Simultaneous measures of contraction and intracellular calcium in single, cultured smooth muscle cells.

Simple methods are presented for quantitating contraction and intracellular calcium simultaneously in single, cultured smooth muscle cells. These methods are the first to demonstrate that reliable velocities of cell shortening can be measured in cultured smooth muscle cells and that cells in vitro exhibit shortening velocities comparable to those measured in the fastest phasic muscles in situ. Temporal relationships between changes in intracellular calcium and shortening within single cells were determined with a resolution of 100 ms and were consistent with measures in more "classical" preparations. Intracellular calcium rose quickly and transiently 10-fold above the basal level of 80-90 nM in response to the muscarinic agonist, carbachol. Shortening of the cells occurred 200 ms after intracellular calcium began to rise. The sensitivity and reliability of these methods allowed the effects of different stimuli to be easily resolved. The present report demonstrates that genuine contractility need not be ignored in cultured smooth muscle cells and that the temporal relations between shortening and intracellular calcium mobilization can be quantitatively assessed in controlled in vitro environments.

Vitronectin regulates smooth muscle contractility via alphav and beta1 integrin.

Previous work from this laboratory has established a method for maintaining physiological contractility of dissociated avian smooth muscle in a defined medium at low density. The present report emphasizes the dramatic potency of serum to alter smooth muscle phenotype and induce a loss of contractility. Vitronectin, a molecule purified from plasma, mimicked these effects of serum via an integrin that is RGD-sensitive. Studies utilizing blocking antibodies against vitronectin demonstrated that the presence of this specific adhesion molecule was necessary for the serum-induced loss of contractility. Based on the actions of function-blocking antibodies and RGD-containing peptides, the integrin alphavbeta1 appears to be the primary receptor involved in vitronectin's ability to induce phenotypic transformation in amniotic smooth muscle. The influence of vitronectin on smooth muscle contractility is particularly relevant, because this molecule is abundant in whole blood and plasma (approx. 400 microg/ml). The results suggest that smooth muscle needs to be continually protected from normal blood constituents in vivo. The implications of these results for smooth muscle-related diseases like atherosclerosis, restenosis and Kaposi's sarcoma are discussed.

Identification of a novel marker for primordial smooth muscle and its differential expression pattern in contractile vs noncontractile cells.

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

Substance P responsiveness of smooth muscle cells is regulated by the integrin ligand, thrombospondin.

The extracellular factors that determine a cell's responsiveness to neurotransmitters are of particular relevance for pharmacologically diverse cell types such as neurons and smooth muscle. We previously demonstrated that matrix-associated factors are capable of dramatically and specifically suppressing the responsiveness of smooth muscle to the neuropeptide, substance P. We now demonstrate that this influence of extracellular matrix on the pharmacological phenotype of smooth muscle cells can be blocked specifically by an Arg-Gly-Asp (RGD)-containing antagonist of integrins. Of a battery of integrin ligands tested, only thrombospondin mimicked the effect of the extracellular matrix on substance P responsiveness. This effect of thrombospondin was dose dependent, RGD sensitive, and blocked by an antibody directed against the RGD-containing region of thrombospondin. Because the mRNA for thrombospondin is present in the cells of the chicken amnion, this extracellular factor may normally suppress substance P responsiveness in amniotic smooth muscle. The results suggest a role for matrix-associated integrin ligands in the regulation of cellular responses to specific neurotransmitters and hormones and in the development and maintenance of tissue-specific pharmacological properties.

Maintenance of contractility in dissociated smooth muscle: low-density cultures in a defined medium.

The loss of contractility in long-term cultures of dissociated smooth muscle is such an established observation that the lack of contractility of cultured smooth muscle cells is often not even noted. This report describes methods of dissociating and culturing smooth muscle cells from the avian amnion that maintain contractility for > 1 mo in a defined medium. Because contractility was assessed by monitoring the contractions of individual cells to neurotransmitter-related substances, it is clear that these cells maintained both contractility and pharmacological responsiveness. However, when amniotic smooth muscle cells were dissociated with enzymes containing impurities or cultured in the presence of serum, they flattened and lost contractility, as reported for many other types of smooth muscle.

Extracellular matrix regulates smooth muscle responses to substance P.

Little is known about the extracellular factors that determine a cell's responsiveness to neurotransmitters. This is a particularly important issue for pharmacologically diverse cell types such as neurons and smooth muscle. This report demonstrates that the contractile responses of amniotic smooth muscle to a specific neuropeptide, substance P, is controlled by a molecule(s) intimately associated with the extracellular basement membrane. This molecule(s) normally represses the expression of substance P responsiveness in this tissue. When the amniotic smooth muscle is separated from the basement membrane by dissociation, normally unresponsive cells exhibit a progressive increase in responsiveness to substance P, beginning within the first 24 hr in culture. The induction of substance P responses was completely inhibited when the cells were plated onto isolated amniotic basement membrane rather than onto polyornithine or collagen I. Similar changes in the responsiveness to another agonist, histamine, did not occur. The data demonstrate that extracellular matrix exerts a major instructive influence in determining the responsiveness of avian amniotic smooth muscle to specific ligands. We suggest that similar regulatory mechanisms may operate in other tissues.

The regulation of synaptogenesis during normal development and following activity blockade.

The mature neuromuscular junction is characterized by the tight spatial colocalization of synaptic vesicles and acetylcholine receptor (AChR) clusters. Although a large body of work exists on the interactions between motoneurons and myotubes leading to synaptogenesis in tissue culture, how the neuromuscular junction acquires its highly specialized structure in vivo is not well understood, particularly during the earliest period of synaptogenesis. In this study, the development of the neuromuscular synapse in chick hindlimb muscles was examined and quantified by simultaneously labeling the pre- and postsynaptic elements from the time the main nerve trunks leave the lumbosacral plexus region to enter the developing limb (St 24) through the end of the motoneuron cell death period (St 36). Based on these results, synaptogenesis can be divided into several distinct stages that are intimately connected to the innervation sequence described in a previous paper (Dahm and Landmesser, 1988). Briefly, as large nerve trunks approach the developing muscles and the first AChR clusters are induced to form on nearby myotubes, none of these initial receptor clusters are in direct contact with a nerve profile. The first appearance of nerve-contacted clusters (synapses) is coincident with the growth of large, unbranched nerve trunks into the muscles. The next step is initiated by the formation of small nerve side branches that grow out from the larger intramuscular nerve trunks to bring most axons and myotubes into contact for the first time. As side branches form, synapses appear around them, and non-nerve-contacted receptor clusters disappear from around the main intramuscular nerve trunks. The next step in synaptogenesis is the restriction of synaptic vesicle antigen to sites of synaptic contact. These early stages of synaptogenesis are also characterized by the growth of the presynaptic terminal to match the length of the postsynaptic receptor cluster. This study showed that AChR cluster formation during early in vivo neuromuscular development does not require close anatomical nerve contact, but that the presence of the nerves is necessary for AChR clusters to form. This suggests that the nerves normally induce AChR clustering via the release of a diffusible substance, a suggestion substantiated by the observation that AChR clusters do not form on aneural myotubes in vivo. In order to assess the role of synapse formation in the regulation of motoneuron number, synaptogenesis was quantitatively examined after chronic neuromuscular blockade, which prevents motoneuron cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

The regulation of intramuscular nerve branching during normal development and following activity blockade.

In vertebrates, approximately 50% of the lumbosacral motoneurons die during a short period of development that coincides with synaptogenesis in the limb. Although it has been postulated that these motoneurons die because they fail to obtain adequate trophic support from the muscles, it is not clear how this factor is supplied. The mechanism by which activity blockade prevents motoneurons cell death is also unknown. In order to begin to understand the nature of these proposed trophic interactions, we have examined the temporal sequence of axonal invasion and ramification within two muscles of the chick hindlimb, the predominantly slow iliofibularis and the fast posterior iliotibialis, during the cell death period. We found striking differences in intramuscular nerve ingrowth and branching between fast and slow muscle. We also observed differences in the molecular composition of fast and slow myotubes that may contribute to the nerve pattern differences. In addition, we observed a progressive increase in the degree of intramuscular nerve fasciculation as well as a precise temporal sequence of nerve branching. The earliest detectable response to chronic curarization was a dramatic decrease in the degree of intramuscular nerve fasciculation. Activity blockade also greatly enhanced nerve branching within the muscles from the time that nerve branches normally formed, and, additionally, interfered with the normal cessation of axon growth. Our results support the idea that nerve endings are the sites of trophic uptake. Furthermore, although our results do not allow us to exclude other activity-dependent influences on motoneuron survival, they suggest the following testable hypotheses: (1) the normal regulation of motoneuron survival may result from the precise control of intramuscular nerve branching, (2) activity blockade may increase motoneuron survival by enhancing intramuscular nerve branching, and (3) anything which affects this complex process of nerve branching may also alter motoneuron survival.

Localization of alpha-bungarotoxin binding sites in the ciliary ganglion of the embryonic chick: an autoradiographic study at the light and electron microscopic level.

Binding sites for [125I]alpha-bungarotoxin were localized in the chick ciliary ganglion by light and electron microscopic autoradiography. Groups of four ganglia were incubated for 4 h with 20 nM [125I]alpha-bungarotoxin alone or with radioactive alpha-bungarotoxin plus either 1 microM unlabeled alpha-bungarotoxin or 100 microM d-tubocurarine. Specific binding to various morphological regions was determined by subtracting the densities of autoradiographic grains in the presence of competing nonradioactive ligands from the densities in the absence of those ligands. Most of the specific binding in the ganglion (91%) was associated with neurons. Seventy-four per cent was found within 1.2 micron of neuronal plasma membranes and 17% was found overlying neuronal cell bodies. Analysis of the specific binding associated with neurons, but not with the neuronal plasma membranes, revealed that lysosomes and multivesicular bodies were 9- and 32-fold more heavily labeled than other cellular organelles. The grain density over choroid cell bodies was significantly higher than that over ciliary neurons. Most (greater than 75%) of the autoradiographic grains within 0.25 micron of neuronal plasma membranes were found in "complex" contact regions of the membranes, which are characterized by extensive membrane evaginations. However, after correcting for the amount of plasma membrane present in the various regions of the membrane studied, alpha-bungarotoxin binding was found to be uniform. Few (less than 10%) of the specialized membranes between pre- and postsynaptic neurons were found in these "complex" contact regions suggesting that the bulk of alpha-bungarotoxin binding to neuronal membranes is located at some distance from the sites of transmitter release. The density of alpha-bungarotoxin binding sites in the neuronal plasma membrane was low (approximately 100-200 sites/micron 2 of neuronal membrane) compared to published values of the density of binding sites at the neuromuscular junction. Since alpha-bungarotoxin does not bind preferentially to specialized synaptic membranes, it seems unlikely that the binding sites for this toxin are the neuronal nicotinic receptors.

The number and distribution of sympathetic neurons that innervate the rat pineal gland.

The sympathetic innervation of the rat pineal gland was examined using a variety of anatomical techniques. Following the injection of horseradish peroxidase into the pineal gland, approximately 250 labeled neurons were found in the ipsilateral superior cervical ganglion. No labeled neurons were found in the middle or inferior cervical ganglia. In animals whose left internal carotid nerve was lesioned prior to the injection of peroxidase, an average of only three labeled neurons was found in the ipsilateral superior cervical ganglion. These data suggest that most, if not all, of the sympathetic neurons innervating the pineal gland exit from the superior cervical ganglia via the internal carotid nerves. The distribution of sympathetic neurons innervating the pineal gland was similar, though slightly more rostrally placed, than the distribution of the entire population of superior cervical ganglion neurons which project into the internal carotid nerve. Both the small number of neurons innervating the pineal gland and their wide distribution in the rostral part of the superior cervical ganglion indicate that their study at the level of the ganglion would be difficult. Sympathetic axons reach the pineal gland via the nervi conarii. Electron microscopic studies indicate that in each nervus conarii there are about 440 axons which make contact with the surface of the pineal gland. In certain cases, bundles of axons from the left and right nervi conarii were found to fuse. Additional evidence for the intermingling of axons from the two nervi conarii was seen in orthograde transport studies with horseradish peroxidase.