Abstract
This study examined the source of Ca2+ mobilized by phorbol esters and its requirement for phorbol-induced contraction of smooth muscle cells isolated from the circular and longitudinal layers of guinea pig intestine. Phorbol-12-myristate-13-acetate caused rapid, sustained, concentration-dependent muscle contraction and increase in cystolic free [Ca2+]i in muscle cells from both layers. Maximal contraction was similar to that elicited by receptor-linked agonists, whereas maximal [Ca2+]i was 50% less. The increase in [Ca2+]i was mediated by Ca2+release in circular, and Ca2+ influx in longitudinal muscle cells; only the latter was abolished by methoxyverapamil and in Ca2+-free medium. [Ca2+]i was essential for contraction in both cell types: contraction in longitudinal muscle cells was abolished by methoxyverapamil and in Ca2+-free medium; contraction in circular muscle cells was abolished only after depletion of Ca2+ stores. Contraction was abolished by the protein kinase C (PKC) inhibitor calphostin C (1 μM), but was not affected by the myosin light chain kinase inhibitor KT5926 (1 μM), suggesting that activation of myosin light chain kinase was suppressed by phorbol-12-myristate-13-acetate or via PKC. Phorbol-induced contraction of permeabilized circular and longitudinal muscle cells was abolished by pretreatment with a common antibody to Ca2+-dependent PKC-α,β,γ, but was not affected by pretreatment with a specific PKC-ε antibody. This study demonstrates the ability of phorbol esters to mobilize Ca2+from different sources in different smooth muscle cell types and establishes the requirement of Ca2+ for phorbol-induced contraction; the latter is exclusively mediated by Ca2+-dependent PKC isozymes.
Footnotes
-
Send reprint requests to: Gabriel M. Makhlouf, M.D., Ph.D., P.O. Box 980711, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0711. E-mail: makhlouf{at}hsc.vcu.edu
-
↵1 This work was supported by Grant DK15564 from the National Institute of Diabetes and Digestive and Kidney Diseases.
- Abbreviations:
- DAG
- diacylglycerol
- PKC
- protein kinase C
- PLA2
- phospholipase A2
- PMA
- phorbol-12-myristate-13-acetate
- PDBu
- phorbol 12,13-dibutyrate
- D600
- methoxyverapamil
- KT5926
- (8R*,9S*,11S*)-(−)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9,10-tetrahydro-8,11-epoxy,1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one
- AACOF3
- arachidonyltrifluoromethyl ketone
- PI
- phosphoinositide
- U73122
- 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrole-2,5-dione
- MLCK
- myosin light chain kinase
- CCK-8
- cholecystokinin octapeptide
- Received January 28, 2000.
- Accepted May 17, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|