George A. Gerencser

Electrical characteristics of isolated Aplysia californica intestine

Abstract 1. 1. Transmural potential difference and short-circuit current of intestinal sheets of Aplysia califonica were stable up to 5 hr. 2. 2. Transmural potential difference was serosa negative relative to the mucosa and the short-circuit current was consistent with a net active anion transport from mucosa to serosa. 3. 3. Transmural potential difference and short-circuit current were dependent upon the presence of sodium and chloride in the bathing medium. 4. 4. Transmural potential difference and short-circuit current were predominantly dependent upon aerobic metabolism, however a finite residual electrical component was dependent upon glycolytic energy. 5. 5. The major portion of the short-circuit current is carried by a net active chloride transfer from the mucosal to serosal compartments while the minor portion is carried by a net active sodium transfer in the same direction.

The Chloride Pump: A CI-Translocating P-Type ATPase

Three widely documented mechanisms of chloride transport across plasma membranes are anion-coupled antiport, sodium-coupled symport, and an electrochemical coupling process. No direct genetic evidence has yet been provided for primary active chloride transport despite numerous reports of cellular Cl(-)-stimulated adenosine triphosphate (ATP)ases coexisting in the same tissue with uphill chloride transport that could not be accounted for by the three common chloride transport processes. Cl(-)-stimulated ATPases are a common property of practically all biological cells, with the major location being of mitochondrial origin. It also appears that plasma membranes are sites of Cl(-)-stimulated ATPase activity. Recent studies of Cl(-)-stimulated ATPase activity and chloride transport in the same membrane system, including liposomes, suggest a mediation by the ATPase in net movement of chloride up its electrochemical gradient across plasma membranes. Further studies, especially from a molecular biological perspective, are required to confirm a direct transport role to plasma membrane-localized Cl(-)-stimulated ATPases.

ATP-dependent chloride transport in plasma membrane vesicles from Aplysia intestine

Abstract A Cl−-stimulated ATPase activity, which is sensitive to both thiocyanate and vanadate, has been localized to the plasma membrane of Aplysia enterocytes. Utilizing plasma membrane vesicles from Aplysia enterocytes, ATP stimulated Cl− uptake to approximately 2.5-times that of control in a Na+, K+ and HCO3−-free medium. This ATP-dependent Cl− uptake was sensitive to both thiocyanate and vanadate. These results are consistent with the hypothesis that the active Cl− absorptive process in Aplysia intestine could be a Cl−-stimulated ATPase found in the enterocyte plasma membrane.

Enhancement of sodium and chloride transport by monosaccharides in Aplysia californica intestine

Abstract 1. 1. Addition of d -glucose, d -galactose, or d -3-o-methyl glucose to the mucosal bathing medium stimulated rapid sustained increases in transmural potential difference (serosa negative) and short-circuit current. 2. 2. The change in short-circuit current increased curvilinearly with increasing concentrations of mucosal d -glucose. 3. 3. Addition of phlorizin to the mucosal bathing medium partially inhibited the d -glucose stimulation of transmural potential difference and short-circuit current. 4. 4. The major portion of the sugar-induced short-circuit current is carried by a net active chloride transfer from mucosa to serosa while the minor portion of the short-circuit current is wholly or predominantly carried by a net active sodium transfer from mucosa to serosa.

Reconstitution of a chloride-translocating ATPase from Aplysia californica gut

Basolateral membranes of Aplysia foregut epithelia contain both a Cl(-)-stimulated ATPase activity and an ATP-dependent Cl- transport. The protein responsible for both of these biochemical activities (Cl- pump) can be solubilized and reconstituted into liposomes with the aid of the detergent digitonin. Proteoliposomal Cl- pump activity was inhibited by vanadate.

Unstirred Water Layers in Rabbit Intestine: Effects of Pectin

Abstract Pectins have been shown to affect the absorption of several different nutrients in clinical studies; however, the mechanisms for decreased absorption have not been defined. A possibility not studied with regards to pectin, but previously demonstrated to be important in absorption, is the effect of change in the unstirred water layer. As the unstirred water layer increases in thickness, the rate of absorption decreases for certain nutrients. The effect of pectin on the unstirred water layer in the lumen of rabbit jejunum was examined by previously described techniques. It was observed that: (1) increases in pectin concentration resulted in an increased thickness of the unstirred water layer; (2) for any stir rate, the addition of pectin increased the thickness of the unstirred water layer; and (3) stir rate is inversely related to the thickness of the unstirred water layer. It was concluded from these results that pectin increases the thickness of the unstirred water layer in rabbit jejunum. This mechanism may explain, in part, the reduction of the rate of absorption of certain nutrients seen following pectin ingestion.

Thiocyanate inhibition of active chloride absortion in Aplysia intestine

This investigation was principally undertaken to examine the mechanism of active chloride absorption across the Aplysia californica intestine by using various inhibitors of ion transport. Isolated intestine, mounted between identical oxygenated sodium-free seawater solutions, maintained stable transmural potential differences (serosa negative) and short-circuit currents for several hours at 25 degrees C. The metabolic inhibitors, 2,4-dinitrophenol and fluoride, reduced both transmural potential difference and short-circuit current; however, the electrical characteristics were predominantly dependent upon glycolytic energy. The addition of thiocyanate to the mucosal solution inhibited both electrical characteristics in parallel, and this inhibition could be titrated according to the thiocyanate concentration. The short-circuit current was carried wholly by a net active chloride transfer from mucosa to serosa as determined by flux measurements. These results suggest that active chloride absorption may be mediated by a primary active transport process.

Chloride ATPase pumps in nature: do they exist?

Five widely documented mechanisms for chloride transport across biological membranes are known: anioncoupled antiport, Na+ and H+‐coupled symport, Cl− channels and an electrochemical coupling process. These transport processes for chloride are either secondarily active or are driven by the electrochemical gradient for chloride. Until recently, the evidence in favour of a primary active transport mechanism for chloride has been inconclusive despite numerous reports of cellular Cl−‐stimulated ATPases coexisting, in the same tissue, with uphill ATP‐dependent chloride transport. Cl−‐stimulated ATPase activity is a ubiquitous property of practically all cells with the major location being of mitochondrial origin. It also appears that plasma membranes are sites of Cl−‐stimulated ATPase pump activity. Recent studies of Cl−‐stimulated ATPase activity and ATP‐dependent chloride transport in the same plasma membrane system, including liposomes, strongly suggest a mediation by the ATPase in the net movement of chloride up its electrochemical gradient across the plasma membrane structure. Contemporary evidence points to the existence of Cl−‐ATPase pumps; however, these primary active transporters exist as either P‐, F‐ or V‐type ATPase pumps depending upon the tissue under study.

Heavy metal detoxification in crustacean epithelial lysosomes: role of anions in the compartmentalization process

SUMMARY Crustacean hepatopancreatic lysosomes are organelles of heavy metal sequestration and detoxification. Previous studies have shown that zinc uptake by lysosomal membrane vesicles (LMV) occurred by a vanadate- and thapsigargin-sensitive ATPase that was stimulated by a transmembrane proton gradient established by a co-localized V-ATPase associated with this organelle. In the present study, hepatopancreatic LMV from the American lobster Homarus americanus were prepared by standard centrifugation methods and 65Zn2+, 36Cl–, 35SO42– and 14C-oxalate2– were used to characterize the interactions between the metal and anions during vesicular detoxification events. Vesicles loaded with SO42– or PO43– led to a threefold greater steady-state accumulation of Zn2+ than similar vesicles loaded with mannitol, Cl– or oxalate2–. The stimulation of 65Zn2+ uptake by intravesicular sulfate was SO42– concentration dependent with a maximal enhancement at 500 μmol l–1. Zinc uptake in the presence of ATP was proton-gradient enhanced and electrogenic, exhibiting an apparent exchange stoichiometry of 1Zn+/3H+. 35SO42– and 14C-oxalate2– uptakes were both enhanced in vesicles loaded with intravesicular Cl– compared to vesicles containing mannitol, suggesting the presence of anion countertransport. 35SO42– influx was a sigmoidal function of external [SO42–] with 25 mmol l–1 internal [Cl–], or with several intravesicular pH values (e.g. 7.0, 8.0 and 9.0). In all instances Hill coefficients of approximately 2.0 were obtained, suggesting that 2 sulfate ions exchange with single Cl– or OH– ions. 36Cl– influx was a sigmoidal function of external [Cl–] with intravesicular pH of 7.0 and 9.0. A Hill coefficient of 2.0 was also obtained, suggesting the exchange of 2 Cl– for 1 OH–. 14C-oxalate influx was a hyperbolic function of external [oxalate2–] with 25 mmol l–1 internal [Cl–], suggesting a 1:1 exchange of oxalate2– for Cl–. As a group, these experiments suggest the presence of an anion exchange mechanism exchanging monovalent for polyvalent anions. Polyvalent inorganic anions (SO42– and PO43–) are known to associate with metals inside vesicles and a detoxification model is presented that suggests how these anions may contribute to concretion formation through precipitation with metals at appropriate vesicular pH.

Reconstituted Cl- pump protein: a novel ion(Cl-)-motive ATPase.

Cl− absorption by theAplysia californica foregut is effected through an active Cl− transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl−-stimulated ATPase and ATP-dependent Cl− transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg2+, Cl−, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl− accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycininduced K+ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl− accumulation and electrical potential while an outwardly-directed valinomycininduced K+ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl− accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate orp-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl− accumulation, intraliposomal negative potential difference, and also Cl−-stimulated ATPase activity. Both aspects of Cl− pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl−-ATPase actively transports Cl− by an electrogenic process.