Effect of outlet obstruction on pyruvate metabolism of the rabbit urinary bladder

Bladder function is dependent upon cellular metabolism of substrates and the adequate generation of high-energy phosphate compounds. Partial outlet obstruction induces a marked decrease in bladder function which is associated with a significant decrease in the oxidative metabolism of glucose.The current investigation was designed to determine whether the time course of the decrease in mitochondrial oxidation in the hypertrophied urinary bladder is similar to the time course of the contractile dysfunction observed. In these studies we determined: 1) the rate of ¹⁴C-pyruvate metabolism to ¹⁴CO₂ in control and obstructed tissue (1, 3, 5 and 7 days), and 2) the mitochondrial enzymatic activities of malate dehydrogenase and citrate synthase.The results can be summarized as follows: 1) The rate of pyruvate metabolism decreases by over 50% within one day following partial outlet obstruction, and remains at this level for the seven day period of study. 2) Kinetic analysis demonstrates that the change in enzymatic activity is related to a decrease in Vmax; the K_d for pyruvate is similar for control and after all time periods of obstruction. 3) The enzymatic activity of malate dehydrogenase and citrate synthase is reduced by over 50% within one day following partial obstruction, and remains at this level throughout the 7 day study period. These metabolic results correlate in time and duration with the decreased ability of the bladder to empty following partial outlet obstruction.     

Cardiovascular adaptations to mechanical overload

The cardiac changes resulting from mechanical overload of the left ventricle have been well documented and a variety of compensatory mechanisms described. These include a decrease in maximum velocity (V₀) of shortening in the absence of reduction in active tension (P₀), and a reversible decrease in myofibrillar adenosine triphosphatase activity resulting from isoenzymic shift from, predominantly, a form of myosin with high ATPase activity (V₁) to another with low (V₃). The thermodynamic advantage of the transition is the hypertrophied muscle possesses a more energy-efficient form of contraction. These reversible transitions resulted from altered gene expression of isoenzymic forms of myosin heavy chain. It must be borne in mind that the adaptational modifications just described appear to occur only in smaller animals such as the rat, that possesses several myosin isozymes. In large mammals it is mainly the V₃ form of myosin that is present, which does not change with altered contractile state. Responses of the large arteries to hypertension have been poorly studied. This is surprising when one recalls that degenerative disease of such vessels, that include the aorta, carotids and ileo-femoral arteries is almost an obligatory concomitant of hypertension. Such studies as have been carried out indicate that hyperplasia is specific for abdominal aortic stenosis while hypertrophy is found in aortic smooth muscle in rats with systemic hypertension. Mechanically, an increase in V₀ with no change in P₀ have been reported; an increase in myofibrillar ATPase activity was also reported. Though two myosin heavy chain isozymes have been found in aortic smooth muscle densitometry did not reveal any difference in distribution between tissues from control and hypertensive rats. The cause of the increased ATPase activity must be in increased phosphorylation of the muscles' 20,000 dalton light chain.     

Alternative splicing and cycling kinetics of myosin change during hypertrophy of human smooth muscle cells

We investigated in vivo expression of myosin heavy chain (MHC) isoforms, 17 kDa myosin light chain (MLC₁₇), and phosphorylation of the 20 kDa MLC (MLC₂₀) as well as mechanical performance of chemically skinned fibers of normal and hypertrophied smooth muscle (SM) of human myometrium. According to their immunological reactivity, we identified three MHC isoenzymes in the human myometrium: two SM-MHC (SM1 with 204 kDa and SM2 with 200 kDa), and one non-muscle specific MHC (NM with 196 kDa). No cross-reactivity was detected with an antibody raised against a peptide corresponding to a seven amino acid insert at the 25K/50K junction of the myosin head (a-25K/50K) in both normal and hypertrophied myometrium. In contrast, SM-MHC of human myomatous tissue strongly reacted with a-25K/50K. Expression of SM1/SM2/NM (%) in normal myometrium was 31.7/34.7/33.6 and 35.1/40.9/24 in hypertrophied myometrium. The increased SM2 and decreased NM expression in the hypertrophied state was statistically significant (P < 0.05). MHC isoform distribution in myomatous tissue was similar to normal myometrium (35.3/35.3/29.4). In vivo expression of MLC_17a increased from 25.5% in normal to 44.2% in hypertrophied (P < 0.001) myometrium. Phosphorylation levels of MLC₂₀ upon maximal Ca²⁰-calmodulin activation of skinned myometrial fibers were the same in normal and hypertrophied myometrial fibers. Maximal force of isometric contraction of skinned fibers (pCa 4.5, slack-length) was 2.85 mN/mm² and 5.6 mN/mm² in the normal and hypertrophied state, respectively (P < 0.001). Apparent maximal shortening velocity (Vmax_app, extrapolated from the force-velocity relation) of myometrium rose from 0.13 muscle length s ¹ (ML/s) in normal to 0.24 ML/s in hypertrophied fibers (P < 0.001). J. Cell. Biochem, 64:171–181. © 1997 Wiley-Liss, Inc.     

Assembly of smooth muscle myosin by the 38k protein, a homologue of a subunit of pre-mRNA splicing factor-2

Smooth muscle myosin in the dephosphorylated state does not form filaments in vitro. However, thick filaments, which are composed of myosin and myosin-binding protein(s), persist in smooth muscle cells, even if myosin is subjected to the phosphorylation- dephosphorylation cycle. The characterization of telokin as a myosin-assembling protein successfully explained the discrepancy. However, smooth muscle cells that are devoid of telokin have been observed. We expected to find another ubiquitous protein with a similar role, and attempted to purify it from chicken gizzard. The 38k protein bound to both phosphorylated and dephosphorylated myosin to a similar extent. The effect of the myosin-binding activity was to assemble dephosphorylated myosin into filaments, although it had no effect on the phosphorylated myosin. The 38k protein bound to myosin with both COOH-terminal 20 and NH(2)-terminal 28 residues of the 38k protein being essential for myosin binding. The amino acid sequence of the 38k protein was not homologous to telokin, but to human p32, which was originally found in nuclei as a subunit of pre-mRNA splicing factor-2. Western blotting showed that the protein was expressed in various smooth muscles. Immunofluorescence microscopy with cultured smooth muscle cells revealed colocalization of the 38k protein with myosin and with other cytoskeletal elements. The absence of nuclear immunostaining was discussed in relation to smooth muscle differentiation.     

Smooth muscle-type myosin heavy chain isoforms in bovine smooth muscle and non-muscle tissues

Summary— The distribution of smooth muscle (SM)-type myosin heavy chain isoforms in several bovine muscular and non-muscular (NM) tissues was evaluated by immunofluorescence tests using monoclonal antibodies SM-E7, reactive with 204 (SM1) and 200 (SM2) kDa isoforms, and SM-F11, specific for SM2 isoform. SM-E7 reacted equally with vascular, respiratory and intestinal SM tissues, whereas SM-F11 stained heterogeneously SM cells in the various muscular systems examined and in some peculiar tissues was unreactive (perisinusoidal cells of hepatic lobule, pulmonary interstitial cells and intestinal muscularis mucosae) or uniquely reactive (nerve cells). On the whole, our findings indicate that SM1 and SM2 isoforms are unequally distributed at the cellular level in various SM and NM tissues and support previous results obtained with tissue extracts and electrophoretic procedures.     

平滑肌肌球蛋白轻链激酶的非激酶作用及对ATP 酶活性的调节

肌球蛋白轻链激酶（myosin light chain kinase, MLCK）具有激酶活性和非激酶活性,在平滑肌收缩过程中起着关键酶调控的作用.为探寻MLCK的非激酶活性区域对MLCK活性的影响,以进一步阐明MLCK的非激酶活性在调节平滑肌收缩过程中的分子机制.采用PCR技术构建MLCK部分氨基酸缺失的重组表达载体pGEX-F6-5/D,经大肠杆菌表达得到可溶性GST融合蛋白,利用SDS-PAGE及Western 印迹鉴定表达的MLCK在细胞中的分布,结果还显示,提取液的上清和沉淀中均有MLCK片段的表达.运用亲和层析技术分离并纯化删除前、后表达的MLCK片段（F6.5和F6-5/D）,经谷胱甘肽琼脂糖凝胶 4B 纯化,SDS-PAGE鉴定显示为单一表达条带.应用EnzChek磷分析试剂盒和孔雀绿两种方法分别测定不同浓度的MLCK对非磷酸化肌球蛋白Mg²⁺-ATP酶活性的影响.两种MLCK的片段均具有激活ATP酶活性的作用,并随MLCK浓度的增加,酶的活性增加.比较删除前后不同MLCK片段对ATP酶活性的影响结果显示,删除MLCK片段1002位丙氨酸至1019位亮氨酸后,对ATP酶的激活作用较删除前明显降低,表明删除的部分氨基酸序列为MLCK非激酶活性所必需的区域.利用电镜技术观察到MLCK片段（F6.5）使非磷酸化肌球蛋白构象发生明显的变化.加入MLCK片段后肌球蛋白的构象由非活性型转化为活性型,并且MLCK片段还具有促进肌球蛋白单体形成肌丝的作用.     

Creatine kinase activity in normal and hypertrophied rabbit urinary bladder tissue (following partial outlet obstruction)

The urinary bladder depends on intracellular ATP to support a number of essential intracellular processes including contraction. The concentration of ATP is maintained by mitochondrial oxidative phosphorylation, cytosolic glycolysis and the cytosolic activity of creatine kinase, the enzyme that catalysis the rapid transfer of a phosphate from creatine phosphate (CP) to ADP resulting in the formation of ATP.Prior studies in this lab and others have demonstrated that mitochondrial respiration is significantly lower in hypertrophied bladder tissue (induced by partial outlet obstruction of the white New Zealand Rabbit). In addition to decreased mitochondrial respiration, there are significant increases in glycolysis and lactic acid formation in the hypertrophied tissue.In view of the increased glycolysis and decreased mitochondrial function in the hypertrophied tissue, and the importance in creatine kinase in maintaining cytosolic levels of ATP, the current study was designed to determine if outlet obstruction induces any changes in the activity of creatine kinase.The following is a summary of the results: 1) The bladder mass increased from 2.2 ± 0.2 gm to 11.5 ±1.6 gm at 7 days following outlet obstruction. 2) The intracellular concentrations of both ATP and CP were significantly reduced in the bladder tissue following 7 days of obstruction. 3) The percent of protein (per tissue mass) was significantly lower in the obstructed bladders, although the percent of soluble protein was similar. 4) Creatine kinase activity of control bladders showed linear kinetics with a V_max = 1120 nmoles/mg protein/4 min and K_m = 147 µM CP. 2) The creatine kinase activity of obstructed bladders also displayed linear kinetics with a V_max = 1125 nmoles/mg protein/4 min tissue, and K_m = 276 µM CP.These studies demonstrate that whereas both control and obstructed bladders have virtually identical maximum creatine kinase activities, the K_m for the obstructed tissue is significantly higher than the K_m for the control tissue. This may indicate that under cellular conditions (at sub-maximum substrate concentrations), the creatine kinase activity of the obstructed bladders may be significantly lower than the activity of the control bladders. In addition, the reduced tissue concentrations of ATP and CP would certainly be consistent with the reduced functional response to bethanechol and field stimulation.     

Effects of exogenous growth hormone on skeletal muscle of young female rats

We examined the effects of exogenous growth hormone (GH) treatment on the soleus and rectus femoris muscles of young female rats. Rat GH (1.8 IU/mg) was administered for 3 weeks by subcutaneous injection, twice a day, at doses of 0.5, 0.6, and 0.8 mg/day during the 1st, 2nd, and 3rd week, respectively. Final body weight, as well as wet and dry weight, of the soleus and rectus femoris muscles were significantly greater in the GH-treated group, compared to controls. Muscle weight to body weight ratios did not differ between the two groups. The fiber type composition of the soleus muscle was determined by histochemical staining for myosin ATPase activity. No statistically significant difference was found between the GH-treated and the control groups in the percentages of fiber types. However, GH treatment significantly increased the cross-sectional area of type II fibers of the soleus muscle. These results suggest that, in young female rats, acceleration of body weight gain by homologous GH administration is accompanied by a proportional hypertrophy of skeletal muscle mass. Increased muscle mass is due to hypertrophy of muscle fibers. Type II muscle fibers appear to be more sensitive to GH stimulation.