Modulation of Vascular O2 Responses by Cytochrome 450‐4A ω‐Hydroxylase Metabolites In Dahl Salt‐Sensitive Rats

Objective: This study evaluated the role of the 20‐HETE/cytochrome P450‐4A ω‐hydroxylase (CYP450‐4A) system in microvascular regulation in the skeletal muscle circulation following short‐term (three‐day) exposure to a high‐salt (HS) diet in Dahl salt‐sensitive (SS) rats. Methods: The effects of inhibiting CYP450‐4A on resting diameter, O₂‐induced constriction, and vasodilator responses to acetylcholine (ACh) and the nitric oxide (NO) donor, sodium nitroprusside (SNP), were evaluated in cremasteric arterioles of SS rats fed a low‐ (LS; 0.4% NaCl) or high‐salt (HS; 4% NaCl) diet for three days. Results: The HS diet upregulated CYP450‐4A mRNA expression and led to an enhanced constriction of arterioles in response to elevated PO₂ in SS rats, which could be blocked by inhibiting CYP450‐4A enzymes with dibromododecenyl methylsulfimide (DDMS). DDMS also inhibited resting tone significantly in SS rats fed the HS, but not the LS, diet, despite similar resting diameters and active tone in the two groups. Arteriolar dilations in response to ACh and SNP were similar in SS rats fed the LS vs. the HS diet and were unaffected by DDMS. Conclusions: These findings suggest that CYP450‐4A enzymes contribute to resting tone and to an enhanced response to elevated PO₂ in arterioles of Dahl‐SS rats fed the HS diet.     

Preparation of clear solutions of reconstituted acetylcholinesterase Effect of ionic strength and lipid/protein ratio

The detergent-soluble globular dimer of acetylcholinesterase from Torpedo californica was reconstituted through dialysis into preformed egg phosphatidylcholine vesicles. The formation of the enzyme-lipid complexes depended on the ionic strength of the dialysis buffer as well as the molar lipid/protein ratio (R). The enzyme was unstable at I < 0.05; increasing the ionic strength increased the size of the complex. A too low R value (e.g. 1000) would promote self-aggregation of the enzyme and produce heterogeneous complexes, especially at high I values. On the other hand, a too high R value (e.g. > 5000) favored the formation of large enzyme-lipid complexes; their solutions were too turbid for optical studies. The enzyme reconstituted at I = 0.07 and R = 4000 gave a clear solution and showed no artifacts due to light scattering. The conformation based on circular dichroism and enzymatic activity of the detergent-soluble enzyme were unchanged upon reconstitution. The reconstituted enzyme in lipid vesicles seemed to be slightly more stable against thermal denaturation than the protein in sodium cholate solution.     

17O and 14 N n.m.r. studies of the Co (II) interaction with cyclo(Ala*-Ala) in aqueous solution

The complexation of cyclo(Ala*-Ala) with the cobaltous ions in aqueous solution was investigated by ¹⁷O and ¹⁴N n.m.r. spectroscopy. The ¹⁷O and ¹⁴N transverse relaxation time (T_2p) and chemical shift (Δω_a) of cyclo(Ala*-Ala) were measured as a function of the temperature at pH = 7.03 ± 0.02, and pH = 6.45 ± 0.02, and as a function of pH at room temperature. No effects of pH on the transverse relaxation time and chemical shift were observed. Complementary ¹⁷O studies of the solvent water molecules were also carried out. The hyperfine coupling constant and the entropy and enthalpy of activation for the exchange of cyclo(Ala*-Ala) and water molecules between the coordinated and noncoordinated states were determined by least-square fit of theoretical equation for the chemical shift Δω_a to experimental data. The hyperfine coupling constant of the peptide bound oxygen was determined to be (– 1.6 ± 0.1) ± 10⁵ Hz and the entropy and enthalpy (32.0 ± 3.0) kJ/mol and (– 12.0 ± 1.0)e.u, respectively. Information obtained from ¹⁷O n.m.r. study allows some inferences concerning the probable coordination sphere of the cobaltous ion. There are three types of complexes: Co(H₂O)²⁺₆, CoL ± 5H₂O and CoL₂ ± 4H₂O, with relative concentrations 19.9%, 2.9%, and 77.2%, respectively. The ¹⁷O n.m.r. line broadening is due to the presence of a minor species, CoL ± 5H₂O, in rapid exchange and not due to the major species, CoL₂ ± 4H₂O, in solution, which has a large ±t_m. ¹⁴N n.m.r. transverse relaxation rates are unaffected by the cobaltous ion, indicating either that the cobaltous ions do not bind to the nitrogen peptide bond or that the rate of exchange is very slow and does not affect the free peptide.     

Subthreshold response to white-noise current input in a tapering cable model of a neuron

^* Author to whom all correspondence should be addressed The expectation (mean) and variance of the depolarization inthe absence of a threshold for action-potential generation isobtained in a neuron model represented by a tapering equivalentcable with a random (white noise) synaptic input current ata point along the dendrites. The results show that the introductionof a taper in the equivalent-cable representation of the neuronproduces larger values for both the expectation and variancewhich are neither insensitive nor symmetrical with respect tothe location of the input. It is also shown that taper extendsthe invariance of the variability in the steady-state somaticresponse to proximally located random inputs, implying thatonly small changes in the noisiness of the somatic responseoccur for a random input located in the dendrites.