Actions of guanine nucleotides and cyclic nucleotides on calcium stores in single patch‐clamped smooth muscle cells from rabbit portal vein

Abstract

1. Single smooth muscle cells were obtained from the rabbit portal vein by enzymic digestion and membrane currents under voltage clamp measured by whole-cell patch clamp technique. 2. When held at depolarized potentials, spontaneous outward currents (STOCs) were discharged; it is likely that these represent the cyclical storage and release within the cell of calcium in relation to Ca-activated K-channels. 3. Application of lower concentrations of carbachol (10(-5)M) or caffeine (10(-3)M) accelerated STOC discharge. Higher concentrations of caffeine (10(-2)M) or carbachol (10(-4)M), or noradrenaline (10(-5)M), produced an outward current of 1-5 nA which disappeared within 5-15s and which was considered to result from the discharge of calcium stores; STOC discharge was abolished for a period. 4. Ryanodine (10(-5)-10(-4)M) or a non-hydrolysable GTP analogue, GTP gamma S (10(-5)-10(-3)M) introduced into the cell abolished STOC discharge within 2-5 min. STOCs were large in cells filled with GDP beta S (10(-3)M) and the action of GTP gamma S introduced at various concentrations was antagonized. 5. GTP gamma S (10(-4)-10(-3)M) in the cell reduced or abolished outward current to caffeine (10(-2)M) noradrenaline (10(-5)M) or carbachol (10(-4)M); the effect on caffeine outward current was antagonized by GDP beta S (10(-3)M) introduced into the cell. GDP beta S reduced noradrenaline outward current but not caffeine outward current implying the existence of a G-protein step in noradrenaline-evoked Ca-store release, possibly regulating phospholipase C enzyme activity and D-myo inositol 1,4,5 trisphosphate formation. 6. If cyclic AMP (10(-3)M) or cyclic GMP (10(-3)M) was introduced into the cell, or 8-bromo cyclic AMP (0.5 x 10(-3)M) or 8-bromo cyclic GMP (0.5 x 10(-3)M) applied to the cell in the bathing solution, STOC discharge was only slightly affected. However, the outward current to caffeine applied after noradrenaline was much enhanced. 7. The results could be explained if cyclic GMP and cyclic AMP enhance calcium storage whereas GTP gamma S depletes calcium stores, an action antagonized by GDP beta S.