Proteoglycans are an important nonfibrous matrix component of the arterial wall. Direct evidence for their role in resistance-sized arteries is lacking, although they likely have an important role in coordinating and regulating vessel behavior, presumably via interactions of their glycosaminoglycan chains or core proteins with other matrix molecules and/or the smooth muscle cell surface. The purpose of this study was to determine whether the removal of specific glycosaminoglycan chains from proteoglycans in resistance-sized mesenteric arteries would change the mechanical properties of the arterial wall, thereby affecting their functional behavior. The major finding of the study was that 65% removal of chondroitin-dermatan sulfate-containing glycosaminoglycans from the arterial wall increased vascular wall stiffness and altered the myogenic behavior of the artery. The significant alterations in myogenic behavior associated with changes in passive mechanics following partial glycosaminoglycan chain removal support our hypothesis that chondroitin-dermatan sulfate-containing proteoglycans contribute significantly to the functional behavior of resistance arteries. We speculate that these alterations are the result of changes in stress transfer between collagen fibrils and/or stress transfer between cells and collagen fibrils under applied pressure.

Immunoelectron microscopic labeling of calsequestrin on ultra-thin sections of rat ventricular muscle prepared by quick-freezing, freeze-drying, and direct embedding in Lowicryl K4M was compared to that observed on ultra-thin sections prepared by chemical fixation, dehydration in ethanol, and embedding in Lowicryl K4M. Brightfield electron microscopic imaging of cryofixed, freeze-dried, osmicated, and Spurr-embedded rat ventricular tissue showed that the sarcoplasmic reticulum was very well preserved by cryofixation and freeze-drying. Therefore, the four structurally distinct regions of the sarcoplasmic reticulum (i.e., the network SR, the junctional SR, the corbular SR, and the cisternal SR) were easily identified even when myofibrils were less than optimally preserved. As previously shown by immunoelectron microscopic labeling of ultra-thin frozen sections of chemically fixed tissue, calsequestrin was confined to the lumen of the junctional SR and of a specialized non-junctional (corbular) SR, and was absent from the lumen of network SR in cryofixed, freeze-dried, Lowicryl-embedded myocardial tissue. In addition, a considerable amount of calsequestrin was also present in the lumen of a different specialized region of the non-junctional SR, called the cisternal sarcoplasmic reticulum. By contrast, relocation of calsequestrin to the lumen of the network SR was observed to a variable degree in chemically fixed, ethanol-dehydrated, and Lowicryl-embedded tissue. We conclude that tissue preparation by cryofixation, freeze-drying, and direct embedding in Lowicryl K4M for immunoelectron microscopic localization of diffusible proteins, such as calsequestrin, is far superior to that obtained by chemical fixation, ethanol dehydration, and embedding in Lowicryl K4M.

1. The mechanical response to drugs and to electrical stimulation of nerves was investigated in isolated strips of intrinsic smooth muscle from the corpora cavernosa penis of the rat. 2. Noradrenaline caused muscle strips to contract in a dose-dependent manner. Contractions could be blocked by pretreatment with the alpha-adrenoreceptor antagonist, phentolamine. 3. Acetylcholine and carbachol had no effect on the baseline tension of muscle strips. Both drugs were relatively ineffective in relaxing noradrenaline-contracted strips. 4. Field stimulation of isolated muscle strips elicited contractions which were blocked by tetrodotoxin and greatly attenuated with phentolamine or reserpine pretreatment. Acetylcholine inhibited the excitatory response to field stimulation. This inhibitory effect of acetylcholine could be blocked with atropine. 5. Field stimulation of noradrenaline-contracted muscle strips caused relaxation. This inhibitory effect, due to nerves which arise from the pelvic plexus, is unaffected by substances which act on cholinergic systems. 6. The results suggest that the erectile muscle of the rat is similar to that of man in that it receives an excitatory noradrenergic innervation and an inhibitory innervation which may have a non-cholinergic component. Although acetylcholine may have a role in penile physiology of the rat, it is unlikely that it has a postsynaptic action.

The techniques of quick freezing and freeze-drying provide an alternative to the more classical methodologies of chemical fixation and dehydration with organic solvents. It is possible to embed freeze-dried tissue in low viscosity resins, either at room temperature or at subzero temperatures in Spurr's resin or Lowicryl K4M, respectively. The choice of embedding medium affords additional flexibility in postdrying and embedding conditions, since Spurr's resin allows vapor fixation with osmium tetroxide and thermal polymerization. Osmium tetroxide is not recommended for Lowicryl resins, but these media permit polymerization at subzero temperatures with ultraviolet light. Both resins have unique advantages that may be utilized, depending upon the purpose of the embedding.In this paper, we discuss the details of preparing smooth muscle, from rabbit renal artery, by quick freezing and freeze-drying, as well as methods for the embedding of the freeze-dried tissue in both Spurr's resin and Lowicryl K4M. Although we have previously reported the ultrastructure of smooth muscle embedded in Spurr's low viscosity resin, the combination of freeze-drying and infiltration in Lowicryl K4M represents a new approach that allows the elimination of chemical fixation, dehydration with organic solvents, and heat polyLowicryl K4M, Smooth muscle, Specimen preparation, Cryofixation, Lyophilization merization of the embedding medium.

In an effort to more clearly elucidate the role of cellular structures as calcium sinks and sources in smooth muscle cells, the intracellular distribution of radioactive calcium was evaluated by a new method based on freeze-drying . The guinea pig vas deferens was exposed to a physiological salt solution that contained "Ca. The muscle was then freeze-dried and prepared for electron microscope autoradiography . The grain density over the plasma membrane, mitochondria, and sarcoplasmic reticulum (SR) was significantly greater than that over the cytoplasmic matrix . The grain density of the nucleus was only slightly greater than that of the matrix . These results suggest that the plasma membrane, mitochondria and SR have the capacity to accumulate calcium . Which of these structures serve as a source of calcium for contraction remains to be determined . A stereological comparison between freeze-dried and conventionally prepared smooth muscles revealed several differences. The cross-sectional area of freeze-dried cells was about twice that of conventionally prepared cells. Moreover, mitochondria and sub-surface vesicles occupied a significantly smaller percentage of the cell in the freeze-dried tissue than they did in the conventionally prepared tissue .