Periodic organization of the contractile apparatus in smooth muscle revealed by the motion of dense bodies in single cells

To study the organization of the contractile apparatus in smooth muscle and its behavior during shortening, the movement of dense bodies in contracting saponin skinned, isolated cells was analyzed from digital images collected at fixed time intervals. These cells were optically lucent so that punctate structures, identified immunocytochemically as dense bodies, were visible in them with the phase contrast microscope. Methods were adapted and developed to track the bodies and to study their relative motion. Analysis of their tracks or trajectories indicated that the bodies did not move passively as cells shortened and that nearby bodies often had similar patterns of motion. Analysis of the relative motion of the bodies indicated that some bodies were structurally linked to one another or constrained so that the distance between them remained relatively constant during contraction. Such bodies tended to fall into laterally oriented, semirigid groups found at approximately 6-microns intervals along the cell axis. Other dense bodies moved rapidly toward one another axially during contraction. Such bodies were often members of separate semirigid groups. This suggests that the semirigid groups of dense bodies in smooth muscle cells may provide a framework for the attachment of the contractile structures to the cytoskeleton and the cell surface and indicates that smooth muscle may be more well-ordered than previously thought. The methods described here for the analysis of the motion of intracellular structures should be directly applicable to the study of motion in other cell types

Ca(2+) Regulation in the Near-Membrane Microenvironment in Smooth Muscle Cells

The microenvironment between the plasma membrane and the near-membrane sarcoplasmic reticulum (SR) may play an important role in Ca(2+) regulation in smooth muscle cells. We used a three-dimensional mathematical model of Ca(2+) diffusion and regulation and experimental measurements of SR Ca(2+) uptake and the distribution of the SR in isolated smooth muscle cells to predict the extent that the near-membrane SR could load Ca(2+) after the opening of single plasma membrane Ca(2+) channels. We also modeled the effect of SR uptake on 1), single-channel Ca(2+) transients in the near-membrane space; 2), the association of Ca(2+) with Ca(2+) buffers in this space; and 3), the amount of Ca(2+) reaching the central cytoplasm of the cell. Our results indicate that, although single-channel Ca(2+) transients could increase SR Ca(2+) to a certain extent, SR Ca(2+) uptake is not rapid enough to greatly affect the magnitude of these transients or their spread to the central cytoplasm unless the Ca(2+) uptake rate of the peripheral SR is an order-of-magnitude higher than the mean rate derived from our experiments. Immunofluorescence imaging, however, did not reveal obvious differences in the density of SR Ca(2+) pumps or phospholamban between the peripheral and central SR in smooth muscle cells

Postnatal changes in caldesmon expression and localization in cardiac myocytes

Caldesmon is a heat-stable protein found in both muscle and non-muscle tissue. It binds to a number of contractile and cytoskeletal proteins and may be involved in regulating acto-myosin interaction in smooth muscle cells and/or the assembly of microfilaments in muscle and non-muscle cells. We have shown previously that caldesmon is localized at the Z-lines in adult cardiac myocytes and that both the low- and high-molecular-weight forms (l-caldesmon and h-caldesmon, respectively) are present in atrial and ventricular myocytes. Here we examined the expression of caldesmon and its localization in freshly isolated cardiac myocytes during postnatal development and when these myocytes were grown in culture. We found that l-caldesmon is expressed in both neonatal and adult rat ventricular myocytes. The expression of h-caldesmon, however, was not detected in myocytes from newborn animals but increased during the first 2 weeks of postnatal development. Caldesmon was generally not co-localized with α-actinin at the Z-lines in neonatal myocytes but became increasingly more so during the first 2 weeks of postnatal development. When myocytes from 5- and 10-day-old rats were grown in primary culture, h-caldesmon expression decreased and caldesmon could not be detected at the Z-lines in the cultured cells. These results indicate that caldesmon plays a role at the Z-lines in adult cardiac myocytes; however, its localization at the Z-lines is not necessary for the prenatal development that occurs at these sites or for the establishment of a contractile phenotype in cultured cardiac myocytes

Inward Rectifier K+ Currents and Kir2.1 Expression in Renal Afferent and Efferent Arterioles

The afferent and efferent arterioles regulate the inflow and outflow resistance of the glomerulus, acting in concert to control the glomerular capillary pressure and glomerular filtration rate. The myocytes of these two vessels are remarkably different, especially regarding electromechanical coupling. This study investigated the expression and function of inward rectifier K+ channels in these two vessels using perfused hydronephrotic rat kidneys and arterioles and myocytes isolated from normal rat kidneys. In afferent arterioles pre-constricted with angiotensin II, elevating [K+]0 from 5 to 15 mmol/L induced hyperpolarization (−27 ± 2 to −41 ± 3 mV) and vasodilation (6.6 ± 0.9 to 13.1 ± 0.6 μm). This manipulation also attenuated angiotensin II-induced Ca2+ signaling, an effect blocked by 100 μmol/L Ba2+. By contrast, elevating [K+]0 did not alter angiotensin II-induced Ca2+ signaling or vasoconstriction in efferent arterioles, even though a significant hyperpolarization was observed (from −30 ± 1 to −37 ± 3 mV, P = 0.003). Both vessels expressed mRNA for Kir2.1 and exhibited anti-Kir2.1 antibody labeling. Patch-clamp measurements revealed prominent inwardly rectifying and Ba2+-sensitive currents in afferent and efferent arteriolar myocytes. Our findings indicate that both arterioles express an inward rectifier K+ current, but that modulation of this current alters responsiveness of only the afferent arteriole. The expression of Kir in the efferent arteriole, a resistance vessel whose tone is not affected by membrane potential, is intriguing and may suggest a novel function of this channel in the renal microcirculation