, but improved coronary microvascular reactivity. Increased oxidative stress in tissues may play a role in the reduced angiogenic response seen with atorvastatin treatment in other studies.

Diffuse coronary atherosclerosis in Ossabaw miniature swine with metabolic syndrome compared to Yucatan swine

Patients with diabetes have diffuse coronary atherosclerosis, also known as three vessel disease. The Ossabaw miniature swine is an excellent large animal model of metabolic syndrome (pre‐diabetes). Here we compare the pervasiveness of coronary atherosclerosis between the Ossabaw swine model of metabolic syndrome and the widely used Yucatan swine that do not show metabolic syndrome. Each breed was fed two separate diets which were calorie matched and divided into two groups each: control (C) and high fat/cholesterol atherogenic diet (H). Intravascular ultrasound was used to quantify atherosclerosis throughout the epicardial coronary vasculature. Ossabaw swine had increased characteristics of metabolic syndrome including: greater weight gain, glucose intolerance, and insulin resistance, compared to Yucatan. Plasma lipids and blood pressure were not different. H diet increased atherosclerosis ~3‐fold in proximal segments of coronary artery compared to C, with no breed differences. However, Ossabaw H compared to Yucatan H had ~2.5‐fold and ~4‐fold greater atherosclerosis in intermediate and distal coronary segments, respectively. Neointima of Ossabaw had less collagen, suggesting more cellular and/or lipid‐laden composition. We conclude that Ossabaw swine, not Yucatan, exhibit diffuse coronary atherosclerosis, a hallmark of patients with diabetes. Support: NIH RR013223, HL062552.

Exercise training decreases the size and alters the composition of the neointima in a porcine model of coronary restenosis

Exercise training reduces adverse events following coronary angioplasty in humans. Therefore, our aim was to determine if exercise reduces the size or alters the composition of the neointima (NI) in a porcine balloon coronary injury (BCI) model. Miniature Yucatan swine were randomly assigned to cage confinement (SED, n=7) or exercise (EX, n=8) training on a treadmill. After 4 months the animals underwent BCI of the left anterior descending (LAD) and left circumflex (LCX) arteries. The animals recovered for 2 days and resumed the previous protocol of SED or EX. Additionally on day 12 following the BCI, each animal received an intravenous injection of BrdU to label proliferating cells. At 28 days post BCI the animals were euthanized and the LAD and LCX were excised. Injured and uninjured sections from each artery underwent standard histological procedures and were stained with VVG, pico Sirius red and antibodies for BrdU and fibronectin. Our results demonstrate that exercise decreased lesion size and NI proliferation (−48%) in the LAD (p&lt;.05) but not the LCX. Exercise significantly increased NI collagen (5.9%) and decreased NI fibronectin (−7.9%) in both arteries (p&lt;.05). However, in the uninjured media collagen decreased in the EX (−5.2%, p&lt;.05) with no effect on fibronectin. In conclusion, exercise may decrease post angioplasty events by reducing lesion size and altering NI composition.Supported by: NIH HL 52490

Effects of Chronic Nitric Oxide Synthase Inhibition on Endothelium-Dependent and -Independent Relaxation in Arteries that Perfuse Skeletal Muscle of Swine

The purpose of this investigation was to test the hypothesis that chronic N(G)-nitro-l-arginine methyl ester (l-NAME) treatment produces differential effects on conduit artery and resistance arteriole relaxation responses to endothelium-dependent and -independent vasodilators in arteries that perfuse skeletal muscle of swine. To test this hypothesis, conduit skeletal muscle arteries and second-order skeletal muscle (2A) arterioles were harvested from 14 Yucatan swine that were chronically administered l-NAME and from 16 controls. In vitro assessments of vasorelaxation to increasing doses of acetylcholine (ACH), bradykinin (BK), and sodium nitroprusside (SNP) were performed in both conduit and 2A arterioles. l-NAME treatment produced a significant reduction in both BK and ACH relaxation responses in the conduit arteries. In contrast, the relaxation response and/or sensitivity to SNP were significantly greater in the intact, but not denuded, conduit arterial rings from chronically l-NAME-treated swine. There were no significant effects of chronic l-NAME treatment on vasodilation of skeletal muscle arterioles. These findings suggest (1) that unlike arterioles, skeletal muscle conduit arteries do not functionally compensate for a lack of NO through the upregulation of alternative vasodilator pathways; (2) that the greater relaxation response in conduit arteries of chronically l-NAME-treated swine to SNP can be explained by alterations to the endothelium.

Endothelium-dependent and -independent relaxation in the forelimb and hindlimb vasculatures of swine

Limb differences in endothelial function exist between arm and leg vasculatures of humans. The current investigation tested the hypothesis that forelimb and hindlimb vasorelaxation are similar in the absence of limb differences in blood pressure. Conduit arteries (brachials/femorals) and second order arterioles were harvested from 22 miniature Yucatan swine. In vitro assessment of vasorelaxation was determined by administering increasing doses of bradykinin (BK), acetylcholine (ACh), and sodium nitroprusside (SNP). The role of the nitric oxide synthase (NOS) and cyclooxygenase (COX) pathways was assessed in conduit arteries but not resistance arterioles through l-NAME (300 μM) and INDO (5 μM) incubation, respectively. The relaxation responses to BK and ACh were similar in brachial and femoral arteries. SNP relaxation response was greater in the brachial compared to femoral arteries. There were also no significant differences in the relaxation responses of second order arterioles of the forelimb and hindlimb to BK, ACh, and SNP. Incubation of conduit arterial rings in l-NAME produced a greater reduction in BK and ACh relaxation in the brachial (∼ 25%) compared to femoral (∼ 13%) arterial rings. The current results of this investigation suggest that the forelimb and hindlimb vasculatures of swine have relatively similar vasorelaxation responses to both endothelium-dependent and -independent vasodilators.

Gender and genetic differences in bladder smooth muscle PPAR mRNA in a porcine model of the metabolic syndrome

The metabolic syndrome and diabetes are associated with bladder dysfunction in many people. Peroxisome proliferator-activated receptors (PPARs) may play a role in the effects of the metabolic syndrome on bladder smooth muscle (BSM). The purpose of this study was to determine if there are gender and genetic differences in PPAR levels in BSM. We measured PPAR levels using quantitative PCR in BSM from male Yucatan swine and male and female Ossabaw Island swine, which is a model for the metabolic syndrome. Male Ossabaw swine had 0.732 +/- 0.111 the amount of PPAR-alpha mRNA as male Yucatan swine (P < 0.05), suggesting a genetic difference in PPAR-alpha levels. This difference may possibly contribute to the incidence of metabolic syndrome in the Ossabaw model compared to the Yucatan model. PPAR-delta mRNA was 2-fold higher in male Ossabaw swine than in female Ossabaw swine, with no significant differences in PPAR-alpha levels. However, PPAR-gamma mRNA was 4.067 +/- 0.134 times higher in female Ossabaw swine than in their male counterparts (P < 0.001). Changing the percentage of calories derived from fat did not alter any PPAR mRNA levels. Thus, PPAR-delta and PPAR-gamma mRNA levels in male and female Ossabaw swine BSM are not only different, but may also result in gender differences in lipid metabolism in bladder smooth muscle. We conclude that PPAR profiles in BSM may contribute to the susceptibility of BSM to lipotoxicity in the metabolic syndrome.

Alpha‐adrenergic responsiveness of porcine forelimb and hindlimb vasculatures

Elevated blood pressure in the legs during upright posture has been hypothesized to contribute to a greater α-adrenergic (α-AR) vasoconstrictor response in the resistance arterioles of the legs compared to the forearms of humans. Therefore, the purpose of this investigation was to test the hypothesis that conduit arteries and resistance arterioles of both the forelimbs and hindlimbs of quadrupeds (Yucatan swine; n = 12) exhibit similar α-AR receptor vasoconstrictor responses. The constrictor response to phenylephrine (PE; 10−9–10−4 M) was determined in vitro and found significantly greater in femoral (FEM) compared to brachial (BR) arteries (Max: 28.0 ± 1.6g, 21.5 ± 1.0g). However, the EC50 for PE was similar in FEM and BR arteries (EC50: −6.54 ± 0.09M, −6.44 ± 0.15M). Additionally, increasing doses of PE produced similar constrictor responses (Max: 0.78 ± 0.04%, 0.81 ± 0.04%) and EC50 (− 5.58 ± 0.34%, −5.94 ± 0.36%) in forelimb and hindlimb 2A arterioles. The current findings of similar α-AR constrictor responsiveness in limb arterioles of swine are consistent with the hypothesis that upright posture contributes to the heterogeneous limb responses reported in humans; in conduit arteries however, our findings suggest that upright posture may not be the only factor contributing to different responses of the forelimbs and hindlimbs. Supported by: RR 18276, AR 048523, HL 36088, HL 52490

Quantitative Mass Spectrometry to Determine Effects of Diabetic Dyslipidemia and Exercise on the plasma Low Density Lipoproteome

There is a predominant role for LDL in atherogenesis. Coronary vascular dysfunction in the Yucatan swine model of diabetic dyslipidemia (DD) was prevented by aerobic exercise training (DDX) without altering total LDL concentration (Mokelke et al. J. Appl. Physiol. 95:, 2003). Attenuation of the DD induced changes in the quantitative and/or qualitative properties of LDL may explain the cardioprotective effects of exercise. We used quantitative mass spectrometry to determine these changes in the complex LDL particle in plasma of healthy control, DD, and DDX pigs. The LDL fraction of the plasma was isolated via FPLC, and the tryptically digested LDL peptides were analyzed serially via linear ion-trap LC-MS/MS. Proteins were identified using SEQUEST and X!Tandem, while protein quantification was carried out based on total ion chromatograms using the proprietary technology of Indiana Centers for Applied Protein Sciences. In this approach, the integral volume under each selected peptide peak was measured, normalized, and compared for relative abundance. In this study, 434 proteins were identified and quantified. Of these, 210 were identified with high confidence, including porcine apolipoproteins B100, D, E, CIII, N, R, AIV and AI. To our knowledge this is the largest number of proteins in the LDL particle ever identified with high confidence. Supported by NIH RR013223, HL062552 (MS) and AA015698 (FAW).

Exercise training prevents Ca2+ dysregulation in coronary smooth muscle from diabetic dyslipidemic yucatan swine

Aerobic exercise training is known to have profound cardioprotective effects in disease, yet cellular mechanisms remain largely undefined. We tested the hypothesis that increased sarcoplasmic reticulum Ca(2+) buffering and increased voltage-gated Ca(2+) channel density underlie coronary smooth muscle intracellular Ca(2+) (Ca(2+)(i)) dysregulation in diabetic dyslipidemia and that exercise training would prevent these increases. Yucatan swine were maintained in 1) control, 2) alloxan-induced hyperglycemic, 3) high fat/cholesterol fed, 4) hyperglycemic plus high fat/cholesterol fed (diabetic dyslipidemic), and 5) diabetic dyslipidemic plus exercise-trained (treadmill running) conditions. After 20 wk, the heart was removed and smooth muscle cells isolated from the right coronary artery. We utilized fura-2 imaging of Ca(2+)(i) levels to separate the functional role of the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) from the Na(+)-Ca(2+) exchanger and the plasmalemmal Ca(2+)-ATPase, and whole-cell patch clamp to examine voltage-gated Ca(2+) channel current density (i.e., Ca(2+) influx). Results indicated that diabetic dyslipidemia impaired plasmalemmal Ca(2+) efflux, increased basal Ca(2+)(i) levels, increased SERCA protein and sarcoplasmic reticulum Ca(2+)(i) buffering, and elicited an approximately 50% decrease in voltage-gated Ca(2+) channel current density. Exercise training concurrent with the diabetic dyslipidemic state restored plasmalemmal Ca(2+) efflux, SERCA protein, sarcoplasmic reticulum Ca(2+)(i) buffering, and voltage-gated Ca(2+) channel current density to control levels. Interestingly, basal Ca(2+)(i) levels were significantly lower in the exercise-trained group compared with control. Collectively, these results demonstrate a crucial role for exercise in the prevention of diabetic dyslipidemia-induced Ca(2+)(i) dysregulation.

Exercise training reverses downregulation of HSP70 and antioxidant enzymes in porcine skeletal muscle after chronic coronary artery occlusion

Oxidative stress is associated with muscle fatigue and weakness in skeletal muscle of ischemic heart disease patients. Recently, it was found that endurance training elevates protective heat shock proteins (HSPs) and antioxidant enzymes in skeletal muscle in healthy subjects and antioxidant enzymes in heart failure patients. However, it is unknown whether coronary ischemia and mild infarct without heart failure contributes to impairment of stress proteins and whether exercise training reverses those effects. We tested the hypothesis that exercise training would reverse alterations in muscle TNF-alpha, oxidative stress, HSP70, SOD (Mn-SOD, Cu,Zn-SOD), glutathione peroxidase (GPX), and catalase (CAT) due to chronic coronary occlusion of the left circumflex (CCO). Yucatan swine were divided into three groups (n = 6 each): sedentary with CCO (SCO); 12 wk of treadmill exercise training following CCO (ECO); and sham surgery controls (sham). Forelimb muscle mass-to-body mass ratio decreased by 27% with SCO but recovered with ECO. Exercise training reduced muscle TNF-alpha and oxidative stress (4-hydroxynonenal adducts) caused by CCO. HSP70 levels decreased with CCO (-45%), but were higher with exercise training (+348%). Mn-SOD activity, Mn-SOD protein expression, and Cu,Zn-SOD activity levels were higher in ECO than SCO by 72, 82, and 112%, respectively. GPX activity was 177% greater in ECO than in SCO. CAT trended higher (P = 0.059) in ECO compared with SCO. These data indicate that exercise training following onset of coronary artery occlusion results in recovery of critical stress proteins and reduces oxidative stress.

Exercise training‐induced increase in coronary blood flow in Yucatan swine is blunted by hypercholesterolemia

We tested the hypothesis that aerobic exercise training would prevent a high-fat/cholesterol diet-induced attenuation of coronary blood flow (CBF) response to a dobutamine stress test in swine. Adult male Yucatan pigs were fed a low-fat control diet (C) or a high-fat/cholesterol diet (H; hypercholesterolemic; 46% kcal from fat, 2% cholesterol) for 20 wk. Pigs fed these diets were either exercise trained (X) or remained sedentary (SED); thus, we studied four groups: CSED, CX, HSED, HX. Exercise was treadmill running up to 75 min/day, 5 days/wk for 16 wk. Pigs were instrumented under anesthesia, and a coronary flow wire determined average peak velocity in response to intravenous dobutamine (5μg/kg/min for 5 min) followed by a 5-min recovery period. Heart rate and blood pressure were continuously recorded. Intravascular ultrasound determined coronary cross sectional area for calculation of CBF (CBF = (1/2) APV*CSA). Rate pressure product (RPP) was used as an assessment of myocardial work, and CBF was standardized to myocardial work (CBF/work). Results: CBF/work ratio of CX was greater than all other groups (p<.05). CBF/work was also higher in the HX compared to CSED and HSED. Surprisingly, the CSED and HSED groups did not differ from each other. Conclusions: Exercise training increased the CBF/work ratio in control and H pigs, relative to sedentary; however, the high fat diet blunted the effects of exercise. Support: NIH HL52490, HL62552.

The Ossabaw swine model of the Metabolic syndrome exhibit greater stenosis after coronary stenting than lean Yucatan swine.

BACKGROUND Patients with the metabolic syndrome have poor prognosis after coronary stenting. Mechanisms underlying this pathology have been elusive because of the lack of a large animal model of the metabolic syndrome. HYPOTHESIS Lean Ossabaw swine predisposed to the metabolic syndrome (n=8; insulin-resistant, propensity to obesity) will exhibit greater in-stent stenosis after coronary stenting vs. lean Yucatan (n=4) swine. METHODS Bare metal stents were placed with 1x apposition (not over-inflation injury) verified by intravascular ultrasound (IVUS). Stenosis was evaluated using IVUS and histology 3–4 weeks later. Stenosis was calculated from the cross sectional neointimal area as a percent of the area within the internal elastic lamina in the stented segment. RESULTS Mean percent stenosis in Ossabaw was greater than Yucatan by IVUS (26±7% vs. 7±3%) and histological analysis (67±8% vs. 34±7%). These data suggest that the risk for increased stenosis already exists in lean Ossabaw pigs with insulin resistance and predisposed to obesity. CONCLUSIONS Ossabaw miniature swine are a novel humanoid model of the metabolic syndrome uniquely suited for the study of coronary interventional devices and mechanisms of accelerated restenosis. Support: NIH RR13223, HL62552, American Diabetes Association.

Training-Induced Sarcoplasmic Reticulum Ca2+ Unloading Occurs without Ca2+ Influx

Aerobic exercise training elicits adaptations in coronary smooth muscle that result in a novel intracellular Ca2+ signaling phenomenon termed sarcoplasmic reticulum (SR) Ca2+ unloading. Sarcoplasmic reticulum Ca2+ unloading is defined as a time-dependent depletion and then repletion of the caffeine-sensitive SR Ca2+ store. To determine whether Ca2+ influx is necessary to elicit SR Ca2+ unloading. Male, Yucatan swine (8 months old) were maintained: 1) sedentary or 2) exercise trained (treadmill running performed 5 d.wk(-1) for 16 wk). Smooth muscle cells were isolated from the right coronary artery and loaded with the intracellular Ca2+-indicator, fura-2. Sarcoplasmic reticulum Ca2+ content was assessed as the change in the caffeine (5 mM)-induced intracellular Ca2+ peak after a 2-, 5-, 8-, 11- or 13-min recovery from high K+ (depolarization)-induced Ca2+ influx in a physiological (2 mM) Ca2+ solution. The effect of Ca2+ influx on SR Ca2+ unloading was assessed by replacing the 2 mM Ca2+ solution with a virtually Ca2+-free (100 nM) solution during the recovery period. Consistent with previous studies, SR Ca2+ unloading was not observed in cells from sedentary swine. In cells from exercise-trained swine, SR Ca2+ depletion was observed in both the 2 mM and Ca2+-free solutions, suggesting that Ca2+-induced Ca2+ release was not initiating SR Ca2+ unloading during the recovery period. In addition, the reloading of the SR Ca2+ store occurred even in the Ca2+-free solution, suggesting that exercise training facilitates an internal cycling of Ca2+ between the SR and another intracellular Ca2+ store. In coronary smooth muscle from male swine, Ca2+ influx is not necessary for the exercise training-induced phenomenon, SR Ca2+ unloading.

Extracellular Cyclic AMP—Adenosine Pathway in Isolated Adipocytes and Adipose Tissue

Our goal was to evaluate the presence and lipolytic impact of the extracellular cyclic adenosine monophosphate (AMP)-adenosine pathway in adipose tissue. Sixteen miniature Yucatan swine (Sus scrofa) were used for these in vitro and in situ experiments. Four microdialysis probes were implanted into subcutaneous adipose tissue and perfused at 2 microL/min with Ringer's solution containing no addition, varying levels of cyclic AMP, 10 microM isoproterenol, or 10 microM isoproterenol plus 1 mM alpha,beta-methylene adenosine 5'-diphosphate (AMPCP), a 5'-nucleotidase inhibitor. Dialysate was assayed for AMP, adenosine, inosine, hypoxanthine, and glycerol. Freshly isolated adipocytes were incubated with buffer, 1 microM isoproterenol, or 1 microM isoproterenol plus 0.1 mM AMPCP, and extracellular levels of AMP, adenosine, inosine, hypoxanthine, and glycerol were measured. Perfusion of adipose tissue with exogenous cyclic AMP caused a significant increase in AMP and adenosine appearance. Perfusion with AMPCP, in the presence or absence of isoproterenol, significantly increased the levels of AMP and glycerol, whereas it significantly reduced the level of adenosine and its metabolites. However, the AMPCP-provoked increase in lipolysis observed in situ and in vitro was not temporally associated with a decrease in adenosine. These data suggest the existence of a cyclic AMP-adenosine pathway in adipocytes and adipose tissue. The role of this pathway in the regulation of lipolysis remains to be clarified.

ROLE OF HBOC-201 RESUSCITATION IN ISCHEMIC-REPERFUSION INJURY IN MAJOR ORGANS FROM SWINE WITH HEMORRHAGIC SHOCK

Purpose: Nitrotyrosine is an indirect marker for free radical-mediated tissue injury. The purpose of this study was to determine whether resuscitation with bovine polymerized hemoglobin (HBOC-201) induces free radical-mediated injury in a swine model of moderately severe hemorrhagic shock. Methods: Yucatan swine underwent 40% hemorrhage, soft tissue injury, and fluid resuscitation with HBOC-201 or Hextend (6% hetastarch in Lactated Ringer’s solution), or no resuscitation. Tissues were harvested following euthanasia at 72 hours or post-early deaths. Nitrotyrosine in frozen sections on slides were detected by immunohistochemistry. Mean staining intensity (in comparison with normal pig tissue) was analyzed statistically using the Kruskal-Wallis Test. Results: No significant difference in staining intensity was detected between the 3 treatment groups in any tissue types (bowel, heart, kidney, liver, and lung). However, different tissues showed varying degrees of staining intensity, regardless of treatment type. The liver displayed the greatest and lung the lowest relative staining intensities, compared to the other tissues analyzed. Conclusions: Overall, the amount of nitrotyrosine was similar in HBOC-201 and Hextend-resuscitated and non-resuscitated swine. These data suggest that HBOC-201 does not increase peroxynitrite production and may not cause significant free radical-mediated reperfusion injury in hemorrhagic shock.

Diabetic dyslipidemia and exercise affect coronary tone and differential regulation of conduit and microvessel K+ current

Spontaneous transient outward K(+) currents (STOCs) elicited by Ca(2+) sparks and steady-state K(+) currents modulate vascular reactivity, but effects of artery size, diabetic dyslipidemia, and exercise on these differentially regulated K(+) currents are unclear. We studied the conduit arteries and microvessels of male Yucatan swine assigned to one of three groups for 20 wk: control (C, n = 7), diabetic dyslipidemic (DD, n = 6), or treadmill-trained DD animals (DDX, n = 7). Circumflex artery blood flow velocity obtained with intracoronary Doppler and lumen diameters obtained by intravascular ultrasound enabled calculation of absolute coronary blood flow (CBF). Ca(2+) sparks were determined in pressurized microvessels, and perforated patch clamp assessed K(+) current in smooth muscle cells isolated from conduits and microvessels. Baseline CBF in DD was decreased versus C. In pressurized microvessels, Ca(2+) spark activity was significantly lower in DD versus C and DDX (P < 0.05 vs. DDX). STOCs were pronounced in microvessel (approximately 35 STOCs/min) in sharp contrast to conduit cells ( approximately 2 STOCs/min). STOCs were decreased by 86% in DD versus C and DDX in microvessels; in contrast, there was no difference in STOCs across groups in conduit cells. Steady-state K(+) current in microvessels was decreased in DD and DDX versus C; in contrast, steady-state K(+) current in conduit cells was decreased in DDX versus DD and C. We conclude that steady-state K(+) current and STOCs are differentially regulated in conduit versus microvessels in health and diabetic dyslipidemia. Exercise prevented diabetic dyslipidemia-induced decreases in baseline CBF, possibly via STOC-regulated basal microvascular tone.