Korean J Physiol Pharmacol.  2009 Jun;13(3):189-194. 10.4196/kjpp.2009.13.3.189.

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

Affiliations
  • 1Department of Internal Medicine, Gangnung Asan Hospital, Gangnung 210-711, Korea.
  • 2Department of Physiology, College of Medicine, Kwandong University, Gangneung 210-751, Korea. skwon2028@kd.ac.kr

Abstract

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca2+ level ([Ca2+]i), and Ca2+ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 micrometer) inhibited high K+ (25 mM and 40 mM)- or histamine (3 micrometer)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K+-evoked contractions. Spontaneous changes in [Ca2+]i and contractions were inhibited by forskolin (1 micrometer) without changing the resting [Ca2+]i. Forskoln (10 micrometer) inhibited muscle tension more strongly than [Ca2+]i stimulated by high K+, and thus shifted the [Ca2+]i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 micrometer) inhibited both [Ca2+]i and muscle tension without changing the [Ca2+]i-tension relationship. In alpha-toxin-permeabilized tissues, forskolin (10 micrometer) inhibited the 0.3 micrometer Ca2+-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca2+-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca2+ sensitivity of contractile elements in high K+-stimulated muscle and a decrease in [Ca2+]i in histamine-stimulated muscle.

Keyword

Fosrkolin; Cytosolic Ca2+; Ca2+ sensitivity; Guinea pig ileum

MeSH Terms

Animals
Contracts
Cytosol
Forskolin
Guanosine Triphosphate
Guinea
Guinea Pigs
Histamine
Ileum
Muscle Tonus
Muscle, Smooth
Muscles
Forskolin
Guanosine Triphosphate
Histamine

Figure

  • Fig. 1. Concentration-response relationship for the inhibitory effect of forskolin on contractions induced by 25 mM KCl, 40 mM KCl, and 3 μM histamine. Forskolin (0.1 nM~10 μM) was cumulatively added after the contractions reached a steady state.

  • Fig. 2. The effect of forskolin (1 μM) on [Ca2+]i (upper trace) and muscle tension (lower trace) in spontaneously active ileum. 100% represents the steady state [Ca2+]i in the presence of 40 mM KCl. Forskolin (1 μM) inhibited rhythmic increases in [Ca2+]i and tension without changing the basal [Ca2+]i. EGTA (4 mM) decreased basal [Ca2+]i below the resting level with no further decrease in muscle tension.

  • Fig. 3. Effects of forskolin on 40 mM KCl (A)- and 3 μM histamine (B)-stimulated [Ca2+]i (upper trace) and muscle tension (lower trace). 100% represents the 40 mM KCl- or histamine-induced increases in [Ca2+]i before addition of forskolin. In panel A, after 40 mM KCl-stimulated [Ca2+]i and muscle tension reached a steady state level, 10 μM forskolin and verapamil were sequentially added. Forskolin partially inhibited contraction without changing [Ca2+]i. Verapamil (10 μM), on the other hand, inhibited [Ca2+]i and tension to the resting level. In panel B, when the [Ca2+]i and muscle tension induced by histamine (3 μM) reached a steady state level, 1 μM forskolin and 10 μM verapamil were sequentially added. Forskolin (1 μM) significantly inhibited [Ca2+]i and contractions. Verapamil (10 μM) inhibited [Ca2+]i and tension to the resting level.

  • Fig. 4. Effect of forskolin (10 μM, open circles) on the relationship between [Ca2+]i (abscissa) and muscle tension (ordinate) in the presence various concentrations of KCl (10, 15, 20, 30, and 40 mM) or histamine (0.03, 0.1, 0.3, 1, and 3 μM). 100% represents 40 mM KCl-induced increases in [Ca2+]i and muscle tension measured before cumulative addition of the stimuli. Each point represents the mean of 7~10 experiments, and the SR mean is shown by vertical and horizontal bars.

  • Fig. 5. (A) Effect of 10 μM forskolin on the contraction evoked by Ca2+ in α-toxin-permeabilized ileum. Ca2+ was cumulatively applied. The experiments were repeated after application of 10 μM forskolin. As a control, 10 μM Ca2+ was applied. (B) The pCa2+-tension relationship observed before (•) and after application of 10 μM forskolin (○). The amplitude of 10 μM Ca2+ was taken as 100%. Each point represents the mean of 6 experiments and the SE mean is shown by a vertical bar. (C) Effects of 10 μM forskolin treated with 0.1 mM GTP. After permebilizing the tissues, 0.3 μM Ca2+ was applied. After the contraction induced by 0.3 μM Ca2+ had reached a steady level, 0.1 mM GTP and forskolin were sequentially applied.


Reference

Adelstein RS., Conti MA., Hathaway DR., Klee CB. Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem. 253:8347–8350. 1978.
Ahn HY., Kang SE., Chang KC., Karaki H. Dibutyryl cyclic AMP and forskolin inhibit phosphatidyl-inositol hydrolysis, Ca2+ influx and contraction in vascular smooth muscle. Jpn J Pharmacol. 59:263–265. 1992.
Andersson R., Nilsson K. Role of cyclic nucleotides: metabolism and mechanical activity in smooth muscle. In Biochemistry of Smooth Muscle (edited by Stephens NL), 263−291. 1977.
Bhalla RC., Webb RC., Singh D., Brock T. Role of cyclic AMP in rat aortic microsomal phosphorylation and calcium uptake. Am J Physiol. 234:H508–H514. 1978.
Article
Bulbring E., Tomita T. Catecholamine action on smooth muscle. Pharmacol Rev. 39:49–96. 1987.
Cooper DM. Molecular and cellular requirements for the regulation adenylate cyclases by calcium. Biochem Soc Trans. 31:912–915. 2003.
Eckly-Michel A., Martin V., Lugnier C. Involvement of cyclic nucleotide-dependent protein kinases in cyclic AMP-mediated vasorelaxation. Br J Pharmacol. 122:158–164. 1997.
Article
Hall IP., Donaldson J., Hill SJ. Inhibition od histamine-stimulated inositol phospholipids hydrolysis by agents which increase cyclic AMP levels in bovine tracheal smooth muscle. Br J Pharmacol. 97:603–613. 1989.
Ise S., Nishimura J., Hirano K., Hara N., Kanaide H. Theophylline attenuates Ca2+ sensitivity and modulates BK channels in porcine tracheal smooth muscle. Br J Pharmacol. 140:939–947. 2003.
Karaki H. Localization and sensitivity in vascular smooth muscle. Trends Pharmacol Sci. 10:320–325. 1989.
Karaki H., Weiss GB. Calcium channels in smooth muscle. Gastroenterology. 87:960–970. 1984.
Article
Kwon SC., Ozaki H., Hori M., Karaki H. Isoproterenol changes the relationship between cytosolic Ca2+ and contraction in guineapig taneia caecum. Jpn J Pharmacol. 61:57–64. 1993.
Kwon SC., Ozaki H., Karaki H. NO donor sodium nitroprusside inhibits excitation-contraction coupling in guinea pig taenia coli. Am J Physiol. 279:G1235–G1241. 2000a.
Article
Kwon SC., Park KY., Ahn DS., Lee HY., Kang BS. The effect of NO donor on contraction, cytosolic Ca2+ level and ionic currents in guinea-pig ileal smooth muscle. Korean J Physiol Pharmacol. 4:33–40. 2000b.
Kushida M., Takeuchi T., Fujita A., Hata F. Dependence of Ca2+- induced contraction in a-toxin-permeabilized preparations of rat femoral artery. J Pharmacol Sci. 93:171–179. 2003.
Lincoln TM., Cornwell TL. Towards an understanding of the mechanism of action of cyclic AMP and cyclic GMP in smooth muscle relaxation. Blood Vessels. 28:129–137. 1991.
Article
Morales S., Camello PJ., Mawe GM., Pozo MJ. Cyclic AMP-mediated inhibition of gallbladder contractility: role of K+ channel activation and Ca2+ handling. Br J Pharmacol. 143:994–1005. 2004.
Murray KJ. Cyclic AMP and mechanism of vasodilation. Pharmacol Ther. 47:329–354. 1990.
Murthy KS. Signaling for contraction and relaxation in smooth muscle of the gut. Annu Rev Physiol. 68:345–374. 2006.
Article
Nishikori K., Maeno H. Close relationship between adenosine3': 5'-monophosphate-dependent endogenous phosphorylation of a specific protein and stimulation of calcium uptake in rat uterine microsomes. J Biol Chem. 254:6099–6106. 1979.
Nishimura J., van Breemen C. Direct regulation of smooth muscle contractile elements by second messengers. Biochem Biophys Res Commun. 163:929–935. 1989.
Article
Ozaki H., Blondfield DP., Hori M., Sanders KM., Publicover NG. Cyclic AMP-mediated regulation of excitation-contraction coupling in canine gastric smooth muscle. J Physiol. 447:351–372. 1992.
Article
Ozaki H., Hori M., Kim YS., Kwon SC., Ahn DS., Nakazawa H., Kobaysahi M., Karaki H. Inhibitory mechanism of xestospongin-C on contraction and ion channels in the intestinal smooth muscle. Br J Pharmacol. 137:1207–1212. 2002.
Article
Pfitzer G., Hofmann F., DiSalvo J., Ruegg JC. cGMP and cAMP inhibit tension development in skinned coronary arteries. Pflugers Arch. 401:277–280. 1984.
Article
Porter M., Evans MC., Miner AS., Berg KM., Ward KR., Ratz PH. Convergence of Ca2+-desensitizing mechanisms activated by forskolin and phenylephrine, but not 8-bromo-cGMP. Am J Physiol. 290:C1552–C1559. 2006.
Ratz PH. Receptor activation induces short-term modulation of arterial sontractions: memory in vascular smooth muscle. Am J Physiol. 269:C417–C423. 1999.
Rembold CM., Chen XL. Mechanisms responsible for forskolin-induced relaxation of rat tail artery. Hypertension. 31:872–877. 1998.
Article
Rembold CM., O'Connor MJ., Clarkson M., Wardle RL., Murphy RA. HSP20 phosphorylation in nitroglycerin- and forskolin-induced sustained reductions in swine carotid artery. J Appl Physiol. 91:1460–1466. 2001.
Seamon KB., Daly JW. Forskolin: its biological and chemical properties. Adv Cyclic Nucleotide Protein Phosphoryl Res. 20:1–150. 1986.
Wellman GC., Santana LF., Bonev AD., Nelson MT. Role of phospholamban in the modulation of arterial Ca2+ sparks and Ca2+-activated K+ channels by cAMP. Am J Physiol. 281:C1029–C1037. 2001.
Woodrum DA., Brophy CM., Wingard CJ., Beall A., Rasmussen H. Phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. Am J Physiol. 277:H931–H939. 1999.
Article
Full Text Links
  • KJPP
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr