Modulation of the calcium-force relationship in smooth muscle by polyamines and metabolic inhibition

Abstract: The relationship between intracellular free calcium ([Ca2+]i) and force in smooth muscle was investigated in receptor-coupled permeabilized preparations and by fluorimetry of the calcium indicator Fura 2 in intact smooth muscle. The time integral of spontaneous [Ca2+]i transients in the intact rat portal vein was decreased by cyanide and, during tonic contraction, cyanide reduced force without changing [Ca2+]i implying metabolic sensitivity of both membrane excitation and [Ca2+]i - activation of the contractile proteins. In permeabilized, receptor-coupled, smooth muscle of guinea pig ileum, the intracellular polyamines spermine and spermidine, ubiquitous cations connected to cellular growth, sensitized force to [Ca2+] at concentrations corresponding to the tissue contents and also inhibited transient contractions induced by release of calcium from intracellular stores. Spermine was found to increase phosphorylation of the regulatory 20 kDa light chains (LC20) by a mechanism involving inhibition of myosin phosphatase activity. Sensitization of force to [Ca2+] depended on this effect. Culture of intestinal smooth muscle with serum reduced responsiveness of force to extracellular Ca2+. This effect was prevented by the calcium channel blocker verapamil and mimicked by the calcium ionophore ionomycin, indicating dependence on chronically raised [Ca2+]i. Stimulated [Ca2+]i transients and calcium current density, as determined with the patch-clamp technique in isolated cells, were both significantly reduced compared to controls whereas the [Ca2+]i -force relationship was unaffected. The adenosylmethionine decarbyxylase inhibitor CGP 48664 reduced the contents of spermine and spermidine by 50 % in cultured guinea pig ileum strips. A high concentration of the inhibitor enhanced phasic spontaneous contractile activity and sensitivity to muscarinic stimulation and reduced the responsiveness of force to [Ca2+]i implying a reduced influence of endogenous polyamines on contractile activation. These results demonstrates influnces of energy- and polyamine-metabolism and Ca2+-homeostasis, on excitation-contraction coupling in smooth muscle.