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.

Superfluous neurotransmitters?

In recent years, studies have suggested that the complexity of eukaryotic gene regulation, with its recurring and interacting motifs of cis and trans-acting regulatory elements, might result in superfluous gene expression. This conclusion is supported by a variety of experimental results that suggest that non-adaptive gene expression might be common. However, with few exceptions, the practical ramifications of unnecessary gene expression for cell biologists have not been addressed directly; this is particularly true for peptidergic neurophysiology, a field that might be plagued more than most with the consequences of this phenomenon. In this article, Chauncey W. Bowers discusses the superfluous expression of neuropeptides in the nervous system in the context of gene regulation extrapolated from studies in Drosophila.

Domain 1.1 of the sigma(70) subunit of Escherichia coli RNA polymerase modulates the formation of stable polymerase/promoter complexes.

The sigma 70 (sigma(70)) subunit of Escherichia coli RNA polymerase specifies transcription from promoters that are responsible for basal gene expression during vegetative growth. When sigma(70) is present within polymerase holoenzyme, two of its domains, 2.4 and 4.2, interact with sequences within the -10 and -35 regions, respectively, of promoter DNA. However, in free sigma(70), DNA binding is prevented by domain 1.1, the N-terminal domain of the protein. Previous work has demonstrated that the presence of domain 1.1 is required for efficient transcription initiation at the lambda promoter P(R). To investigate whether this is a general property of domain 1.1, we have used five promoters to compare polymerases with and without domain 1.1 in in vitro transcription assays, and in assays assessing the formation and decay of stable, pretranscription complexes. We find that the absence of domain 1.1 does not render the polymerase defective at all of these promoters. Depending on the promoter, the absence of domain 1.1 can promote or inhibit transcription initiation by affecting the formation of stable pretranscription complexes. However, domain 1.1 does not affect the stability of these complexes once they are formed. For polymerases containing domain 1.1, the efficiency of stable complex formation correlates with how well the -10 and -35 regions of a promoter match the ideal sigma(70) recognition sequences. However, when domain 1.1 is absent, having this match becomes less important in determining how efficiently stable complexes are made. We suggest that domain 1.1 influences initiation by constraining polymerase to assess a promoter primarily by the fitness of its -10 and -35 regions to the canonical sequences.

Localization of neurons in the rat superior cervical ganglion that project into different postganglionic trunks.

Horseradish peroxidase (HRP) was used to determine whether neurons in the rat superior cervical ganglion (SCG) are localized in regions of the ganglion as a function of the postganglionic trunk they utilize. In separate experiments, each of the two major postganglionic trunks was cut 1-3 mm from the SCG and solid HRP was applied to the cut end proximal to the ganglion. The results demonstrated that the cell bodies of neurons whose axons project out the internal carotid nerve (ICN) were located primarily in the rostral part of the ganglion. Cell bodies of neurons whose axons project out the external carotid nerve (ECN) were located primarily in the caudal part. The total percentages of neuronc with axons in the ICN and ECN were about 35% and 45%, respectively. When HRP was applied to both these trunks, 73% of the neurons in the SCG were labeled. In the caudal portion of the ganglion, an additional group of neurons was observed whose axons project into the cervical sympathetic trunk. Control studies indicated that the neuronal labeling observed in our experiments was due to retrograde axonal transport rather than the direct uptake of HRP by neuronal cell bodies. Thus, neuronal subpopulations exist in specific regions of the rat SCG. The significance of these results to biochemical and electrophysiological studies is discussed.

Localization of neurons in the rat spinal cord which project to the superior cervical ganglion.

Horseradish peroxidase (HRP) was used to determine the location in the spinal cord of neurons projecting to the superior cervical ganglion of the rat. HRP was applied to the proximal cut end of the cervical sympathetic trunk, close to its entry into the superior cervical ganglion. After survival times of 3, 6, or 9 days, the animals were sacrificed and their spinal cords were processed to visualize HRP using diaminobenzidine, benzidine dihydrochloride, or tetramethylbenzidine. Labeled neurons were found only ipsilateral to the site of HRP application and were restricted to spinal segments C8-T5. Ninety percent of these neurons were located in segments T1-T3. Similar numbers of labeled neurons were found at survival times of 3 and 6 days and the mean number +/- S.E.M. for 11 experiments at these two survival times was 1575 +/- 89. Nine days after application of HRP the mean number of labeled cells and the density of label per cell were reduced. Labeled neurons were found in four regions of the spinal cord: the intermediolateral nucleus (75%), the lateral funiculus (23%), the central autonomic area (1%), and the intercalated region (1%). The cells of the intermediolateral nucleus did not form a continuous column along the rostrocaudal axis of the spinal cord, but instead were often found in clusters, several clusters being present per spinal segment.

The efferent role of sensory axons in nerve-evoked contractions of bullfrog bladder.

The neural input to the frog bladder was characterized in vitro. The nerve-evoked bladder contraction consists primarily of an early parasympathetic cholinergic component and a later, longer-lasting non-adrenergic non-cholinergic component. This slow non-adrenergic non-cholinergic contraction is not only resistant to cholinergic and adrenergic antagonists, but also to H1 and H2 histaminergic antagonists and to the serotoninergic antagonist, methysergide. It is concluded that the non-adrenergic non-cholinergic contraction is mediated by an efferent action of the sensory system because it is resistant to ganglionic nicotinic antagonists and because it is elicited specifically by stimulation of the peripheral cut end of the dorsal root. 5-Hydroxytryptamine is a potent and specific inhibitor of the sensory non-adrenergic non-cholinergic contraction. Although the bladder smooth muscle is innervated by terminals containing a somatostatin-like substance, somatostatin does not cause a bladder contraction. Luteinizing hormone-releasing hormone, enkephalin, histamine, 5-hydroxytryptamine, adenosine and adenosine 5 monophosphate are also unlikely candidates for the non-adrenergic non-cholinergic transmitter because they do not produce bladder contractions and/or their antagonists are ineffective on the nerve-evoked contraction. A putatively sensory network of fibers containing a substance P-like material is located within the wall of the bladder. Substance P produces bladder contractions at concentrations as low as 10(-9) M and so it, or a related substance, is a viable transmitter candidate in this system. Adenosine 5'-triphosphate (ATP)(10(-5) M) also causes a bladder contraction and remains a possible candidate as well. The data demonstrate that the bladder contraction resulting from electrical stimulation of the bladder nerves is due in large part to the "antidromic" stimulation of sensory axons. The likely presence therefore of potent and releasable substances in the peripheral sensory terminals of the bladder suggests that this sensory system may exert significant local, efferent control of bladder smooth muscle (i.e. independent from the central nervous system).

A substance P-like peptide in bullfrog autonomic nerve terminals: anatomy biochemistry and physiology.

Substance P immunoreactivity was localized to dense-cored vesicles in parasympathetic preganglionic nerve terminals in the bullfrog cardiac ganglion. Analysis with radioimmunoassay and high pressure liquid chromatography indicated that the immunoreactive substance was more similar to substance P than to substance K but was not identical to either. The bullfrog substance P was also shown to be different than kassinin, eledoisin, ranatensin, bombesin, neuromedin K and physalaemin. Although the peptide was present in most of the terminals in the ganglion and appeared to be releasable in a calcium-dependent manner, intracellular recordings from ganglion neurons during stimulation revealed no electrophysiological events that might be mediated by an endogenous peptide. In addition, the direct application of substance P to ganglion neurons generally produced no changes in membrane potential, membrane conductance, somal calcium spikes or nerve-evoked release of acetylcholine. High concentrations of substance P did cause an apparent increase in the rate of desensitization of the nicotinic receptors but there was little indication that this phenomenon would occur under physiological conditions. It is suggested that bullfrog substance P is released as a neurotransmitter but is involved in electrically silent events in the postsynaptic neurons or is acting on non-neuronal cells near the terminals. The possible implications of these results for peptidergic systems in general are discussed.

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.

Structure and receptor binding of PYY analogs.

Differences in the structure of PYY and two important analogs, PYY [3-36] and [Pro34]PYY, are evaluated. Y-receptor subtype ligand binding data are used in conjunction with structural data to develop a model for receptor subtype selective agonists. For PYY it is proposed that potent binding to Y1, Y4 and Y5 receptors requires the juxtaposition of the two termini while Y2 binding only requires the C-terminal helix. Further experiments that delineate between primary and tertiary structure contributions for receptor binding and activation are required to support the hypothesis that tertiary structure is stable enough to influence the expression of PYY's bioactivity.

A cadmium-sensitive, tetrodotoxin-resistant sodium channel in bullfrog autonomic axons.

The major component of the compound action potential of the bullfrog cardiac nerve was not blocked by TTX (10(-5) M) although the conduction velocity of these fibers was decreased by 50%. The TTX-resistant compound action potential was completely blocked by 100-300 microM CdCl2 but not by 5 mM CoCl2 or NiCl2. This cadmium-sensitive action potential was only partially reduced by removal of calcium from the medium but was abolished by replacement of sodium with sucrose, tetramethylammonium or choline. Therefore, in addition to the classical TTX-sensitive sodium channels, the parasympathetic axons of the bullfrog may also utilize a TTX-insensitive, cadmium-sensitive sodium channel.

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.

Transforming electrocortical mapping data into standardized common space.

Subdural grid electrodes are implanted routinely for the pre-surgical work up of epilepsy. While different approaches are available, many centers, including ours, visualize electrode locations by co-registering pre-operative 3-D MR images with post-implantation 3-D CT images. This method allows the determination of the electrode positions in relation to the individual patient's anatomy, but does not easily allow comparison across patients. The goal of this study was to develop and validate a method for transforming electrode positions derived from 3-D CT images into standardized space. We analyzed data from twelve patients with subdurally implanted electrodes. Volumetric CT and MRI images were co-registered and then normalized into common stereotactic space. Electrode locations were verified statistically by comparing distances between the anterior commissure and a representative sampling of 8 electrode sites per patient. Results confirm the accuracy of our co-registration method for comparing electrode locations across patients.

Expression of functional neurotransmitter receptors in an uninnervated tissue: avian amnion.

The smooth muscle of the avian amnion is unusual because it is normally never innervated. However, as assessed by contractile response, this tissue expressed at least 11 different types of receptor for neurotransmitter substances including acetylcholine, norepinephrine, histamine, 5-hydroxytryptamine, vasoactive intestinal peptide, urotensin II, neurotensin, and somatostatin-28. Three neurotransmitters, histamine, 5-hydroxytryptamine, and norepinephrine, each acted via 2 separate and antagonistic types of receptors. The amnion also responded to prostaglandin E2. On the other hand, the tissue did not respond to substance P or bradykinin, 2 peptides that are known to affect smooth muscle contractility in a variety of other systems. Studies with organ-cultured amnion demonstrated that the smooth muscle can be cultured early in development and will differentiate in vitro. Some, but not all, of the amniotic responses developed in a defined medium. The results indicate that this novel smooth muscle preparation will be useful for identifying epigenetic factors that control the expression of functional receptors.

Development and implementation of evidence-based guidelines: a multisite demonstration project.

The development of alternative patient care delivery systems is being explored by health care providers, managed care corporations, insurance companies, and the government. Pathways, guidelines, care maps, and algorithms are techniques that assist in the development of patient care delivery systems with the potential to both decrease cost and ensure quality. This article reviews our experience with a program designed to transport clinical guidelines and pathways developed from evidence-based scientific knowledge to 7 acute care facilities located throughout the United States.

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.

Mechanisms of synaptic fatigue in a developing autonomic ganglion.

Synaptic transmission in developing systems has often been noted to exhibit depression or failure at moderate frequencies of stimulation. While this is often presumed to be a transient, nonspecific inability of developing systems to meet the demands of synaptic transmission, this report demonstrates that such failure in the choroidal neurons of the embryonic ciliary ganglion is due to muscarinically mediated inhibition. Although the ganglion is composed of both choroid and ciliary neurons, only the choroid neurons exhibit the muscarinic depression, and only during embryonic development. The pharmacological properties of the relevant receptor are different from those of the muscarinic receptor involved in presynaptic inhibition in adult autonomic systems. Receptor-mediated, synaptic failure during development may serve to protect immature postsynaptic neurons from potentially toxic overstimulation.

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.