Kyle W. Yancey

A murine model of obesity implicates the adipokine milieu in the pathogenesis of severe acute pancreatitis

Obesity is clearly an independent risk factor for increased severity of acute pancreatitis (AP), although the mechanisms underlying this association are unknown. Adipokines (including leptin and adiponectin) are pleiotropic molecules produced by adipocytes that are important regulators of the inflammatory response. We hypothesized that the altered adipokine milieu observed in obesity contributes to the increased severity of pancreatitis. Lean (C57BL/6J), obese leptin-deficient (LepOb), and obese hyperleptinemic (LepDb) mice were subjected to AP by six hourly intraperitoneal injections of cerulein (50 microg/kg). Severity of AP was assessed by histology and by measuring pancreatic concentration of the proinflammatory cytokines IL-1beta and IL-6, the chemokine MCP-1, and the marker of neutrophil activation MPO. Both congenitally obese strains of mice developed significantly more severe AP than wild-type lean animals. Severity of AP was not solely related to adipose tissue volume: LepOb mice were heaviest; however, LepDb mice developed the most severe AP both histologically and biochemically. Circulating adiponectin concentrations inversely mirrored the severity of pancreatitis. These data demonstrate that congenitally obese mice develop more severe AP than lean animals when challenged by cerulein hyperstimulation and suggest that alteration of the adipokine milieu exacerbates the severity of AP in obesity.

Influence of ADP on Cross-Bridge-Dependent Activation of Myofibrillar Thin Filaments

Contraction of skeletal muscle is regulated by calcium at the level of the thin filament via troponin and tropomyosin. Studies have indicated that strong cross-bridge binding is also involved in activation of the thin filament. To further test this, myofibrils were incubated with a wide range of fluorescent myosin subfragment 1(fS1) at pCa 9 or pCa 4 with or without ADP. Sarcomere fluorescence intensity and the fluorescence intensity ratio (non-overlap region/overlap region) were measured to determine the amount and location of bound fS1 in the myofibril. There was lower sarcomere fluorescence intensity with ADP compared to without ADP for both calcium levels. Similar data were obtained from biochemical measures of bound fS1, validating the fluorescence microscopy measurements. The intensity ratio, which is related to activation of the thin filament, increased with increasing [fS1] with or without ADP. At pCa 9, the fluorescence intensity ratio was constant until 80-160 nM fS1 without ADP conditions, then it went up dramatically and finally attained saturation. The dramatic shift of the ratio demonstrated the cooperative character of strong cross-bridge binding, and this was not observed at high calcium. A similar pattern was observed with ADP in that the ratio was right-shifted with respect to total [fS1]. Saturation was obtained with both the fluorescence intensity and ratio data. Plots of intensity ratio as a function of normalized sarcomere intensity (bound fS1) showed little difference between with and without ADP. This suggests that the amount of strongly bound fS1, not fS1 state (with or without ADP) is related to activation of the thin filament.

Influence of microtubules on vascular smooth muscle contraction

Microtubules are ubiquitous in eukaryotic cells and play key roles in many cellular activities. The purpose of this study was to investigate the influence of microtubules on vascular smooth muscle contraction. Quantitative immunocytochemical analysis of rat aortic tissue revealed that, relative to the control group, colchicine (15 μM, 90 min) and nocodazole (15 μM, 90 min) decreased the microtubule density by 40–50% while taxol (10 μM, 90 min) increased the microtubule density by 33%. Isometric contraction studies demonstrated that both colchicine and nocodazole caused an upward shift in the phenylephrine (10−8 to 10−5 M) dose–response curve while taxol caused no significant change when compared to the control group. Potassium chloride (30 mM) induced 55 ± 5% P0 contraction in DMSO treated vessel rings. The active tension increased to 73 ± 5% P0 and 71 ± 6% P0 after pretreatment of the aortic rings with colchicine or nocodazole, respectively. Taxol did not cause a significant change in the active tension (56 ± 7% P0). These results indicate that microtubule depolymerization enhances isometric contraction of vascular smooth muscle and this enhanced contraction is not receptor dependent. Pretreatment of the aortic rings with an inhibitor of nitric oxide synthase (NOS) (Nω-nitro-L-arginine) did not change the increased contractile response to phenylephrine due to microtubule depolymerization suggesting that this phenomenon is not mediated by endothelium dependent relaxation.

Microtubule disruption modulates the Rho-kinase pathway in vascular smooth muscle

Microtubules constitute one of the main cytoskeletal components in eukaryotic cells. Recent studies have shown that microtubule disruption induced significant vasoconstriction or enhanced agonist-induced contraction in vascular smooth muscle. However, the underlying mechanisms are not clear. We hypothesize that microtubule disruption may affect contractile signaling in vascular smooth muscle and lead to the enhanced contraction. The present study demonstrates that both colchicine and nocodazole induced a small but sustained contraction (4–6% P0) in rat aortic rings. This microtubule disruption-induced contraction was abolished by co-treatment with either HA 1077 or Y-27632, both of which are relatively specific Rho-kinase inhibitors. However, co-treatment with ML-9, an inhibitor of myosin light chain kinase, (MLCK) did not have a significant effect on the colchicine-induced contraction. The enhanced KCl-induced contraction due to treatment with colchicine was also blocked by inhibition of Rho-kinase, but not by inhibition of MLCK. These results indicate that microtubule disruption modulates contractile signaling in vascular smooth muscle, mainly through the Rho-kinase pathway, but not MLCK. Interestingly, the colchicine-enhanced, phenylephrine-induced contraction was not completely blocked by inhibition of Rho-kinase suggesting that other signaling pathways might also be involved.

Cross bridge-dependent activation of contraction in cardiac myofibrils at low pH

Striated muscle contracts in the absence of calcium at low concentrations of MgATP ([MgATP]), and this has been termed rigor activation because rigor cross bridges attach and activate adjacent actin sites. This process is well characterized in skeletal muscle but not in cardiac muscle. Rigor cross bridges are also thought to increase calcium binding to troponin C and play a synergistic role in activation. We tested the hypothesis that cross bridge-dependent activation results in an increase in contractile activity at normal and low pH values. Myofibrillar ATPase activity was measured as a function of pCa and [MgATP] at pH 7.0, and the data showed that, at pCa values of ≥5.5, there was a biphasic relationship between activity and [MgATP]. Peak activity occurred at 10-50 μM MgATP, and [MgATP] for peak activity was lower with increased pCa. The ATPase activity of rat cardiac myofibrils as a function of [MgATP] at a pCa of 9.0 was measured at several pH levels (pH 5.4-7.0). The ATPase activity as a function of [MgATP] was biphasic with a maximum at 8-10 μM MgATP. Lower pH did not result in a substantial decrease in myofibrillar ATPase activity even at pH 5.4. The extent of shortening, as measured by Z-line spacing, was greatest at 8 μM MgATP and less at both lower and higher [MgATP], and this response was observed at all pH levels. These studies suggest that the peak ATPase activity associated with low [MgATP] was coupled to sarcomere shortening. These results support the hypothesis that cross bridge-dependent activation of contraction may be responsible for contracture in the ischemic heart.

The proteome of normal pancreatic juice

Objectives The aims of this study were to characterize the proteome of normal pancreatic juice, to analyze the effect of secretin on the normal proteome, and to compare these results with published data from patients with pancreatic cancer. Methods Paired pancreatic fluid specimens (before and after intravenous secretin stimulation) were obtained during endoscopic pancreatography from 3 patients without significant pancreatic pathology. Proteins were identified and quantified by mass spectrometry-based protein quantification technology. The human RefSeq (NCBI) database was used to compare the data in samples from patients without pancreatic disease with published data from 3 patients with pancreatic cancer. Results A total of 285 proteins were identified in normal pancreatic juice. Ninety had sufficient amino acid sequences identified to characterize the protein with a high level of confidence. All 90 proteins were present before and after secretin administration but with altered relative concentrations, usually by 1 to 2 folds, after stimulation. Comparison with 170 published pancreatic cancer proteins yielded an overlap of only 42 proteins. Conclusions Normal pancreatic juice contains multiple proteins related to many biological processes. Secretin alters the concentration but not the spectrum of these proteins. The pancreatic juice proteome of patients without pancreatic disease and that of patients with pancreatic cancer differ markedly.

Increased in vivo levels of neurotransmitters to trigeminal motoneurons: Effects on craniofacial bone and TMJ

The results of chronic, in vivo delivery of excitatory and inhibitory neurotransmitter substances upon the craniofacial skeleton are of ongoing interest to clinician and basic scientist alike. Our purpose was to document and compare the effects of biodegradable glycine, glutamate, and thyrotropin‐releasing hormone (TRH) microspheres upon the craniofacial skeleton and TMJ of actively growing rats. Glycine, glutamate, TRH, and blank microspheres were stereotactically implanted in proximity to motoneurons within the trigeminal motor nucleus in order to test the following null hypotheses: (1) neurotransmitter microspheres implanted near trigeminal motoneurons of growing rats have no significant effect on the craniofacial skeleton and temporomandibular joints of implanted animals, and (2) there are no significant differences between the relative effects of glutamate, TRH (excitatory to trigeminal motoneurons), and glycine (inhibitory to trigeminal motoneurons) implants upon the craniofacial skeleton and temporomandibular joint. Fifty male Sprague‐Dawley rats underwent stereotactic neurosurgery at 35 days; five rats each were killed at 14 and 21 days postoperative for data collection and comparison between glycine‐, glutamate‐, TRH‐, blank‐microsphere, and sham‐surgery rats. Glycine rats had significantly (P ≤ 0.05, 0.01) smaller implant‐side cranial dimensions and mandibular condyles, all glycine rats showed increased gracility of implant‐side bones, and deviation of their facial skeleton away from the implant‐side; this was in contrast to the generally larger implant‐side bony structures in both glutamate and TRH rats. The two null hypotheses were both rejected. Due to their inhibitory and excitatory effects upon trigeminal motoneurons, masticatory muscles, and their neuromuscular generation of biomechanical forces that affect bone, the neurotransmitter substances glycine, glutamate, and TRH appear to play an important role in the growth and development of the mammalian craniofacial skeleton and TMJ. Anat Rec 258:369–383, 2000.

Natural history of implantable cardioverter-defibrillator implanted at or after the age of 70 years in a veteran population a single center study

Background The median age of patients in major Implantable Cardioverter-defibrillator (ICD)trials (MUSTT, MADIT-I, MADIT-II, and SCD-HeFT) was 63-67 years; with only 11% ≥70 years. There is little follow-up data on patients over 70 years of age who received an ICD for primary/secondary prevention of sudden cardiac death, particularly for veterans. Objective The aim of this study was to study the natural history of ICD implantation for veterans over 70 years of age. Methods We retrospectively reviewed single center ICD data in 216 patients with a mean age at implantation 76 ± 4 years. The ICD indication was primary prevention in 161 patients and secondary prevention in 55 patients. The ICD indication was unavailable in 4 patients. Results Mean duration of follow up was 1686 ± 1244 days during which 114 (52%) patients died. Of these, 31% died without receiving any appropriate ICD therapy. Overall, 60/216 (28%) received appropriate therapy and 28/216 (13%) received inappropriate therapy. Patients who had ICD implantation for secondary prophylaxis had statistically more (p= 0.02) appropriate therapies compared to patients who had ICD implantation for primary prevention. Indication for implantation and hypertension predicted appropriate therapy, while age at the time of implantation and presence of atrial fibrillation predicted inappropriate therapies. Overall, 7.7% had device related complications. Conclusions Although 28% septuagenarians in this study received appropriate ICD therapy, they had high rates of mortality, inappropriate therapy, and device complications. ICD implantation in the elderly merits individualized consideration, with higher benefit for secondary prevention.