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Background The investigation examined whether primary alcohols could be used as tools to explore the mechanism of anesthetic actions in airway smooth muscle (ASM). The hypothesis was that, like volatile anesthetics, the primary alcohols relax intact ASM by decreasing intracellular Ca2+ concentration ([Ca2+]i) and by inhibiting agonist-induced increases in the force developed for a given [Ca2+]i (Ca2+ sensitivity). Method The effects of butanol, hexanol, and octanol on isometric force in canine tracheal smooth muscle were examined. The effects of hexanol on [Ca2+]i (measured with fura-2) and the relationship between force and [Ca2+]i were studied during membrane depolarization provided by KCl and during muscarinic stimulation provided by acetylcholine. Results The primary alcohols relaxed ASM contracted by KCl or acetylcholine in a concentration-dependent manner, with potency increasing as chain length increased. The alcohols could completely relax the strips, even during maximal stimulation with 10 microM acetylcholine (median effective concentrations of 28 +/- 12, 1.3 +/- 0.4, and 0.14 +/- 0.05 mM [mean +/- SD] for butanol, hexanol, and octanol, respectively). Hexanol decreased both [Ca2+]i and force in a concentration-dependent manner. Hexanol decreased Ca2+ sensitivity during muscarinic stimulation but had no effect on the force-[Ca2+]i relationship in its absence. Conclusions Primary alcohols produce reversible, complete relaxation of ASM, with potency increasing as chain length increases, by decreasing [Ca2+]i and inhibiting increases in Ca2+ sensitivity produced by muscarinic receptor stimulation. These actions mimic those of volatile anesthetics on ASM, a circumstance suggesting that the primary alcohols may be useful tools for further exploring mechanisms of anesthetic effects on ASM.

1. The effects of the NH2-terminal fragments of M130, a 130 kDa regulatory subunit of smooth muscle myosin phosphatase, on contraction and myosin light chain phosphorylation were investigated in Triton X-100-permeabilized porcine renal artery. 2. Incubation of the permeabilized fibres with M1301-633 (a fragment containing amino acid residues 1-633) or M13044-633 enhanced the Ca2+-induced contraction and shifted the [Ca2+]i-force relationship to the left (EC50 of Ca2+: 330 nM, control, without fragment; 145 nM, M1301-633; 163 nM, M13044-633). Pre-incubation for 1-3 h was needed for these long constructs. 3. M1301-374, M130304-511 and M130297-374, i.e. relatively short constructs compared with M1301-633 and M13044-633, also induced leftward shifts of the [Ca2+]i-force relationship (EC50 of Ca2+: 65 nM, 72 nM and 180 nM, respectively). However, these required no pre-incubation. 4. Deletion of residues 304-374 from the most potent construct, M1301-374, abolished the Ca2+-sensitizing effect. 5. Wortmannin inhibited the enhancement of contraction induced by M130 fragments when added before contraction was initiated and partially inhibited the effects when added after steady-state contraction. 6. M1301-374 slowed the rate of relaxation in Ca2+-free medium. The time for 50 % relaxation with this fragment was 510 +/- 51 s, compared with 274 +/- 14 s for control. 7. The levels of myosin light chain phosphorylation (22.4 %) and force (34. 5 %) obtained with 300 nM Ca2+ were increased by 3 microM M1301-374 to 35.7 and 92.2 %, respectively. However, M1301-374 had no effect on the phosphorylation-force relationship. 8. In conclusion, the NH2-terminal M130 fragments containing residues 304-374 inhibited myosin phosphatase, increased myosin light chain phosphorylation and increased the Ca2+ sensitivity of the contractile apparatus in permeabilized porcine renal artery.

It has recently been indicated that rho A and rho kinase may be involved in the mechanism for the increase in Ca2+sensitivity in smooth muscle myofilaments. In the present study, we investigated the mRNA expression of rho A and rho kinase in porcine vascular smooth muscle cells (aorta, coronary artery, pulmonary artery, and pulmonary vein). In reverse transcription–polymerase chain reaction (RT–PCR) experiments, using total RNA from these tissue specimens and the primers designed in the conserved regions of each mRNA in human and bovine, rho A and rho kinase mRNAs were detected in all of these smooth muscle cells. An analysis of these PCR products by direct sequencing indicated that they were derived from each mRNA. The finding that both rho A and rho kinase mRNAs were expressed in the various porcine vascular smooth muscle cells strongly supports the idea that these proteins are involved in the mechanism dealing with the increase in Ca2+sensitivity in the myofilaments of vascular smooth muscle cells.

Background Anesthetics inhibit airway smooth muscle contraction in part by a direct effect on the smooth muscle cell. This study tested the hypothesis that the anesthetics halothane and hexanol, which both relax airway smooth muscle in vitro, inhibit acetylcholine-promoted nucleotide exchange at the alpha subunit of the Gq/11 heterotrimeric G protein (Galphaq/11; i.e., they inhibit muscarinic receptor-Galphaq/11 coupling). Methods The effect of halothane (0.38 +/- 0.02 mm) and hexanol (10 mm) on basal and acetylcholine-stimulated Galphaq/11 guanosine nucleotide exchange was determined in membranes prepared from porcine tracheal smooth muscle. The nonhydrolyzable, radioactive form of guanosine-5'-triphosphate, [S]GTPgammaS, was used as the reporter for Galphaq/11 subunit dissociation from the membrane to soluble fraction, which was immunoprecipitated with rabbit polyclonal anti-Galphaq/11 antiserum. Results Acetylcholine caused a significant time- and concentration-dependent increase in the magnitude of Galphaq/11 nucleotide exchange compared with basal values (i.e., without acetylcholine), reaching a maximal difference at 100 microm (35.9 +/-2.9 vs. 9.8 +/-1.2 fmol/mg protein, respectively). Whereas neither anesthetic had an effect on basal Galphaq/11 nucleotide exchange, both halothane and hexanol significantly inhibited the increase in Galphaq/11 nucleotide exchange produced by 30 microm acetylcholine (by 59% and 68%, respectively). Conclusions Halothane and hexanol interact with the receptor-heterotrimeric G-protein complex in a manner that prevents acetylcholine-promoted exchange of guanosine-5(')-triphosphate for guanosine-5'-diphosphate at Galphaq/11. These data are consistent with the ability of anesthetics to interfere with cellular processes mediated by heterotrimeric G proteins in many cells, including effects on muscarinic receptor-G-protein regulation of airway smooth muscle contraction.

Background Chlorpromazine has been widely used by anesthesiologists to take advantage of its anesthesia-potentiating and vasorelaxing actions. However, the mechanisms of vasorelaxation induced by chlorpromazine are still not fully understood. Methods Using front-surface fluorometry of fura-2 and porcine pulmonary arterial strips, we investigated the effects of chlorpromazine on the intracellular Ca2+ concentration ([Ca2+]i) and force of vascular smooth muscle. The affinities of chlorpromazine and other neuroleptics to vascular alpha(1)-adrenergic receptors were then determined by a radio-ligand binding study. Results Chlorpromazine (as much as 1 microM) inhibited both the elevation of [Ca2+]i and force in pulmonary arterial smooth muscle induced by 80 mM K+-depolarization and 1 microM norepinephrine in a concentration-dependent manner. The extent of inhibition by chlorpromazine in norepinephrine-induced contraction was much greater than that in 80 mM K+-induced contraction. In contrast, as much as 1 microM chlorpromazine had no effect on the increases in [Ca2+]i or force induced by U46619, a thromboxane A2 analogue. Chlorpromazine also had no effect on the intracellular Ca2+ release induced by U46619. In a radio-ligand displacement study, chlorpromazine, haloperidol, phentolamine, trifluoperazine, and imipramine inhibited the specific binding of [3H]prazosin to the porcine aortic membranes, in this order of potency. Conclusions Chlorpromazine induces vasorelaxation through an alpha-adrenergic blocking action as well as a calcium antagonistic action; the former action may, therefore, play a major role in chlorpromazine-induced vasorelaxation.

Using reverse transcription polymerase chain reaction (RT-PCR), we determined the expression of the mRNAs for basic calponin isoforms, namely h 1 and h 2 forms, in the porcine aortic endothelial cells (PAECs) and aortic smooth muscle cells (PASMCs). The mRNAs for both h 1 and h 2 calponin isoforms were expressed in PAECs as well as in PASMCs. However, the expression of h 2 isoform mRNA was more abundant than h 1 isoform in PAECs, while h 1 isoform was more abundant than h 2 in PASMCs. These findings indicated the existence of calponin mRNA and the predominance of h 2 over h 1 calponin isoform in PAECs. It is suggested that h 2 as well as h 1 , calponin isoform might have a physiological role in vascular endothelial cell. This is the first report which describes the existence of calponin h 2 -dominant cells.