This study examined the role of Ca2+ in regulatory volume decrease by Necturus erythrocytes. Hypotonic shock (50% tonicity)stimulated an increase in cytosolic free Ca2+, detected using epi-fluorescence microscopy and the fluorescent Ca2+ indicator fluo-4-AM (10 μM). A similar increase in fluorescence did not occur under isosmotic conditions, unless cells were exposed to the Ca2+ionophore A23187 (0.5 μM). In addition, a low Ca2+ medium(amphibian Ringer solution with 5 mM EGTA), hexokinase (2.5 U/ml, an ATP scavenger), suramin (100 μM, a P2 receptor antagonist) and gadolinium (10μM, a stretch-activated channel blocker) each inhibited the swelling-induced increase in Ca2+. Consistent with these studies, a low Ca2+ Ringer solution increased osmotic fragility, whereas volume recovery following hypotonic shock (measured with a Coulter counter)was potentiated with A23187 (0.5 μM). By contrast, a low Ca2+extracellular medium or buffering intracellular Ca2+ with BAPTA-AM(100 μM) reduced the rate of volume recovery following hypotonic challenge. Finally, a low Ca2+ extracellular Ringer solution inhibited whole-cell currents that are activated during cell swelling (measured with the whole-cell patch clamp technique). Our results are most consistent with hypotonic shock causing an increase in cytosolic free Ca2+, thereby stimulating subsequent volume decrease.

We have carried out a series of experiments in which the lipid composition of the photosynthetic membrane has been altered by the addition of lipid from a defined source under experimental conditions . Liposomes prepared by sonication are mixed with purified photosynthetic membranes obtained from spinach chloroplasts and are taken through cycles of freezing and thawing. Several lines of evidence, including gel electrophoresis and freezefracture electron microscopy, indicate that an actual addition of lipid has taken place. Structural analysis by freeze-fracture shows that intramembrane particles are widely separated after the addition of large amounts of lipid, with one exception: large hexagonal lattices of particles appear in some regions of the membrane . These lattices are identical in appearance with lattices formed from a single purified component of the membrane known as chlorophyllprotein complex II . The suggestion that the presence of such lattices in lipid-enriched membranes reflects a profound rearrangement of photosynthetic structures has been confirmed by analysis of the fluorescence emission spectra of natural and lipid-enriched membranes. Specifically, lipid addition in each of the cases we have studied results in the apparent detachment of chlorophyll-protein complex II from photosynthetic reaction centers. It is concluded that specific arrangements of components in the photosynthetic membrane, necessary for the normal functioning of the membrane in the light reaction of photosynthesis, can be regulated to a large extent by the lipid content of the membrane .The photosynthetic membrane provides an ideal system for the study of the relationship between membrane structure and function. It is characterized by light-driven reactions which are easily assayed, as well as by a defined and highly ordered ultrastructure . The membranous sacs, or thylakoids, of the chloroplasts of higher plants have been extensively investigated by a number of workers (3,4,19). These membranes contain the machinery necessary for the light reaction of photosynthesis, involving the transport of electrons from water to appropriate acceptor molecules via a chain of electron carriers . The fundamental components of the system are two reaction centers, termed photosystem I and photosystem II . Associated with each photosystem are chlorophyll, carotenoids, and other accessory pigments.Based on freeze-fracture electron microscopy and the use of mutants or deficient plants, a rough model of the organization of the photosynthetic membrane has been constructed . For example, tobacco plants deficient in photosystem 11 lack the large (140-180 Á) particles normally observed on the so-called