title: Arachidonic acid and diacylglycerol release associated with inhibition of myosin light chain dephosphorylation in rabbit smooth muscle.
abstract: 1. Exogenous arachidonic acid (AA) inhibits the protein phosphatase that dephosphorylates smooth muscle myosin, thus sensitizing the contractile response to Ca2+; it also inhibits voltage-gated Ca2+ channels in smooth muscle. The purpose of the present study was to determine whether endogenous AA is increased by agonists in a manner consistent with its role as a messenger regulating myosin phosphatase and Ca2+ channels. Both AA and diacylglycerol (DAG) were measured in [3H]AA-labelled intact and permeabilized (with staphylococcal alpha-toxin) rabbit femoral arteries stimulated with the alpha 1-adrenergic agonist phenylephrine (PE) (intact and permeabilized smooth muscles) or by guanosine-5'-O-(3-thiotriphosphate (GTP gamma S; permeabilized smooth muscles in which the [Ca2+] was maintained constant). Arachidonic acid mass was determined with gas chromatography and mass spectrometry (GC-MS). 2. In intact smooth muscle, PE increased both AA and DAG levels significantly, to 210 and 145% of baseline values, respectively. Another Ca2+-sensitizing agent, the thromboxane analogue U46619, caused a similar increase in AA and DAG levels in rabbit pulmonary artery. 3. In permeabilized smooth muscle at constant [Ca2+](pCa 6.5) GTP gamma S-induced AA and DAG release preceded force development and GTP gamma S (50 microM, 10 min) increased AA mass to 61-88 microM. 4. Phorbol-12,13-dibutyrate (PDBu), another Ca2+-sensitizing agent, also increased both AA and DAG levels in permeabilized smooth muscle at pCa 6.5, whereas the inactive analogue, 4 alpha-phorbol, did not have a Ca2+-sensitizing effect, nor did it increase AA and DAG levels. 5. In the virtual absence of Ca2+ (pCa > 8) GTP gamma S also increased AA and DAG levels by 3.5- and 1.6-fold, respectively. The effect of free Ca2+ itself on AA and DAG release was modest in the physiological range (pCa 7.0 to pCa 6.0), but pCa 4.5 caused an approximately 3- to 4-fold increase in AA and DAG levels, compared with the levels at pCa 8. In permeabilized ileum smooth muscle maintained at constant [Ca2+] (pCa 6.0), carbachol also significantly increased AA to 1.75 times its original value within 1 min of its application. 6. Our results are consistent with, although do not prove, the roles of AA and DAG as second and/or co-messenger(s) in smooth muscle, while the increases in AA and DAG levels induced by PDBu raise the possibility that they contribute to some of the cellular effects of phorbol esters.
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia Medical Center, Charlottesville, 22908, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Gong
authors.forename: M T
authors.initials: MT
authors.lastname: Kinter
authors.forename: A V
authors.initials: AV
authors.lastname: Somlyo
authors.forename: A P
authors.initials: AP
authors.lastname: Somlyo
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D004075:Diglycerides / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000502:physiology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D010656:Phenylephrine / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011817:Rabbits; D010738:Type C Phospholipases / Q000494:pharmacology; D014662:Vasoconstrictor Agents / Q000494:pharmacology
chemical_list: D002120:Calcium Channel Agonists; D004075:Diglycerides; D018994:Myosin Light Chains; D014662:Vasoconstrictor Agents; D010656:Phenylephrine; D016718:Arachidonic Acid; D015240:Phorbol 12,13-Dibutyrate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases