Effects of octopamine on miniature excitatory junction potentials from developing and adult moth muscle

Intracellular recordings of excitatory junction potentials (EJPs) and miniature EJPs (MEJPs) were made from the dorsal longitudinal muscle of Manduca sexta to determine the sites of action of octopamine. MEJPs increased in amplitude and frequency as the moth developed during the 3 days before eclosion. DL-Octopamine (5 X 10(-6) M) increased the amplitude of excitatory junction potentials in both immature moths (one day before eclosion) and adults. Octopamine (10(-5) M) also increased the amplitude and frequency of MEJPs from immature animals (one and two days before eclosion) but had the opposite effect on adults and pharate adults ready to eclose. Treatment with octopamine (10(-5) M) resulted in a decrease in input resistance and a hyperpolarization in both immature and adult muscle fibers. The results suggest that octopamine acts both presynaptically and postsynaptically but that the increase in the amplitude of the evoked response is due primarily to influences on presynaptic processes.     

氯离子通道阻断剂对豚鼠耳蜗螺旋动脉平滑肌细胞兴奋性接头电位的影响

血管平滑肌细胞膜上存在氯离子通道,不仅参与调节平滑肌细胞的肌原性紧张,而且参与多种血管床的神经平滑肌细胞之间的信息传递,但氯离子通道及其阻断剂对耳蜗螺旋动脉（spiral modiol arartery,SMA）平滑肌细胞兴奋性接头电位（excitatory junction potential,EJP）是否有影响,尚不清楚。本实验运用细胞内微电极记录技术,在豚鼠耳蜗SMA离体标本上,研究氯通道阻断剂（niflumic acid,NFA,indanyloxyacetic acid 94,IAA-94;disodium 4,4’-diisothiocyanatostilbene-2．2’-disulfonate,DIDS）对去甲肾上腺素（norepinephrine,NE）引起SMA平滑肌细胞去极化反应和平滑肌细胞EJP的影响。结果显示,多数SMA平滑肌细胞在适宜的刺激下,通过神经兴奋传递产生EJP（75％,n=49）。在联合使用α1（prazosin,0．1-1 μmol／L）,α2（idazoxan,0．3-1μmol／L）和P2x（PPADS,10-100μmol／L）受体拮抗剂时,所产生的EJP幅值仅有30％-80％被抑制。在使用上述拮抗剂的基础上,NFA（10-1000μmol/L）能进一步抑制EJP,而且缩短EJP的时程。减少细胞外氯离子浓度（由135．6mmol／L减少到60mmol／L）,在同样刺激强度下激起的EJP的幅度和时程均增加,低氯的这一作用可被IAA-94和DIDS所反转。NFA和IAA-94也可进一步抑制α1、α2和β受体拮抗剂联合使用不能消除的NE（1—50μmol／L）引起的去极化反应。结果提示：NE可能通过激活一类非α、非β肾上腺能受体（可能属于γ肾上腺能受体）引起氯离子通道开放,增加氯离子电导,调节耳蜗SMA平滑肌细胞的生理活动。     

Integrin on developing and adult skeletal muscle

Avian integrin is a complex of integral membrane glycoproteins that appears to function as a dual receptors for both intracellular cytoskeletal and extracellular matrix components. Antibodies were raised against this complex and used to (1) immunolocalize integrin on cryosections of developing and adult muscle tissue and on developing myotube cultures in vitro and (2) immunoaffinity purify integrin from various fiber-type specific muscles. Integrin localization was compared with that of its putative cytoskeletal-associated and extracellular matrix ligands, talin and vinculin and fibronectin and laminin, respectively. The goal was to identify putative sites of interaction between the muscle sarcolemma and the cytoskeleton and the extracellular matrix and to reveal any differences in the molecular composition at these sites. Integrin's distribution on the sarcolemma of early (Day 12) embryonic limb muscle was random and punctate. On late embryonic (Days 17-19) limb muscle tissue its distribution was generally uniform but with occasional increased densities at specific sites along the sarcolemma. Posthatch (greater than 3 weeks) fast twitch muscle showed a highly regionalized distribution. These regions of integrin concentration coincided with densities of acetylcholine receptors, revealed by TRITC alpha-bungarotoxin labeling, and regions of muscle-tendon interaction, identified by morphological criteria. Tissue culture studies also demonstrated integrin densities at analogous sites in vitro, e.g., acetylcholine receptor clusters and sites at which myofibrils terminate at the sarcolemma. These integrin-rich sites were also shown to be Triton X-100 insoluble and therefore presumably are linked to the cytoskeleton or extracellular matrix. The localization of integrin on developing and adult muscle tissue was compared with that of fibronectin, laminin, vinculin, and talin using double, immunofluorescently labeled cryosections. In general, integrin did not colocalize exclusively with any one of its putative ligands. In the embryo, discrete densities of both talin and vinculin were observed at the myotendinous junction, whereas integrin immunoreactivity was widely distributed on muscle, vasculature, nerve, and connective tissue with no discernible sites of increased density. Laminin was primarily associated with muscle and nerve whereas fibronectin was prominent on connective tissue. On posthatch tissue, the distributions of talin, vinculin, laminin, and fibronectin were similar to those in the embryo, whereas the distribution of integrin was restricted to specific sites. The distribution of integrin was also examined for fiber-type specific differences on adu     

Immunolocalization of tight junction proteins in the adult and developing human brain

The formation of endothelial tight junctions (TJs) is crucial in blood-brain barrier (BBB) differentiation, and the expression and targeting of TJ-associated proteins mark the beginning of BBB functions. Using confocal microscopy, this study analyzed endothelial TJs in adult human cerebral cortex and the fetal telencephalon and leptomeninges in order to compare the localization of two TJ-associated transmembrane proteins, occludin and claudin-5. In the arterioles and microvessels of adult brain, occludin and claudin-5 form continuous bands of endothelial immunoreactivity. During fetal development, occludin and claudin-5 immunoreactivity is first detected as a diffuse labeling of endothelial cytoplasm. Later, at 14 weeks, the immunosignal for both proteins shifts from the cytoplasm to the interface of adjacent endothelial cells, forming a linear, widely discontinuous pattern of immunoreactivity that achieves an adult-like appearance within a few weeks. These results demonstrate that occludin and claudin-5 expression is an early event in human brain development, followed shortly by assembly of both proteins at the junctional areas. This incremental process suggests more rapid establishment of the human BBB, consistent with its specific function of creating a suitable environment for neuron differentiation and neurite outgrowth during neocortical histogenesis.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00418-004-0665-1Daniela Virgintino and Mariella Errede contributed equally to this work     

Glial cells in the developing and adult olfactory lobe of the moth Manduca sexta

The antennal lobe of the moth contains several classes of glial cells that are likely to play functional roles in both the developing and mature lobe. In this study, confocal and electron microscopy were used to examine in detail the morphology of two classes of glial cells, those associated with olfactory receptor axons as they course to their targets in the lobe and those that form borders around the synaptic neuropil of the olfactory glomeruli. The former, the nerve-layer glia, have long processes with multiple expansions that enwrap axon fascicles; the latter, the neuropil glia, constitute two subgroups: complex glia with large cell bodies and branching, vellate arbors; and simple glia, with multiple, mostly unbranched processes with many lamellate expansions along their lengths. The processes of complex glia appear to be closely associated with axon fascicles as they enter the glomeruli, while those of the simple glia surround the glomeruli as part of a multi-lamellar glial envelope, their processes rarely invading the synaptic neuropil of the body of the glomerulus. The full morphological development of antennal-lobe glial cells requires more than two-thirds of metamorphic development. During this period, cells that began as cuboidal or spindle-shaped cells that were extensively dye-coupled to one another gradually assume their adult form and, at least under nonstimulated conditions, greatly reduce their coupling. These changes are only weakly dependent on the presence of olfactory receptor axons. Glial processes are somewhat shorter and less branched in the absence of these axons, but basic structure and degree of dye-coupling are unchanged.     

Glial cells in adult and developing prothoracic ganglion of the hawk moth Manduca sexta

The glial cells of the prothoracic ganglion of the hawk moth Manduca sexta were studied in histological sections of several postembryonic stages and classified according to cell morphology, size, staining properties, and topographical relationships. In general, each glial cell type was found to be confined to one of the major ganglionic domains and each of these domains (i.e., perineurium, cell body rind, glial cover of the neuropil, and neuropil) was found to comprise specific cell types. Some types of glia were recognized in both larval and later stages, but other types were found exclusively from late pupal stages. It is proposed that the higher morphological diversity expressed by the glia of the pharate adult is attained by differentiation of new cell types during metamorphosis. Before the differentiation of new cell types, extensive cell death and cell proliferation seem to occur within some glial subpopulations.     

Two classes of spontaneous miniature excitatory junction potentials and one synaptic vesicle class are present in the ray electrocyte

Summary Cross sections (1–2 mm thick) of the ray (Raja) tail were secured to a dish and immersed in elasmobranch saline. Spontaneous miniature excitatory junction potentials (MEJPs) were recorded by advancing a 50 k, KCl filled electrode into the electric organ (20 V peak-to-peak baseline noise). Data were filmed, and/or recorded on magnetic tape for computer analyses. Intracellularly recorded MEJP amplitude histograms showed a peak at 60 V and had a right-hand skew with MEJPs up to 0.5 mV. The small peak amplitude and the skewed amplitude distribution of intracellularly recorded MEJPs result from the relatively low input resistance and the short space constant of the electrocyte coupled with the dispersed synapses on the electrocyte. At 23 °C the intracellularly recorded MEJP frequency ranged from 1–10 MEJPs/s. The MEJPs became larger and became focally recorded as the electrode was advanced against the intracellular surface of the innervated membrane of the electrocyte. Focal extracellular MEJPs (reversed polarity) were also recorded with the electrode positioned against the outside surface of the innervated side of the electrocyte. The frequency of focally recorded intracellular MEJPs was increased (up to 40/s) when the electrode was pushed against the membrane. Focal MEJP frequencies decreased to a few/min within 5–10 min but the mean amplitude of 3–5 mV remained constant. Decreases in amplitude and frequency in focally recorded intracellular MEJPs are attributed to changes in electrode pressure against the membrane. Amplitude histograms were constructed from focally recorded intracellular or extracellular MEJPs which showed the same time characteristics. The focal MEJP amplitude histograms have two distinct classes, each forming a bell-shaped distribution. It is concluded that both classes are generated at the electrode tip. The smaller class of MEJPs has a mean 1/10th that of the larger class and composes about 2% of the MEJPs. The small class is analogous to the sub-MEPP class found in the frog sartorius (Kriebel and Gross 1974) and mouse diaphragm (Kriebel et al. 1976, 1982). Distributions of synaptic vesicle diameters are slightly log normal (right hand skew) such that the mean diameter (57 nm) is slightly larger than the modal value (52 nm). Vesicles touching the membrane were of the same size and diameter distribution as the entire vesicle population. The profiles of the distributions are smooth and suggest only 1 class of synaptic vesicle based on diameter.     

Effects of hyaluronidase on miniature endplate potentials and currents at the frog neuromuscular junction

The effects of 0.1% testicular hyaluronidase on miniature endplate potentials and currents (MEPP and MEPC) were investigated in frog pectorocutaneous muscle. The action of hyaluronidase on preparations with armine-induced blockade of acetylcholinesterase was associated with decreased amplitude and duration of MEPP and MEPC half-decay time and rising phase. The correlation between amplitude and half-decay time of MEPP and MEPC declined at the same time, while MEPC decay remained exponential. Treating preparations having intact acetylcholinesterase with hyaluronidase increased the length of MEPC halfdecay, with duration of the rising phase and amplitude remaining constant. It is suggested that enzymatic breakdown of a proportion of the glycocalix of cells forming the neuromuscular junction and a portion of the extracellular matrix at the synaptic cleft leads to attenuation of nonspectific acetylcholine binding, thus facilitating acetylcholine diffusion into the synaptic cleft.A. A. Zhdanov State University, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 113–119, January–February, 1988.     

Effects of forskolin and cilostazol on clot retraction

We examined the effects of two inhibitors of aggregation of human platelets the, Forskolin and Cilostazol on clot retraction. Both substances suppressed clot retraction in a dose-dependent way. Both suppress platelet aggregation because of an increase in intercellular cyclic AMP, but there was no close correlations were shown between suppression rate for clot retraction and cyclic-AMP content in platelets in the clot in each substance. Furthermore, although it has been considered that actomyosin in platelets is a major contractile source for clot retraction and that failure of actin polymerization results suppression of clot retraction. As it was difficult to obtain active actin from platelets of the reagents on the polymerization. Cilostazol accelerated actin polymerization, whereas Forskolin did not. From these findings, it was considered that the effects of both substances on clot retraction could not be interpreted directly just by the increasing effect for intracellular cyclic-AMP. Clot retraction is consider to be a in vitro model of hemostasis and its contractile force is supplied from platelets (1,2,3). Experiments used prostaglandin E-1 revealed that elevation of cyclic-AMP (c-AMP) would regulate the clot retraction, and experiments used cytochalasin B demonstrated that actomyosin is responsible to the retraction (4,5). Many date demonstrate that elevation of c-AMP level suppresses platelet aggregation (6,7). c-AMP, therefore, should play a key role on platelet activation. Forskolin and Cilostazol are newly-developed reagents as a suppress for platelet functions. Pharmacological action of these substances have been interpreted to process increase effect for intracellular c-AMP of platelets(8,9). If so, both substances should show some effect on clot retraction. Under this assumption, we examined the effects.     

Effects of bicarbonate on resting potentials in mammalian skeletal muscle

In the absence of external HCO3, resting membrane potentials (Vm) in extensor digitorum longus muscle were depolarized as compared to the normal Vm in the presence of HCO3. Removal of Na or Cl form the HCO3-free media induced repolarization. In muscle in HCO3 buffer at 20 degrees C, internal K, Na, and Cl activities were analyzed with liquid ion selective microelectrodes. The averages were respectively, 119.7 +/- 2.1, 6.69 +/- 0.3, and 3.41 +/- 0.06 mM. In a high proportion of cells analyzed, the equilibrium potential for Cl was negative to Vm. Removing external HCO3, decreased internal K while internal Na and Cl increased. An increase in temperature and the application of HCO3 significantly lowered internal activities of both Na and Cl. Removal of HCO3 with temperature held constant caused a rapid depolarization, an increase in internal Na and Cl, and a decrease in internal K. Furosemide (10 microM) induced a repolarization of cells that were previously depolarized in the HCO3-free state, but the drug does not decrease internal Na.     

Alpha smooth muscle actin expression in developing and adult human lung

Abstract. Myofibroblast-like cells containing smooth muscle actin have been identified in lung injury and repair. These cells differ from typical smooth muscle cells by architectural configuration, location and lack of smooth muscle myosin. Their progenitors are unknown. We hypothesized that these cells might have a developmental analog critical to lung morphogenesis. Lung tissue from developing and adult human lungs was studied using a highly specific monoclonal antibody directed against alpha smooth muscle actin (ASMA). Cells im-munoreactive for ASMA (ASMA cells) were identified prenatally in the form of smooth muscle investing the developing vasculature and airway structures. ASMA was not expressed in undifferentiated mesenchymal cells at any prenatal stage. Late in development, ASMA cells within the lung acinus increased proportionally to terminal airway and vascular complexity. In the early postnatal period, the specific distribution of ASMA cells within inflated lung became clearer, and three populations were identified: (1) typical smooth muscle investing the large airways and blood vessels; (2) small clusters of cells with in the acinus distributed at the tips of septa protruding into the alveolar duct; (3) individual cells within the alveolar sac sparsely distributed near the junctions of individual alveoli, frequently in association with small blood vessels. We conclude that ASMA cells appear only in developing small and large airways and pulmonary vessels and that they may play a critical role in branching morphogenesis during development.     

Myosin heavy-chain isoforms in adult and developing rabbit vascular smooth muscle

Two monoclonal antibodies specific for smooth muscle myosin (designated SM-E7 and SM-A9) and one monoclonal anti-(human platelet myosin) antibody (designated NM-G2) have been used to study myosin heavy chain composition of smooth muscle cells in adult and in developing rabbit aorta. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting experiments revealed that adult aortic muscle consisted of two myosin heavy chains (MCH) of smooth muscle type, named MHC-1 (205 kDa), and MHC-2 (200 kDa). In the fetal/neonatal stage of development, vascular smooth muscle was found to contain only MHC-1 but not MHC-2. Non-muscle myosin heavy chain, which showed the same electrophoretic mobility as the slower migrating MHC, was expressed in an inverse manner with respect to MHC-2, i.e. it was detectable only in the early stages of development. The distinct pattern of smooth and non-muscle myosin isoform expression during development may be related to the different functional properties of smooth muscle cells during vascular myogenesis.