Suresh K. Joseph

Neomycin: a specific drug to study the inositol-phospholipid signalling system?

Neomycin, an antibiotic previously thought to interact specifically with inositol‐containing phospholipids, was found to inhibit IP3‐mediated Ca2+ release from the intracellular stores of permeabilized insulinoma and liver cells. This inhibition could be relieved by increasing the IP3 concentration. Radiolabelled IP3 was found to bind tightly to columns prepared from neomycin covalently attached to glass beads. ATP was also bound by these colums. It is concluded that neomycin acts in biological systems as a weak anion exchanger and is therefore unsuitable for use as a specific tool to study the role of inositol phospholipids in intracellular signalling.

Subcellular localization and some properties of the enzymes hydrolysing inositol polyphosphates in rat liver

The hydrolysis of inositol [32P]trisphosphate (IP3) and inositol [32P]bisphosphate (IP2) has been examined in subcellular fractions of rat liver. IP3 was degraded by an enzyme located in the plasma membrane which did not degrade IP2. Cytosolic fractions were found to degrade both IP3 and IP3. The IP3 phosphatase activity of liver plasma membranes displayed a neutral pH optimum, was Mg2+ dependent and was not inhibited by high concentrations of Li+. Half‐maximal activity of the enzymes hydrolysing IP3 and IP2 was obtained with substrate concentrations in the range 1–2μM. The significance of these observations to the proposed Ca2+ ‐mobilizing role of IP3 is discussed.

Polyethylene glycol-stimulated microsomal GTP hydrolysis: Relationship to GTP-mediated Ca2+ release

It has recently been observed that GTP mediates Ca2+ release from internal Ca2+ stores. In contrast to effects on permeabilized cells, GTP‐dependent Ca2+ release in isolated microsomes requires the presence of polyethylene glycol (PEG). We have investigated the effects of PEG on microsomal GTPase activity and report that PEG stimulates a high‐affinity (K m = 0.9 μM) GTPase. The effects of PEG reflect an increase in the V max of this activity; no effects on K m were observed. The concentration dependence for PEG‐dependent stimulation of the high‐affinity GTPase exactly mimicked that for GTP‐dependent Ca2+ release. The stimulation of GTP hydrolysis by PEG was specific for the microsome fraction; only small effects were obtained with plasma membrane or cytosol fractions. As observed for GTP‐dependent Ca2+ release, the microsomal PEG‐stimulated GTPase was competitively inhibited by the GTP analog GTPγS (K i = 60 nM). It is proposed that the PEG‐stimulated GTPase may represent an intrinsic activity of the guanine nucleotide binding protein involved in the regulation of reticular Ca2+fluxes.

Hormone-Induced Inositol Lipid Breakdown and Calcium-Mediated Cellular Responses in Liver

Although it has been recognized for many years that changes of the intracellular free Ca2+ concentration by a variety of agonists form an important signaling device for regulation of cell function, the source of the Ca2+ and the molecular events regulating receptor-mediated changes of cellular calcium homeostasis have remained recalcitrant problems despite much effort directed towards their elucidation. However, advances made along a number of different lines have contributed towards the rapid increase of knowledge in this area. These include on the one hand the development of fluorescent Ca2+ indicators such as Quin 2 (Tsien, 1983) and more recently Fura 2 (Grynkiewicz et al., 1985), which allow kinetic measurements of changes in the cytosolic free Ca2+ concentration of isolated cells, and on the other hand the elucidation of the signaling roles of two new intracellular second messengers, namely, inositol trisphosphate and diacylglycerol (for reviews see Nishizuka et al., 1984; Nishizuka, 1984a; Berridge and Irvine, 1984; Williamson et al., 1985; Williamson, 1986).

Intracellular Calcium Homeostasis with Extrapolations to Cardiac Ischemia

The processes involved in the maintenance of intracellular calcium homeostasis are complex and still poorly understood. Most cells contain between 3 and 6 umol total calcium per g dry wt, are exposed to medium containing 1.25 mM free Ca2+ and yet maintain an intracellular free Ca2+ concentration of 50 to 200 nM in the resting stated1–7. Apart from calcium bound to the glycocalyx and head groups of externally facing phospholipids of the plasma membrane, which rapidly exchanges with the extracellular Ca2+8, most of the intracellular calcium is present in bound form in mitochondria and sarcoplasmic reticulum. 45Ca2+ flux studies have shown that there is a dynamic equilibrium between extracellular and intracellular Ca2+ and between the different intracellular calcium pools8, 9.