Identification of thoracic intervertebral spaces by means of surface anatomy: a magnetic resonance imaging study.

BACKGROUND: Successful thoracic epidural analgesia depends on the sensory blockage of specific dermatomes following appropriate placement of the epidural catheter. This study aimed to ascertain how accurately anaesthesiologists identify thoracic intervertebral spaces, and whether counting from the prominent vertebra is easier than using the iliac crest as an anatomical landmark. METHODS: Five anaesthesiologists attempted to locate one out of five consecutive intervertebral spaces (Th7-Th8 to Th11-Th12) on patients referred for magnetic resonance imaging of the vertebral column. The intended thoracic interspace and the counting reference point (C7-Th1 or L3-L4) were marked with oil capsules. The body mass index, gender and position of the patient were recorded. The exact capsule positions were determined by a radiologist after the study. RESULTS: In 92 patients, 26.7% of the thoracic interspaces were correctly identified. The counting reference point was the only variable studied with a significant influence on error. The accuracy increased when the iliac crest was used as an anatomical landmark rather than the prominent vertebra (odds ratio, 0.29). The majority (76.4%) of all the incorrectly placed capsules were found cephalad to the intended level. CONCLUSION: We recommend that the caudal of two to three possible interspaces should be used when placing an epidural catheter in the thoracic spine. Because of the inaccurate localization of the thoracic intervertebral spaces, documentation should state the site of puncture as being in the upper or lower thoracic spine instead of claiming to be in an exact interspace.

Delineation of an endogenous zinc-binding site in the human dopamine transporter.

The molecular basis for substrate translocation in the Na+/Cl--dependent neurotransmitter transporters remains elusive. Here we report novel insight into the translocation mechanism by delineation of an endogenous Zn2+-binding site in the human dopamine transporter (hDAT). In micromolar concentrations, Zn2+ was found to act as a potent, non-competitive blocker of dopamine uptake in COS cells expressing hDAT. In contrast, binding of the cocaine analogue, WIN 35,428, was markedly potentiated by Zn2+. Surprisingly, these effects were not observed in the closely related human norepinephrine transporter (hNET). A single non-conserved histidine residue (His193) in the large second extracellular loop (ECL2) of hDAT was discovered to be responsible for this difference. Thus, Zn2+ modulation could be conveyed to hNET by mutational transfer of only this residue. His375 conserved between hDAT and hNET, present in the fourth extracellular loop (ECL4) at the top of transmembrane segment VII, was identified as a second major coordinate for Zn2+ binding. These data provide evidence for spatial proximity between His193 and His375 in hDAT, representing the first experimentally demonstrated proximity relationship in an Na+/Cl--dependent transporter. Since Zn2+ did not prevent dopamine binding, but inhibited dopamine translocation, our data suggest that by constraining movements of ECL2 and ECL4, Zn2+ can restrict a conformational change critical for the transport process.

Calcium regulation of porcine aortic myosin.

Calcium regulation of actin-activated porcine aortic myosin MgATPase was studied. The MgATPase of the purified actomyosin was stimulated about 10-fold by 0.1 mM Ca2+. The 20,000 molecular weight light chain subunit (LC20) of myosin was phosphorylated by an endogenous kinase that required Ca2+. Half-maximal activation of both kinase and ATPase occurred at about 0.9 microM Ca2+. Phosphorylated and unphosphorylated myosins, free of actin, kinase, and phosphatase, were purified by gel filtration. The MgATPase of phosphorylated myosin was activated by rabbit skeletal muscle actin; unphosphorylated myosin was actin activated to a much lesser extent. Actin activation was maximal in the presence of Ca2+. Regulation of the aortic myosin MgATPase seems to involve both direct interaction of calcium with phosphorylated myosin and calcium activation of the myosin kinase. The MgATPase of trypsin-treated actomyosin did not require Ca2+ for full activity. The trypsin-treated actomyosin was devoid of LC20. When purified unphosphorylated aortic myosin was treated with trypsin, the LC20, was cleaved and the MgATPase, which was not appreciably actin activated before exposure to protease, was increased and was activated by skeletal muscle actin. After incubation of this light chain-depleted myosin with light chain from rabbit skeletal muscle myosin, the actin activation but not the increased activity, was abolished. Unphosphorylated LC20 seems to inhibit actin activation in this smooth muscle.

Physical and enzymatic properties of myosin from porcine brain.

Porcine brain myosin is a cytoplasmic protein similar to, but distinct from, its muscle counterpart. It has a high K+-ATPase activity at high ionic strength in EDTA and a low Mg+2-ATPase activity that is activated fivefold by either porcine brain or rabbit skeletal muscle actin. The molecule consists of three classes of subunits, with molecular weights of approximately 195,000 , 19,000, and 16,000. Brain myosin contains less glutamic acid, less lysine, and more threonine, serine, proline, and tyrosine than skeletal muscle myosin. The brain myosin extinction coefficient at 278 nm is 0.810 cm2/mg. Hydrodynamic studies yield an S020,w of 4.95S, a D020,w of 1.07 x 10(-7) cm2/s for brain myosin, and indicate that the molecules aggregate at high ionic strength. The molecular weight of the molecule, as calculated from extrapolation of D020,w/S20,w to zero concentration, is 444,000. The intrinsic viscosity of brain myosin is 0.191 ml/mg. These data are consistent with a highly asymmetric molecular species. Circular dichroism spectroscopy indicates that brain myosin is 58-60% alpha-helical in the presence of Ca+2 ions, and that removal of Ca+2 causes a small change in the spectrum.

Physical properties of myosin from aortic smooth muscle.

Porcine aortic myosin is a smooth muscle contractile protein similar to its striated muscle counterpart. Electrophoresis in sodium dodecyl sulfate indicates that the molecule consists of three classes of subunits with polypeptide chain molecular weights of 192,000, 19,000, and 15,000. At 277 nm the absorption spectrum gives an extinction coefficient for aortic myosin of 0.558 cm2/mg; the circular dichroism spectrum of the protein indicates that aortic myosin contains about 70% of its residues in the alpha-helical configuration. Amino acid analysis shows that the smooth muscle myosin has significantly more arginine and leucine and significantly less valine and isoleucine than rabbit skeletal muscle myosin. Other studies yielded these data: Vapp = 0.716 mL/g [eta] = 0.213 mL/mg, S20, w = 5.84 x 10(-13)S. Similar studies with rabbit skeletal muscle myosin indicate that Vapp = 0.711 mL/g and S20, w = 6.36 x 10(-13)S. These properties suggest that aortic myosin, like skeletal muscle myosin, behaves hydrodynamically like a rigid rod.

Myosin-mediated Ca++-regulation of actomyosin-adenosinetriphosphatase from porcine aorta.

The nature of the Ca++-sensitive regulatory system for contraction of vascular smooth muscle is considered in detail. Smooth muscle actomyosin prepared from the medial layer of porcine aorta is analyzed chemically and its ATPase (adenosinetriphosphatase, EC 3.6.1.14) activities are investigated. The Mg++-ATPase of this vascular actomyosin is sensitive to the concentration of calcium in the range from 0.1 mM to 10 nM. The calcium sensitivity is maintained in the presence of excess pure actin from skeletal muscle and is abolished in the presence of pure skeletal myosin. It is concluded that the regulatory properties of this natural actomyosin from smooth muscle are in the myosin portion of the protein complex and are not bound to actin-tropomyosin as in skeletal muscle.