Quantification of ANP mRNA in primary cultures of adult rat atrial myocytes by image processing: in situ hybridization to multiple parallel samples using single-stranded cDNA probes.

In primary cultures of adult rat atrial myocytes, we quantified the accumulation of atrial natriuretic peptide (ANP) mRNA in parallel with ANP secretion. ANP mRNA was quantified by image analysis of myocytes hybridized in situ with single-stranded cDNA probes generated by two successive thermal cycling procedures. In situ analysis permitted measurement of many small experimental samples in tandem while avoiding the possibility of differential extraction and processing of mRNA from sample to sample. The single-step application of 32P-labeled probes allowed processing of many parallel samples and generated intense punctate autoradiographic signals that were readily countable by image processing. Biotin-labeled probes, in conjunction with gold-labeled anti-biotin antibodies and silver intensification, gave an apparently equivalent specific signal but presented more difficulty in uniform processing of many samples and was harder to quantify by our image processing system. Measurement of ANP mRNA during atrial myocyte culture showed that ANP mRNA accumulated from undetectable levels after 1 day of culture to maximal levels by Day 8. In contrast, secretion of ANP (which is stored in atrial granules) slowly decreased, but was not abolished, during the first 5 days of culture. Subsequently, ANP secretion increased, with the increase trailing ANP mRNA accumulation by at least 24 hr.

Atomic force microscopy of freeze-fracture replicas of rat atrial tissue.

Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.

Rat atrial myocyte plasmalemmal caveolae in situ. Reversible experimental increases in caveolar size and in surface density of caveolar necks.

The structure, size, and surface density of the conspicuous flask-shaped structures called caveolae that are located under the plasma membrane of cardiac myocytes in intact rat atria were studied by electron microscopy after physiological perturbations designed to examine whether caveolae and/or their necks are fixed or mobile and whether the caveolar lumen is always open or can close off from the interstitial space. We showed that, in stretched and unstretched atria, horseradish peroxidase could enter or be washed out of caveolae at 37 degrees, 18 degrees, and 4 degrees C, but this finding does not rule out that caveolae and/or their necks can cycle rapidly between states closed and open to the interstitial space. Electron microscopy of thin sections revealed that exposure of atria at 37 degrees or 18 degrees C to physiological salt solutions made hypertonic by adding 150 mM sucrose or mannitol resulted in a striking enlargement of caveolar profiles within 1 to 5 minutes. Caveolar enlargement was rapidly reversible on return to control saline. After freeze fracture of atria exposed to these hypertonic solutions, quantitative analysis of electron micrographs of the fracture faces revealed statistically significant increases in cross-sectional diameter of cross-fractured caveolar necks and in mean number of caveolar necks penetrating per unit area of plasmalemmal fracture face. These results suggest that atrial myocyte caveolae are dynamic structures whose necks may be reversibly inserted into and withdrawn from the plasmalemma, possibly (but not necessarily) corresponding to states in which caveolae are, respectively, open and closed to the interstitial spaces.

Expression of phospholamban mRNA during early avian muscle morphogenesis is distinct from that of alpha-actin.

We have studied the expression of phospholamban during the early development of chick embryos by in situ hybridization and have compared it to that of alpha-cardiac and alpha-skeletal actin. In adult cross-striated muscles there is only one phospholamban gene and it is expressed exclusively in the heart and slow muscles. In the heart phospholamban transcripts were first detected at stage 14 in the region of presumptive ventricle and at stage 20 in the atrium. In the myotomal portion of the somites phospholamban mRNA was first detected at stage 20, which lagged behind the appearance of the alpha-actins. In the limb rudiments all three mRNAs were barely detectable through stage 24, but increased by stage 28+. However, quantitative analysis of signal intensity at stage 28+ indicated that less phospholamban mRNA is present in the limb bud than in the myotome since for phospholamban the ratio of the signal density in the myotome to that in the limb rudiments was about twice the value of the ratio determined for the alpha-actins. Northern blot analysis of embryonic day 11 chick fast pectoralis muscle showed that phospholamban mRNA was not detected in vivo while alpha-cardiac actin mRNA was. Moreover, no phospholamban mRNA was detected in primary cultures derived from pectoralis muscle of the same age. In concert with previous observations that phospholamban is not detectable at stage 30-32 in wing or thigh muscle, these results suggest that phospholamban mRNA is expressed independently of the alpha-actins in the limb buds during early myogenesis.

Accumulation of indium-111-labeled human low density lipoprotein in the rabbit aorta: Implications for nuclear imaging of vascular lesions.

Nuclear imaging of atheromata must distinguish lesions from both blood pool and normal arterial tissue. We have examined spatial and temporal variations of indium-111-labeled human low density lipoprotein (LDL) accumulation in rabbit aortas. LDL-derived In-111 activity was time-independent in lesion-resistant regions of aortas from normal and hypercholesterolemic animals (mean 2.9 × 10(-6) percent injected activity per milligram tissue [%IA/mg]) and in lesion-prone regions of normal aortas (mean 7.1 × 10(-6) %IA/mg). In contrast, activity in sudanophilic lesions of hypercholesterolemic rabbit aortas reached a peak of 31 × 10(-6) %IA/mg at 92 hours postinjection. The mean ratio between activity in lesions versus lesion-resistant regions described a broad convex curve with minima of 4:1 at 14 hours and 136 hours and a peak of 14:1 measured at 72 hours postinjection. The mean ratio between In-111 in lesions and blood followed a sigmoid curve, rising exponentially from 1:25 at 14 hours to 1:3 by 72 hours postinjection. We conclude that optimal signal-to-noise ratios for monitoring atheroma-associated LDL-derived radioactivity occur late, not before about 3 days postinjection. Therefore, LDL labeled with In-111 or even longer-lived radionuclides holds the greatest promise for effective clinical nuclear imaging of atherosclerosis.

Histochemistry and isomyosins of tail musculature in Xenopus.

The main bulk of the larval tail in Xenopus laevis is composed of thick muscle fibres which are succinate dehydrogenase (SDH) negative and show strong positive ATPase activity only at alkaline preincubation (pH 10.4). The thin muscle fibres (which cover the surface of the myotomes and extend to the tail tip) show positive SDH activity as well as strong positive ATPase activity after both alkaline and acid preincubations (pH 10.4, 4.5, 4.4, and 4.3). The pattern of myosin isoenzymes does not change in the tail muscle in the course of development of Xenopus: the same three bands of larval isomyosins were found in all the examined developmental stages: 40, 47, 50 and 56. However, the larval bands were distinct from three bands of myosin isoforms in the musculus longissimus dorsi (MLD) and musculus gastrocnemius (MG) of the metamorphosed Xenopus.

Developmental changes in P-face and E-face particle densities of Xenopus cardiac muscle plasma membrane.

P- and E-face particle densities (PPD and EPD) were measured in electron micrographs of freeze-fractured cardiac sarcolemma from eight developmental stages of Xenopus laevis (stages 33/34 (33 post-otic somite embryos) to 66 (fully metamorphosed juvenile toad], using stereo-imaged replicas. We found striking progressive increases in PPD and EPD, most rapid between stages 33/34 and 37/38; that PPD was significantly greater than EPD at all stages; that both PPD and EPD of stereo-imaged replicas were about X2 greater than corresponding values not stereo-imaged; and that sarcolemmal PPD of late anuran embryonic and post-metamorphosis hearts were significantly greater than our previously determined PPD values for chick late embryo and adult mammalian sarcolemma. We suggest that PPD and EPD depend on how membrane particles segregate during freeze-fracture and on the relative contributions of membrane-spanning and non-membrane-spanning integral membrane protein complexes to each fracture face.

Developmental changes in internal structure of chick heart plasma membrane.

Although changes in electrophysiologically measurable membrane properties of chick embryo cardiac plasma membrane have been repeatedly documented during embryonic development, ultrastructural techniques were heretofore too insensitive to detect developmental changes in internal structure of this membrane. We report here significant structural changes detected by applying the quantitative analysis of Kordylewski, Karrison, and Page (Amer. J. Physiol. 245, H992-H997, 1983 and 248, H297-H304, 1985) to stereo imaged electron microscopic negatives of glutaraldehyde-fixed chick embryo hearts, freeze fractured and photographed with a goniometer stage. Between Hamburger-Hamilton stages 12+ (about 48 hr incubation) and 24 (about 96 hr incubation), plasmalemmal P-face particle density of ventricular myocytes increased from 2228 +/- 139 to 3063 +/- 109 (P less than 0.01); thereafter, measurements at stages 30, 37, 40, and 45 (7, 11, 15, and 19 days incubation) showed a slower significant linear increase which gave a least-squares line with a slope of 41 +/- 13 particles/day (P less than 0.01). Just before hatching, (stage 45) the value of 3762 +/- 234 was similar to, though slightly smaller than, the values of 4122 +/- 153 (8 days after hatching) and 4281 +/- 218 (adult chicken). These results indicate striking stage-dependent changes in the population of integral membrane proteins (channels, carriers, receptors, etc.), especially marked during early embryogenesis.

Measurements on the internal structure of freeze-fractured cardiac plasma membrane.

We describe a quantitative analysis of the internal structure of cardiac plasma membranes freeze fractured in situ, including the P-face particle density (lambda), the P-face particle diameter (d), the percent of fracture face area occupied by particles (Ap), and the spatial distribution of particles (random, clustered, or ordered). This analysis has been applied to seven sheep hearts to compare the plasmalemmal internal structures in ventricular and atrial myocytes and Purkinje strands of the same hearts and also to myocytes of frog, chicken, rabbit, and rat ventricles (to compare internal plasmalemmal structure of different vertebrate classes). Measurements were made on tissues conventionally prepared for freeze fracture by glutaraldehyde fixation and cryoprotection. We found that, in the same sheep hearts, lambda and Ap for ventricular plasmalemma significantly exceeded those for atrial plasmalemma and that the distribution of atrial P-face particles was more clustered than that for ventricle. d and Ap for frog ventricular plasmalemmal P-face significantly exceeded values for some of the other vertebrates.

P-face particle density of freeze-fractured vertebrate cardiac plasma membrane.

P-face particle density of freeze-fractured cardiac plasmalemma provides an estimate of the total population of membrane channels, carriers, and receptors that insert into or traverse unit area of lipid bilayer. We have determined the size of this population of integral membrane protein assemblies by counting the number of P-face particles per square micron of plasmalemma in freeze-fractured hearts, using stereoimaged replicas tilted with a goniometer stage in the electron microscope. Particle numbers per square micron were 4,525 +/- 231, 4,799 +/- 235, 4,122 +/- 153, 4,281 +/- 218, and 5,848 +/- 300 for ventricular myocyte plasmalemmas of rat, rabbit, 8-day chick, adult chicken, and frog, respectively. These values are at least two times greater than published values, in which stereo imaging was not used. Published ligand-binding studies indicate that only the surface density of the Na+-K+ pump sites can account for a significant fraction of P-face particles; the rest so far lack functional correlates. Particle density of frog heart plasmalemma significantly exceeded particle densities of chicken and mammalian plasma membranes.

Experimental evidence for the accumulation of egg pigment in the brain cavities of Xenopus tadpoles.

The origin and fate of darkly pigmented clusters of cells that float freely in the brain cavities of the tadpoles of Xenopus laevis have been experimentally investigated. The results point to the conclusion that the clusters are the sites of egg pigment accumulation, which remain within the brain cavities or at its walls until metamorphosis.

An ultrastructural study of the melanophages in the cerebrospinal fluid of the tadpoles of Xenopus.

Melanin deposits in the brain ventricles of Xenopus tadpoles were studied with light and electron microscopy (TEM and SEM). They appeared to be aggregations of melanophages which accumulated free pigment granules excreted by ependymal cells into the cerebrospinal fluid. Whereas the meningeal melanophores contained oval melanosomes of various sizes, the melanosomes in the scavenger cells were all spherical, large (0.6-1.1 microns) and fairly uniform in size. Moreover, they were arranged in spherical groups which were never seen in the cytoplasm of the melanophores. The melanosomes within the cells were identical to the free melanosomes found in the cerebrospinal fluid and those which occurred within the ependymal cells in the young larva, suggesting a common origin from the egg cytoplasm. The number of the melanosomes in the melanophages increased with age. Fine cytoplasmic projections were involved in catching and engulfing the melanosomes. Some other features of the cytoplasm, e.g., large deposits of cell detritus, also indicated that the cells were macrophages. In the later stages, (48, 49) no projections were observed, but the cells were totally filled with melanosomes.

Light and electron microscopic observations of the development of intestinal musculature in Xenopus.

In the developing intestine of Xenopus the inner circular muscle layer is always thicker and is set up earlier than the outer, longitudinal one. First undifferentiated myoblasts appear between the intestinal epithelium and serosa in the stage 41 (NIEUWKOOP and FABER, 1967). Two crossed muscle layers appear not earlier than stage 50. Then the outer, longitudinal layer consists of single undifferentiated cells, while the inner one is already built of several rows of myocytes. Their ultrastructure and distribution were examined with TEM and SEM. The differentiation of myocytes examined at ultrastructural level was found to be more advanced in the inner layer than in the outer one. However, only after metamorphosis the smooth myocytes of each layer display all the features of mature muscle cells and contain large numbers of the characteristic elements: dense bodies and caveolae. The latter were not found in the larvae and tadpoles. The increase of collagen contents in the submucosa but not between the layers was also observed. Its possible role in the arrangement of the muscular coat is discussed.

Morphology of motor end-plate and its size relation to the muscle fibre size in the amphibian submandibular muscle.

The morphology of neuromuscular junction in the amphibian submandibular muscle has been examined both light and electron microscopically. Despite the differentiation of muscle fibres into types, both the structure and ultrastructure of the motor end plates was found fairly uniform in the examined species. The strongly positive correlation between the motor end plate surface area and muscle fibre diameter was found in Triturus and Rana. Some aspects of the motor end plate morphology are discussed in connection with the results of the author's physiological and morphological observations obtained formerly.

Morphology of muscle fibres in amphibian submandibular muscle.

The microscopic organization and ultrastructure of the submandibular muscle of 10 species of Amphibia were compared. Among other fibre features the diameter of fibres, their content of mitochondria and fat, organization of sarcomeres: morphology of Z-line, M-band and sarcoplasmic reticulum were taken into consideration and 4 main types of muscle fibres were distinguished. They correspond to tonic (slow) and phasic (red, white and intermediate) ones. Slight variety of fibre morphology and of fibre elements among the examined species was found. Special attention to the variety of fibre morphology among the established types has been paid and the existence of continuous "spectrum" of fibres was suggested. The correlation of frequency of fibres of particular types with the body size, gular oscillation frequency, and some other characteristics of the submandibular muscle in the examined species was discussed. Also the zonal arrangement of muscle according to the fibre types, as well as possible dynamic nature of muscle fibres were emphasised.

Scanning electron microscopic observations of the development of the somites and their innervation in anuran larvae.

The development of the paraxial mesoderm in tadpoles of Xenopus, Bufo and Rana was observed with a scanning electron microscope. In addition to examination of the differentiation of the surface and the interior of the somites, some attention was also paid to the transformation of the material of the neural crests and to the innervation of the developing myotome.