It is controversial whether changes in adenosine 3',5'-cyclic monophosphate (cAMP) and in the cAMP-to-guanosine 3',5'-cyclic monophosphate (cGMP) ratio are involved with cell swelling and in the activation of volume-regulatory mechanisms. We examined whether these nucleotides are involved in cell volume regulation in skeletal muscle. Isolated (intact and internally perfused) barnacle muscle cells were used because these cells, when exposed to a hyposmotic environment, undergo an extracellular Ca2+ (Cao)-dependent regulatory volume decrease (RVD). Using intact cells we found that dibutyryl cAMP and forskolin significantly promoted RVD in cells exposed to Cao-free solutions and that dibutyryl cGMP significantly inhibited RVD in cells exposed to Cao-containing solutions. In perfused cells in which the intracellular free Ca2+ concentration ([Ca2+]i) was heavily buffered [with 8 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)], cAMP induced a volume loss that was inhibited by presence of cGMP. Furthermore, if perfused cells were exposed to hyposmotic conditions, they swelled and underwent RVD provided that [Ca2+]i buffering was low (with 2 mM EGTA). This effect was inhibited by presence of the cAMP antagonist, [R]-p-adenosine 3',5'-cyclic monophosphorothioate.

Isosmotic removal of extracellular Na+ (Nao) is a frequently performed manipulation. With the use of isolated voltage-clamped barnacle muscle cells, the effect of this manipulation on isosmotic cell volume was studied. Replacement of Nao by tris(hydroxymethyl)aminomethane produced membrane depolarization (approximately 20 mV) and cell volume loss (approximately 14%). The membrane depolarization was verapamil insensitive but depended on extracellular Ca2+ (Cao) and was probably due to activation of intracellular Ca2+ (Cai)-dependent nonselective cation channels. The cell volume loss did not require membrane depolarization but depended on Cao. This was probably due to an increase in Cai, mediated by activation of Ca2+ influx via Na+/Ca2+ exchange. Nao replacement by Li+ also promoted membrane depolarization (approximately 20 mV) and cell volume loss (20%). Both effects were reduced (approximately 73%) but were not abolished by Cao removal. Under this condition, the remaining membrane depolarization was probably due to a higher membrane permeability of Li+ over Na+. The remaining cell volume loss was due to membrane depolarization, which probably induced Ca2+ release from intracellular stores.

Isosmotic removal of extracellular Ca2+ (Cao) and changes in membrane potential (Vm) are frequently performed manipulations. Using isolated voltage-clamped barnacle muscle cells, we studied the effect of these manipulations on isosmotic cell volume. Replacing Cao by Mg2+ induced 1) verapamil-sensitive extracellular Na(+)-dependent membrane depolarization, 2) membrane depolarization-dependent cell volume reduction in cells whose sarcoplasmic reticulum (SR) was presumably loaded with Ca2+ [intracellular Ca2+ (Cai)-loaded cells], and 3) cell volume increase in cells whose SR was presumably depleted of Ca2+ (Cai-depleted cells) or in Cai-loaded cells whose Vm was held constant. Membrane depolarization induced 1) volume reduction in Cai-loaded cells or 2) verapamil-sensitive volume increase in Cai-depleted cells. This suggests tha, in Cai-loaded cells, membrane depolarization induces SR Ca2+ release, which in turn promotes volume reduction. Conversely, in Cai-depleted cells, the depolarization activates Na+ influx through a verapamil-sensitive pathway leading to the volume increase. This pathway is also revealed when Cao is removed in either Cai-depleted cells or in cells whose Vm is held constant.