Short chain fatty acids stimulate feline colonic smooth muscle contraction

The effect of short chain fatty acids (SCFA) on feline colonic smooth muscle contraction was evaluated in vitro. Colonic tissue was obtained from seven healthy male and female adult cats and seven healthy male and female kittens. Longitudinal and circular colonic smooth muscle strips from proximal and distal colon were incubated with SCFA (acetate, butyrate and propionate; 1-100mM). SCFA-induced contractions were compared to responses obtained using maximal concentrations (10(-4)M) of acetylcholine (ACh). The calcium dependence of the SCFA response was investigated by incubating with nifedipine (1 microM) or verapamil (1 microM). Acetate, butyrate and propionate elicited isometric stress responses (0.25-1.98 x 10(4)N/m(2)) in longitudinal, but not circular, smooth muscle from both the proximal and distal colon of adult cats. Maximal responses were attained at 50 and 100mM SCFA. Maximal butyrate and propionate responses were 29 and 19% of the maximal ACh response (10(-4)M), respectively. Acetate was least effective in stimulating contractile responses. Nifedipine and verapamil abolished all responses. Contractile responses in kittens were similar to those observed in adult cats, but were smaller in amplitude.Results of these studies have shown that SCFA stimulate longitudinal colonic smooth muscle contractions in kittens and adult cats in vitro. These SCFA-induced contractions involve activation of calcium influx. These in vitro findings may account for some of the effects of dietary fiber on feline colonic motility in vivo.     

猪骨骼肌肌球蛋白轻链激酶单克隆抗体制备及鉴定

为制备猪骨骼肌肌球蛋白轻链激酶(skMLCK)的特异性单克隆抗体,设计合成多肽并与载体蛋白偶联,经细胞融合、培养基筛选、阳性细胞的检测与单克隆化后,获得3株能稳定分泌抗skMLCK单克隆抗体的杂交瘤细胞株;种植BALB/c小鼠腹腔,收集腹水,纯化后得到skMLCK的单克隆抗体。分别应用高效液相色谱、质谱和Western blot对纯化单克隆抗体进行抗体纯度、效价和敏感性检测。结果显示:获得的猪skMLCK单克隆抗体,能特异性识别skMLCK纯化蛋白、猪肌肉组织的内源性蛋白。本试验成功制备了能特异性结合猪skMLCK的单克隆抗体,这为今后进一步研究探讨skMLCK对猪肌肉发育的影响提供物质基础。     

Effect of bombesin and substance P on the smooth muscle of the chicken crop

The effect of bombesin and of substance P was investigated in smooth muscle strips of the chicken crop. Bombesin in picomolar concentration (0.1×10^-12–5×10^-12mol/l) caused a concentration-related contraction of the muscle strips. Substance P in nanomolar concentration (0.1×10^-9–10×10^-9mol/l) was effective in the same manner. Tetrodotoxin (2×10^-7mol/l) did not influence the contraction responses to either bombesin or substance P. The excitatory effect of bombesin and of substance P did not follow activation of cholinergic receptors since their effect on the crop smooth muscle was not antagonized by atropine (10^-4mol/l) or by hexamethonium (10^-4mol/l). Strips stored for 24 hours in the Tyrode's solution at 4°C without a supply of oxygen maintained their full sensitivity to bombesin and to substance P. These results are consistent with a direct action of bombesin and substance P on the crop smooth muscle.     

Similarities of cow mammary gland cytosolic 21-kDa protein, the substrate for protein kinase C, to the 20-kDa myosin light chain from smooth muscle

In the cytosol of cow mammary gland, several proteins are phosphorylated in the presence of the protein kinase C (PKC) cofactors 1-oleoyl-2-acetyl-sn-glycerol (OAG), phosphatidylserine (PS) and Ca2+. Of the substrates, the 21-kDa protein is inferred to be a 20-kDa regulatory myosin light chain (MLC20) from smooth muscle because of its molecular mass, its distribution in the cytosol, its association with melittin and sphingosine (the PKC modulators), and phosphorylation by PKC as well as by Ca2+/calmodulin-dependent myosin light chain kinase (MLCK). The present study was undertaken to examine whether the 21-kDa protein could be identified as MLC20, by adding cow uterine MLC20 to the reaction mixture containing cytosol with or without the PKC cofactors and/or calmodulin. In the absence of MLC20, the 21-kDa protein was phosphorylated when the PKC cofactors and calmodulin were added to the reaction mixture. Phosphorylation of the 21-kDa protein was inhibited by melittin or sphingosine, and the inhibition was reversed by PS, but not by calmodulin. When MLC20 was included in the reaction mixture, it was phosphorylated in the presence of the PKC cofactors, and the phosphorylated MLC20 band overlapped that of the 21-kDa protein. The indistinguishably overlapped band of the two proteins was inhibited by melittin and by sphingosine, and their inhibition was reversed by PS, not by calmodulin. It is suggested that the 21-kDa protein is the smooth muscle MLC20 and also that the 21-kDa MLC20 is phosphorylated by PKC, but not by MLCK.     

Relationships between tropomyosin and myosin heavy chain isoforms in bovine skeletal muscle

The composition of tropomyosin (TPM) and myosin heavy chain (MyHC) isoforms was analyzed in 10 physiologically different bovine muscles ( masseter , diaphragm, tongue, semispinalis, pectoralis profundus , biceps femoris, psoas major , semimembranosus, longissimus thoracis and semitendinosus ) to clarify the relationships between TPM and MyHC isoforms in different muscle fiber types. The content of TPM1 and TPM3 was different in muscles according to their function in muscle contraction, although the content of TPM2 was constantly about 50% of the total TPM in all muscles. The content of TPM1 was higher in semimembranosus , longissimus thoracis and semitendinosus, while that of TPM3 was higher in masseter and diaphragm. The high positive correlation between MyHC-slow content and TPM3 content ( r  = 0.92) suggested a coexpression of TPM3 and MyHC-slow isoforms in a muscle fiber. MyHC-slow and TPM3 were expressed at the same level in masseter and diaphragm, whereas there was more TPM3 than MyHC-slow in tongue and semispinalis , so it appears that the excess TPM3 in tongue and semispinalis is expressed with other MyHC isoforms. MyHC-2a was the only fast type isoform expressed in tongue and semispinalis . Therefore, the excess TPM3 was composed of myofibrils with MyHC-2a. The results suggested that a fiber expressing MyHC-2a would be regulated delicately by changing the TPM isoform types.     

Response of equine airway smooth muscle to acetylcholine and electrical stimulation in vitro

Smooth muscle strips from the midcervical portion of the trachea and bronchial smooth muscle strips from third-generation airways of horses were placed in tissue baths, and isometric contractile force was measured. Active force was measured in response to electrical stimulation and exogenous acetylcholine. Square-wave electrical stimuli were applied at various voltages (10, 12, 15, 18, 20, 25 V), frequencies (3, 5, 10, 15, 20, 25, 30 Hz), and pulse durations (0.2, 0.5, 1.0, 1.5, 2.0 ms). Isometric contractile force increased as voltage, frequency, and pulse duration increased. Maximal contractile response to electrical stimulation was obtained at 18 V, 25 Hz, and 0.5 ms. Atropine (10(-6)M) or tetrodotoxin (3 x 10(-6)M) blocked the contraction, indicating that the contractile response was attributable to the release of neurotransmitter from cholinergic nerves. Cumulative concentration-response curves to acetylcholine (10(-9)M through 10(-4)M) were determined. Isometric contractile force increased as acetylcholine concentration increased. There was a significant (P less than 0.05) difference in the 50% effective dose for acetylcholine in tracheal smooth muscle and bronchial smooth muscle. The mean (+/- SD) contractile response to maximal electrical stimulus was 89% (+/- 7.4%) of that in response to 10(-4)M acetylcholine in tracheal smooth muscle and was 68% (+/- 10.4%) of the response to 10(-4)M acetylcholine in bronchial smooth muscle.     

Endometrial stromal sarcoma with smooth muscle and glandular differentiation of the feline uterus

The intra-abdominal tumor developing in the uterus and lung of a domestic Shorthair cat was examined histopathologically and immunohistochemically. The tumor showed a proliferation of both endometrial stromal and smooth muscle cells accompanied by prominent vasculature. There were well-differentiated endometrial glands, and tubuli made up a monolayer of eosinophilic cuboidal epithelium. Immunohistochemically, the spindle-shaped cells and half of the stromal-like cells reacted to caldesmon and desmin antibodies. The neoplastic epithelium expressed AE1/AE3 cytokeratin. Feline endometrial stromal tumor has, to the best of our knowledge, not been reported previously and has smooth muscle and glandular components that are a unique variant to the human counterpart.     

Spontaneous contractions of intestinal smooth muscle re-aggregates from the new-born rat triggered by thromboxane A2

Isolated smooth muscle cells from the small intestine of new-born rats were prepared by enzymatic digestion. These cells re-aggregate after 1 day in culture to clusters. The re-aggregates show spontaneous rhythmical contractions at 37 degrees C with a frequency (13.1 +/- 0.8 min-1, n = 49), which is similar to that of the intact smooth muscle layer. The cholinergic agonist carbachol (5 x 10(-5) mol l-1) caused an increase in the frequency of the spontaneous contractions often ending in a permanent contraction. A similar effect was achieved with the thromboxane A2 (TXA2) agonist, U-46619 (10(-5) mol l-1). In contrast, both the TXA2 receptor blocker, Bay u3405 (5 x 10(-4) mol l-1), as well as the Ca2+ channel blocker, verapamil (5 x 10(-5) mol l-1), suppressed the spontaneous contractions. The observed contractility was insensitive against the neuronal blocker tetrodotoxin (10(-6) mol l-1). These analyses of video images were supported by the measurement of relative changes in the intracellular Ca2+ concentration with the Ca(2+)-sensitive dye, fura-2. Spontaneous contractions were paralleled by spikes in the intracellular Ca2+ concentration, which were abolished by Bay u3405, but stimulated by U-46619 or carbachol. In summary, these results obtained at re-aggregates of intestinal smooth muscle cells support the hypothesis of a role of TXA2 in the generation of spontaneous intestinal smooth muscle contractions in vitro.     

Inhibition of spontaneous smooth muscle contractions in rat and rabbit intestine by blockers of the thromboxane A2 pathway

The effect of inhibitors of the thromboxane A2 pathway on spontaneous contractions of intestinal smooth muscle preparations was studied. The thromboxane A2 antagonists Bay u3405, SK and F 88046 and KW-3635 concentration-dependently inhibited both the amplitude and the frequency of spontaneous contractions of the longitudinal muscle from the rat proximal colon. A concentration-dependent inhibition of the myogenic contractions was also observed with the thromboxane A2 synthase inhibitor U-51605, and with the combined cyclooxygenase/lipoxygenase inhibitor nordihydroguaiaretic acid, whereas indomethacin, a pure cyclooxygenase inhibitor, was ineffective. None of these inhibitors affected the contractile response evoked by the cholinergic agonist carbachol, excluding non-specific actions on intestinal motility. A similar response was observed for the rabbit jejunum, which, in contrast to the rat colon, exhibits more regular, high-frequency spontaneous contractions, which were inhibited by Bay u3405, SK and F 88046 and KW-3635 in a concentration-dependent manner, whereas the response to carbachol remained unaffected. These results suggest a role for thromboxane A2 in the generation and/or facilitation of spontaneous smooth muscle contractions in the gut.     

Effects of muscarinic receptor antagonists on acetylcholine-induced contractions of jejunal smooth muscle in horses

This study investigated the effects of a muscarinic type 1 (M(1)), 2 (M(2)), and 3 (M(3)) antagonists (4-DAMP, pirenzepine, and methoctramine, respectively) on acetylcholine (Ach)-induced contractions of longitudinal jejunal muscle strips of horses. Strips were irrigated with Krebs-Henseleit solution gassed with 95% O(2) and 5% CO(2), and the developed tension in response to Ach was recorded before and after incubation with increasing concentrations of 4-DAMP (10(-8)-10(-6) M), pirenzepine (10(-6)-10(-4) M), and methoctramine (10(-5)-10(-3) M). When competitive antagonism was characterized, the affinity constant (pA(2)) was calculated by Schild plots. A parallel rightward shift in the concentration-response curves was observed after 4-DAMP and pirenzepine. Methoctramine presented a dual effect on the concentration-response curves: lower concentrations induced a parallel rightward shift without altering the maximum intensity of contraction (E(max)), while the highest concentration increased slope of the concentration-response curve and increased E(max). The pA(2) for 4-DAMP and pirenzepine was 9.18 and 7.13, respectively. Acetylcholine-induced contractions of longitudinal jejunal smooth muscle are mediated mainly via M(3) receptors. The complex role of M(2) receptors in jejunal smooth muscle contractions was evident because methoctramine potentiated the contractile response to higher doses of Ach.     

Myosins 1 and 6, myosin light chain kinase,actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes

Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.     

Changes in muscle fiber type and expression of mRNA of myosin heavy chain isoforms in porcine muscle during pre‐ and postnatal development

The purpose of this study is to elucidate developmental changes in muscle fiber type in the pig during pre‐ and postnatal development. For this purpose, we performed a histochemical analysis for myosin adenosine triphosphatase activity to assess muscle fiber type and determined abundances of messenger RNA (mRNA) of myosin heavy chain (MHC) isoforms. Samples of Longissimus dorsi (LD) muscle were taken from fetuses on day 90 of the fetal stage. Further, samples of LD, Rhomboideus and Biceps femoris (B. femoris) muscles were taken from pigs when they were 1, 12, 26, 45 or 75 days old. Expression of MHC 2b mRNA in the LD and the B. femoris muscles rapidly and considerably increased from the late fetal stage to the early postnatal stage and this increase was associated with the development of type 2b fibers at least in the LD muscle. As shown by the rapid and considerable changes in expression of MHC 2b mRNA, it seems that a certain plasticity of muscle fiber type still remains in this developmental stage.     

Identification of Pentatrichomonas hominis in feline fecal samples by polymerase chain reaction assay

Pentatrichomonas hominis is considered to be a commensal protozoan of the vertebrate digestive tract. On the basis of light microscopic examination of feces, some investigators presumptively identified P. hominis as a causative agent of feline diarrhea. However, molecular identification of P. hominis infection in the cat has not been reported. Another trichomonad, Tritrichomonas foetus, is recognized as an intestinal pathogen in cats and often presumptively diagnosed on the basis of the presence of trichomonads in diarrheic feces. It is of importance to determine if cats are natural hosts for P. hominis, as the presence of this organism could result in inaccurate assumption of T. foetus infection. In this study, we used a species-specific PCR assay to identify P. hominis 18S rRNA genes in fecal samples collected from a convenience population of cats in which a high prevalence of T. foetus infection had been previously identified (cat show) or suspected (submitted for T. foetus diagnostic testing). The prevalence of T. foetus infection in these samples was 31% and 28.6%, respectively. P. hominis infection was identified by PCR of DNA extracted from feces of five cats (1.9% and 2.1% of fecal samples, respectively). All cats in which P. hominis was identified were also infected with T. foetus. PCR identification of P. hominis infection in the cat should facilitate future studies to determine the pathogenicity of this species and enable differentiation of P. hominis from other known or as-yet unidentified species of trichomonads that may infect cats.     

Influence of halothane genotype and body-weight on myosin heavy chain composition in pig muscle as related to meat quality

In an effort to understand the relationship between muscle fiber type, live weight, genotype, and PSE development, enzyme-linked immunosorbent analyses were used to evaluate myosin heavy chain (MyHC) isoform content in the longissimus muscle of pigs differing in halothane gene status (nn, homozygous mutant; Nn, heterozygous; NN, homozygous normal) that were slaughtered at three different weights (100, 120 and 140 kg). Pigs carrying the n gene (Nn and nn) exhibited more IIB MyHC and less slow type I MyHC than those pigs free of the n gene, while NN pigs had greater amounts of IIAX MyHC. The relative abundance of IIB and IIAX MyHC in muscle of all pigs studied was strongly negatively correlated (r=−0.834). Heavier pigs (140 kg) had the greatest amounts of slow and IIA MyHC. Across all genotypes, the relative abundance of IIB MyHC and muscle pH at 45 min postexsanguination (pH45) was negatively correlated (r=−0.418). In addition, the relative amount of slow was positively correlated with pH45 (r=0.386). Because muscle of homozygous nn positive pigs exhibited similar IIB/slow MyHC ratios to that of heterozygous Nn pigs, yet less desirable pH45 values and ultimate meat quality scores argues against a role of MyHC content per se in contributing to PSE development. However, these data do not preclude that those pigs with greater amounts of IIB MyHC are more ‘susceptible’ to adverse pork quality development than those pigs with less IIB MyHC.     

Lack of detection of feline leukemia and feline sarcoma viruses in diffuse iris melanomas of cats by immunohistochemistry and polymerase chain reaction.

Diffuse iris melanoma was confirmed by light-microscopic examination in 10 formalin-fixed, paraffin-embedded globes from 10 cats. To determine if feline leukemia virus or a replication defective feline leukemia virus, feline sarcoma virus, was present in these anterior uveal melanomas, immunohistochemistry and polymerase chain reaction for feline leukemia virus were utilized. Immunohistochemical staining for feline leukemia virus glycoprotein 70 was performed on all 10 tumors using an avidin-biotin complex technique. The DNA was extracted from each specimen and a 166-base pair region of the feline leukemia virus long terminal repeat was targeted by polymerase chain reaction. Immunohistochemical staining for feline leukemia virus glycoprotein 70 and polymerase chain reaction amplification of a feline leukemia virus long terminal repeat region were negative in all cases. Feline leukemia virus/feline sarcoma virus was not detected in any neoplasms and therefore was unlikely to play a role in the tumorigenesis of these feline diffuse iris melanomas.     

Mechanisms of NO-resistant relaxation induced by acetylcholine in rabbit renal arteries

The effects of K+ channel blockers and P2Y receptor agonist/antagonist on the vasorelaxation mediated by endothelium-derived hyperpolarizing factor (EDHF) were investigated in the rabbit renal artery. Acetylcholine (ACh, 1 nM-10 microM) induced endothelium-dependent relaxation of arterial rings precontracted with norepinephrine (NE, 1 microM) in a concentration-dependent manner. NG-nitro-L-arginine (L-NAME. 0.1 mM), an inhibitor of NO synthase, partially inhibited the ACh-induced endothelium-dependent relaxation. The ACh-induced relaxation was only partially inhibited by L-NAME whereas combined addition of L-NAME and 30 mM KCl completely inhibited the relaxation. The ACh-induced relaxation observed in the presence of L-NAME was significantly reduced by a combination of iberiotoxin (0.1 microM) and apamin (1 microM), and almost completely blocked by 4-aminopyridine (5 mM). The ACh-induced relaxation was antagonized by P2Y receptor antagonist, cibacron blue (10 and 100 microM) in a concentration-dependent manner. Furthermore, ADPbetaS, a potent P2Y agonist, induced the endothelium-dependent relaxation, and this relaxation was markedly reduced by either the combination of iberiotoxin and apamin or by cibacron blue alone. In conclusion, ACh may activate the release of ATP from endothelial cells which in turn activates a P2Y receptor on the endothelial cells followed by a release of EDHF, resulting in a vasorelaxation via a mechanism that involves activation of both the voltage-gated K+ channels and the Ca2+-activated K+ channels. EY WORDS: ATP, K+ channel, rabbit renal artery.     

Proliferation of pulmonary artery smooth muscle cells in the development of ascites syndrome in broilers induced by low ambient temperature

Pulmonary vascular remodelling, mainly characterized by arterial medial thickening, is an important pathological feature of broiler ascites syndrome (AS). Since vascular smooth muscle cells (VSMC) form the major cellular component of arterial medial layer, we speculate that VSMC proliferation is one of the causes of pulmonary arterial medial thickening in ascitic broilers. Hence, the present study was designed to investigate the role of VSMC proliferation in pulmonary vascular remodelling in development of AS induced by low ambient temperature. Broilers in control group (22 +/- 1.5 degrees C) and low temperature group (11 +/- 2 degrees C) were sampled every week at 15-50 days of age. Proliferative indexes of VSMC in pulmonary arteries were assessed with proliferating cell nuclear antigen, and the relative medial thickness (RMT) and relative wall area (RWA), as indexes of pulmonary vascular remodelling, were examined by computer-image analysing system. The results showed that the high incidence (18.75%) of AS was induced by low temperature, and a significantly increased VSMC proliferation was observed in pulmonary arteries in the low temperature group at 22-50 days of age (P < 0.05). In addition, RMT and RWA in pulmonary arteries were significantly elevated in the low temperature group from 36 days of age (P < 0.05), indicating that pulmonary vascular remodelling occurred following VSMC proliferation in AS. Our data suggest that proliferation of VSMC may facilitate pulmonary vascular remodelling and have a pivotal role in AS induced by low ambient temperature.     

The use of immunohistochemistry and the polymerase chain reaction for detection of feline leukemia virus and feline sarcoma virus in six cases of feline ocular sarcoma

Ocular sarcoma was diagnosed by light microscopic examination in enucleated globes ( n  = 4), orbital tissue biopsy ( n  = 1) and ocular evisceration contents ( n  = 1) from six cats. To determine if feline leukemia virus (FeLV) or a replication-defective FeLV, feline sarcoma virus (FeSV), was present in these ocular sarcomas, immunohistochemistry (IHC) and polymerase chain reaction (PCR) for FeLV were utilized. Immunohistochemical staining for FeLV glycoprotein 70 (gp70) was performed on all six formalin-fixed, paraffin-embedded tumors using an avidin–biotin complex technique. DNA was extracted from each specimen and a 166 bp region of the FeLV long-terminal repeat (LTR) was amplified by PCR. All tumors were composed primarily of spindle cells; two neoplasms had PAS-positive basement membrane enveloping areas of spindle cells. All tumors involved the uvea and five of six tumors showed transcleral extension, one of which invaded the optic nerve. Immunohistochemical staining for FeLV gp 70 was negative. PCR to amplify a portion of the FeLV LTR was negative. Based on these findings of these limited number of cases, FeLV/FeSV may not play a role in the tumorigenesis of feline ocular sarcomas. However, additional tumors representing all morphological subtypes should be investigated for the presence of viral antigen and DNA. It is important to determine the etiology and pathogenesis of these malignant ocular sarcomas. If the cell of origin and pathogenesis involve ocular and lenticular injury, and FeLV/FeSV is not present, then the clinical management of cases of feline ocular trauma, uveitis and glaucoma may prevent the development of this tumor.