Distribution and properties of cAMP-dependent protein kinase isozymes in the myocardium of spontaneously hypertensive rat

cAMP-dependent protein kinase isozymes were isolated from the soluble fraction of cardiac tissue of 12 to 16-week spontaneously hypertensive rat (SHR) and Kyoto Wistar Normotensive rat (WKY). No differences were observed in the relative distribution and elution profile of isozyme I and II between SHR and WKY. In both types of rat isozyme I was the major enzyme comprising approximately 65% of total enzyme activity. Specific activity of cAMP-dependent protein kinase was significantly decreased in the soluble fraction as well as isozymes I and II of SHR as compared to WKY. V_max of cAMP stimulation was decreased for Types I and II isozymes of SHR as compared to WKY, with no differences in the K_a value (concentration of cAMP required for half maximal activation). K_m values for ATP and Mg²⁺ were not altered for isozymes I and II of SHR, but V_max was significantly decreased in SHR as compared to WKY. Thermostability and salt dissociation experiments did not reveal any differences between SHR and WKY isozymes I and II. These data would suggest that cAMP-dependent protein kinase activity could be decreased in SHR myocardium due to a reduction in the number of enzyme molecules.     

Architecture of myocardial cells in human cardiac ventricles with concentric and eccentric hypertrophy as demonstrated by quantitative scanning electron microscopy

Summary Scanning electron microscopy (SEM) was used to compare the shapes, size, and connections of cardiocytes in the midwall myofibers in the left ventricles of 5 normal hearts (266±16 g), 5 hearts with concentric hypertrophy (564±99 g) and 5 with eccentric hypertrophy (651±114 g), obtained at autopsy and fixed in formalin. In the myofibers from normal and hypertrophied hearts, intercalated discs demarcated cardiocytes which consisted of a cylindrical trunk and one or more series and/or lateral branches; cell connections at the intercalated discs had 6 common basic patterns. The length (L), width (W) and L/W ratio of the cells and the size and number of the series and lateral branches per cell were measured in 50 cells from each heart and averaged for comparison studies. The cells in the concentrically hypertrophied ventricles were much thicker than normal (33.0 ± 2.8 vs 18.2±1.4µm,P<0.01) but not significantly longer (81.2±9.5 vs 71.2±9.6µm, NS), so that the L/W ratio was greatly decreased (2.6±0.3 vs 4.1±0.7,P<0.01). The cells in the eccentrically hypertrophied ventricles were markedly thickened (25.9±2.4µm,P<0.01) and elongated (102.3±10.5µm,P<0.01), so that the L/W ratio (4.2±0.4, NS) remained the same as in normal hearts. In both types of hypertrophy, series and lateral branches were significantly thicker and longer than in normal hearts; the number of series branches per cell was also significantly increased. The number of lateral branches per cell did not differ between the normal and concentrically hypertrophied hearts (2.2±0.7 vs 2.3±0.6, NS), but it was decreased by approximately one-half in the eccentrically hypertrophied hearts (1.2±0.3,P<0.05). The potential significance of these differences in SEM findings of cardiocytes is discussed with special reference to the differences in the cause, anatomy, and pathophysiology of concentric and eccentric hypertrophy in adult human hearts.This work was supported in part by a Research Grant for Cardiovascular Diseases (62 C-4) from the Ministry of Health and Welfare and a Research Grant for the Pathogenesis and Prevention of Myocardial Cell Injury (# O1624006) from the Ministry of Education, Culture and Science, Japan.     

Ultrastructural changes during myocardial hypertrophy and its regression: Long-term effects of nifedipine in adult spontaneously hypertensive rats

Summary Nifedipine (20mg/kg/day) was given to 15-week-old spontaneously hypertensive rats for 20 weeks (SHR-N,n=8). Comparison was done with sex-matched 15-week-old SHR (SHR-15,n=7), untreated 35-week-old SHR (SHR-C,n=10), 15-week-old normotensive Wistar-Kyoto rats (WKY-15,n=15), and 35-week-old WKY (WKY-15,n=5). Light and electron microscopic data on the subepicardial, middle, and subendocardial layers and papillary muscles of the left ventricle were compared among the five rat groups. In SHR-N, blood pressure was significantly reduced by nifedipine, but was higher than in WKY-35 (199±11 mmHg vs 121±13mmHg). The left ventricular weight/body weight ratio was much lower in SHR-N than in SHR-C, and was even below the baseline value in SHR-15. In addition, cardiac myocyte diameter was much smaller in each myocardial layer of SHR-N than in SHR-C, and was similar to the findings in SHR-15, but still larger than in WKY-35. The interstitial area ratio was markedly reduced in SHR-N and did not differ from that in SHR-15 or even WKY-15, while capillary density was significantly greater than in SHR-C and comparable to that in WKY-35. In SHR-C, large fibrotic foci were common, and many hypertrophic cardiac myocytes showed various degenerative changes including those of mitochondria and widening of the intermyofibrillar spaces. These changes were rarely seen in SHR-N. The intracellular volume ratio of myofibrils did not differ between SHR-N and WKY-35, but was significantly decreased in SHR-C, whereas that of mitochondria did not differ between SHR-N and SHR-C or WKY-35. These findings indicate that despite only a moderate suppression of hypertension, long-term nifedipine treatment caused regression of left ventricular hypertrophy, with cardiocyte hypertrophy, interstitial fibrosis, degenerative changes, and subcellular remodeling being reversed to the baseline levels in SHR-15. In addition, the capillary density was increased to that seen in WKY-35.     

Scanning electron microscope examination of pancreatic ducts in stroke-prone spontaneously hypertensive rats (SHRSP)

Summary Conclusion Ischemic injury to the pancreatic ductal cells causes stasis of pancreatic juice, which in turn induces chronic pancreatitis. Background The pathogenesis of chronic ischemic pancreatitis is still unclear. Methods In 12-, 24-, and 36-wk-old, male stroke-prone spontaneously hypertensive rats (SHRSP), which have sclerotic arterioles with a marked narrowing lumen in the pancreas, pancreatic tissue was examined using light and scanning electron microscopy. Corrosion casts of pancreatic ducts and blood vessels were examined using scanning electron microscopy. Results Ductular proliferation, tortuous ductal channels, crater-like depressions of ductal inner surfaces, and long cilia of ductal cells were found to be sporadically distributed throughout the pancreatic tissue. Similar pancreatic abnormalities had been found in previous studies using WBN/Kob rats that exhibited pancreatic juice stasis. A few interlobular ducts in the pancreatic tissue of the SHRSP had an inner surface that was gyrus-like in appearance, a feature not found in WBN/Kob rats.     

Intermediate filaments of hepatocytes and biliary epithelial cells in bile duct obstruction: Transmission and scanning electron microscopy study

The cytoskeletons of hepatocytes and biliary epithelial cells in bile duct ligated rate livers were investigated by transmission and scanning electron microscopy. The three dimensional organization of the intermediate filaments (IFs) of hepatocytes and biliary epithelial cells was clearly demonstrated by scanning electron microscopy. Cell borders and dilated bile canaliculi were well preserved after perfusion with detergent solution. A very dense filamentous network of IFs was seen throughout the cytoplasm, especially around the dilated bile canaliculi and at the cell borders. IFs in biliary epithelial cells were more numerous compared with hepatocytes. Morphometric analysis showed that the IFs of hepatocytes significantly (p>.001) increased in amount in bile duct ligated rats. The IFs of biliary epithelial cells showed no significant changes in bile duct ligated rats compared to controls. These results suggest that the increase in IFs in hepatocytes results from the adaptation of the hepatocytes to the stress imposed by bile duct ligation. It may be that the resulting intracanalicular pressure and back diffusion of bile induces a metaplastic change in hepatocytes so that they acquire more IFs to function like the bile duct epithelium to conduct bile flow. This study was supported in part by a grant no. 62480198 from the Ministry of Education, Japan. We are thanksful to Miss Noriko Iwanami and Miss Atsuko Fujimoto for their secretarial help.     

Analysis of microvessels in pancreatic cancer: by light microscopy,confocal laser scan microscopy,and electron microscopy

Background/Purpose

The microvessel density (MVD) of most malignant tumors is considered to be strongly related to metastasis and prognosis. Weidner’s “hot spot method” for determining MVD is in general use, but it is possible that cells other than endothelial cells will also be stained. In our previous study, no correlations were observed between MVD determined by the “hot spot method” and prognosis/metastasis. But, using the “lumen method,” we found a correlation with the number of vessel structures only. In the present study, we analyzed the staining of microvessels in pancreatic cancer, using light microscopy, confocal laser scan microscopy (CLSM), and transmission electron microscopy (TEM).

Methods

Microvessel staining of pancreatic cancer with CD34, factor VIII, and CD45 antibodies was examined in consecutive slices by light microscopy. For CLSM, freshly resected specimens were immunostained with factor VIII and fluorescein isothiocynate. For TEM, specimens were fixed with 2.5% glutaraldehyde, treated with 1% osmium tetroxide, and embedded in epoxy resin.

Results

Staining of vessels with CD34 and factor VIII antibodies appeared similar under light microscopy. However, CD34-stained consecutive slices were judged not to reveal vessel structures, and some cells stained with CD45 antibody were similar in appearance to CD34-stained cells. Under CLSM, irregular arrangements of neovascularization, consisting of many branches, were observed, but many positively stained cells not identified as vessels were also seen. Microvessels were distinctly identified under TEM, but the types of individual cells could not be determined.

Conclusions

An integrated, reproducible method for the measurement of MVD is vital. For pancreatic cancer, the “lumen method” is recommended.