Sidney K. Pierce

INVERTEBRATE CELL VOLUME CONTROL MECHANISMS: A COORDINATED USE OF INTRACELLULAR AMINO ACIDS AND INORGANIC IONS AS OSMOTIC SOLUTE

All cells have some capacity for cell volume regulation when confronted with a hypoosmotic stress. The basis of this physiological response is an extrusion of intracellularosmotic solute. The cells of euryhaline osmoconforming invertebrates are capable of regulating volume over a wide range of external osmotic concentra tions. Most of the existing data indicate that these cells utilize free amino acids from a substantial intracellular pool as the solute source. However, recent studies indicate that these invertebrate cells utilize inorganic ions as osmotic solute as well. The relative contribution of each solute type varies from species to species and, perhaps, from cell type to cell type. The two solute types are regulated by different mechanisms and often with different time courses, but both solute control systems function in a coordinated manner to regulate cell volume. In addition, evidence is appearing demonstrating a role for organic solutes in the volume regulatory processes of ver tebrate cells. At present, it seems that the volume regulatory mechanisms utilized by all cells may be more similar than currently thought, differing in relative con tributions of the two solute types rather than kind of solute utilized.

TWO CELL VOLUME REGULATORY SYSTEMS IN THE LIMULUS MYOCARDIUM: AN INTERACTION OF IONS AND QUATERNARY AMMONIUM COMPOUNDS

The horseshoe crab Limulus polyphemus is extremely euryhaline. Previous studies have shown it surviving in salinities ranging from 6% to 200% sea water. Blood osmotic concentration is hyperregulated in low salinities, but above 65% sea water Limulus is an osmoconformer. Limulus regulates cell volume when exposed to low salinity, despite a small intracellular free amino acid pool. Instead, the quaternary ammonium compound glycine betaine is the major nitrogenous osmotic solute in Limulus heart tissue. However, volume regulation is complete before intracellular glycine betaine concentrations change. Isolated heart tissue exposed to low salinity shows no change in glycine betaine levels in 24 h though volume regulation occurs. During the initial phase of volume regulation intracellular Na+ and Cl- content in the isolated tissue decreases markedly with exposure to low salinity. Therefore, Limulus utilizes two osmotic solute types during cell volume regulation: Na+ and Cl- initially and glycine betaine later.

The time course of intracellular free amino acid accumulation in tissues of Modiolus demissus during high salinity adaptations

Abstract 1. 1. The total amino acid pool increased in ventricles and gills removed from M. demissus during adaptation to high salinity. However, each major amino acid had a unique time course of accumulation. 2. 2. Initially, alanine concentration increased rapidly followed by slower accumulations of glycine and taurine. Alanine declined as the other amino acids increased. Thus the pool size remained constant following the initial elevation. 3. 3. Proline, undetected at first, increased after 1 day in the high salinity. Eventually, proline concentrations peaked, declined and disappeared again. 4. 4. Mantle tissue showed a different pattern of amino acid accumulation from the other tissues. Although alanine accumulated first, followed by glycine and proline, taurine did not increase. 5. 5. A pattern of amino acid accumulation similar to that in tissues from the whole animal was found in isolated gills and ventricles during high salinity stress.

Transcriptomic evidence for the expression of horizontally transferred algal nuclear genes in the photosynthetic sea slug, Elysia chlorotica.

Analysis of the transcriptome of the kleptoplastic sea slug, Elysia chlorotica, has revealed the presence of at least 101 chloroplast-encoded gene sequences and 111 transcripts matching 52 nuclear-encoded genes from the chloroplast donor, Vaucheria litorea. These data clearly show that the symbiotic chloroplasts are translationally active and, of even more interest, that a variety of functional algal genes have been transferred into the slug genome, as has been suggested by earlier indirect experiments. Both the chloroplast- and nuclear-encoded sequences were rare within the E. chlorotica transcriptome, suggesting that their copy numbers and synthesis rates are low, and required both a large amount of sequence data and native algal sequences to find. These results show that the symbiotic chloroplasts residing inside the host molluscan cell are maintained by an interaction of both organellar and host biochemistry directed by the presence of transferred genes.

Cellular volume regulation in salinity stressed molluscs: The response ofNoetia ponderosa (Arcidae) red blood cells to osmotic variation

SummarySalinity stressed marine molluscs volumeregulate utilizing intracellular free amino acids. However, the actual changes in cell volume associated with free amino acid regulation are usually nerver measured or only indirectly measured since volume regulation has only been studied in whole animals or isolated tissues. The blood cells ofNoetia ponderosa (Mollusca: Arcidae) were used to specify the relationship between cell size changes and free amino acid permeability during volume regulation. Over the nonlethal salinity range,N. ponderosa is an osmotic conformer that volume regulates using free amino acids. Furthermore, when changes in blood cell size were measured with hematocrits, isolatedN. ponderosa blood cells placed into hypoosmotic seawater, volume regulated with a free amino acid efflux. Thus, the isolatedN. ponderosa blood cells provide a suitable preparation for studying the control of membrane amino acid permeability during volume regulation.

Free amino acid mediated volume regulation of isolatedNoetia ponderosa red blood cells: Control by Ca2+ and ATP

SummaryThe ionic and metabolic requirements of cellular volume regulation and the free amino acid (FAA) efflux from hypoosmotically stressedNoetia ponderosa (Mollusca: Arcidae) red blood cells was studied. Deletion of Ca2+ from 50% ASW prevented cell volume regulation and decreased the FAA efflux. Addition of Co2+, Mn2+, or La3+ to 50% ASW increased volume regulation and the FAA efflux, while verapamil, a Ca2+ antagonist, inhibited volume regulation and the FAA efflux. Volume regulation by the blood cells has a metabolic component also since DNP or incubation of the cells at 4°C both inhibited volume regulation and the FAA efflux. Thus, the FAA permeability ofN. ponderosa blood cell membranes can be manipulated by altering seawater [Ca2+] or by indirectly modifying intracellular levels of ATP.

Hypoosmotic Cell Volume Regulation in Marine Bivalves: The Effects of Membrane Potential Change and Metabolic Inhibition

Molluscan cells volume regulate in dilute salinities by releasing amino acids from an intracellular pool. The magnitude of this efflux depends upon external divalent cation concentration. Incubation of isolated Modiolus demissus ventricles in a K⁺ solution isosmotic to 100% seawater (SW) results in membrane depolarization and an increased amino acid efflux. In the presence of Ca²⁺-Mg²⁺-free SW, the myocardial cell membrane is depolarized and an amino acid efflux occurs. Additional removal of Na⁺ repolarizes the membrane and reduces the amino acid release. Cyanide or 2,4-DNP in 50% SW result in a prolonged amino acid efflux from the isolated hearts. Decreased temperature markedly reduces the hypoosmotically induced amino acid efflux. These results indicate that the control mechanism of the amino acid release from molluscan cells stressed by hypoosmotic salinities depends upon external divalent cation concentration, membrane permeability, and ATP.

Annual Viral Expression in a Sea Slug Population: Life Cycle Control and Symbiotic Chloroplast Maintenance

In a few well-known cases, animal population dynamics are regulated by cyclical infections of protists, bacteria, or viruses. In most of these cases, the pathogen persists in the environment, where it continues to infect some percentage of successive generations of the host organism. This persistent re-infection causes a long-lived decline, in either population size or cycle, to a level that depends upon pathogen density and infection level (1-4). We have discovered, on the basis of 9 years of observation, an annual viral expression in Elysia chlorotica, an ascoglossan sea slug, that coincides with the yearly, synchronized death of all the adults in the population. This coincidence of viral expression and mass death is ubiquitous, and it occurs in the laboratory as well as in the field. Our evidence also suggests that the viruses do not re-infect subsequent generations from an external pathogen pool, but are endogenous to the slug. We are led, finally, to the hypothesis that the viruses may be involved in the maintenance of symbiotic chloroplasts within the molluscan cells.

Synthesis of Several Light-Harvesting Complex I Polypeptides Is Blocked by Cycloheximide in Symbiotic Chloroplasts in the Sea Slug, Elysia chlorotica (Gould): A Case for Horizontal Gene Transfer Between Alga and Animal?

The chloroplast symbiosis between the ascoglossan (=Sacoglossa) sea slug Elysia chlorotica and plastids from the chromophytic alga Vaucheria litorea is the longest-lived relationship of its kind known, lasting up to 9 months. During this time, the plastids continue to photosynthesize in the absence of the algal nucleus at rates sufficient to meet the nutritional needs of the slugs. We have previously demonstrated that the synthesis of photosynthetic proteins occurs while the plastids reside within the diverticular cells of the slug. Here, we have identified several of these synthesized proteins as belonging to the nuclear-encoded family of polypeptides known as light-harvesting complex I (LHCI). The synthesis of LHCI is blocked by the cytosolic ribosomal inhibitor cycloheximide and proceeds in the presence of chloramphenicol, a plastid ribosome inhibitor, indicating that the gene encoding LHCI resides in the nuclear DNA of the slug. These results suggest that a horizontal transfer of the LHCI gene from the alga to the slug has taken place.

PROLINE BETAINE: A UNIQUE OSMOLYTE IN AN EXTREMELY EURYHALINE OSMOCONFORMER

The extremely euryhaline mollusc, Elysia chiorotica, does not utilize intracellular free amino acids for cell volume regulation during osmotic stress. Instead, Elysia utilizes an osmolyte previously unknown from animals, proline betaine. Although proline betaine occurs in some plants and Elysia forms a symbiosis with an algae, the proline betaine in Elysia seems to be a product of the animal.