Paula Q. Barrett

Effect of dietary phosphate on transport properties of pig renal microvillus vesicles

Dietary phosphate manipulation results in stable adaptive changes in the transport functions of microvillus membrane vesicles isolated from pig renal cortex. When assayed under sodium gradient conditions, phosphate uptake is enhanced 200--400% in vesicles prepared from animals maintained on a low-phosphate diet (0.22%) compared to high-phosphate diet controls (0.82%). When transport is assayed in sodium preequilibrated vesicles, a 100% enhancement of phosphate uptake is demonstrable. Stimulation of phosphate uptake into low-phosphate diet vesicles after the imposition of a sodium chloride gradient is equivalent if uptake is measured at pH 6.0 or 8.0 and can be kinetically characterized as resulting from a Vmax alteration in the phosphate transport system. Microvillus membrane vesicle phosphate transport is maximally stimulated after only 2 days of dietary deprivation. Although a longer period (1 and 2 wk) of phosphate restriction does not further stimulate phosphate transport, it does result in an inhibtion of other sodium gradient-dependent transport systems (glucose, alanine).

Protein kinase C activity in renal microvillus membranes

Protein kinase C activity was found in rabbit renal microvillus membrane vesicles. C-kinase activity was assayed by examining H1 histone phosphorylation using microvillus membrane vesicles dispersed with Triton X. Calcium-activated protein kinase activity was only demonstrable in the presence of phosphatidylserine (PS). With PS (15 micrograms/ml) the Ka for activation by calcium was 1.04 microM. This was reduced to 0.38 microM by addition of diolein (3.75 micrograms/ml). These activations were dose-dependent and their combined synergistic activation could be reproduced by the combination of PS (15 micrograms/ml) and the phorbol ester, TPA (1.17 ng/ml). During microvillus membrane purification, protein kinase C activity enriched 5-fold relative to its activity in the homogenates. The activity was not due to trapped cytosol or adventitious association with microvillus membranes during homogenization. During further purification on sucrose gradients, the C-kinase activity coenriched with brush border and not with basolateral enzyme markers. We conclude that protein kinase C is a normal component of the renal microvillus membrane.

Signal transduction mechanisms involved in carbachol-induced aldosterone secretion from bovine adrenal glomerulosa cells.

In cultured bovine adrenal glomerulosa cells, diacylglycerol content remains elevated for up to 75 min following the removal of angiotensin II. This maintained increase could provide a mechanism by which angiotensin II pretreatment may prime cells to secrete aldosterone in response to the calcium channel agonist Bay K 8644. In the present study we find that carbachol failed both to produce this persistent diacylglycerol elevation and to exert a priming effect. In addition, because carbachol was also a less potent activator of phospholipase D than angiotensin II, our results implicate phospholipase D in the maintained increase in diacylglycerol content observed following stimulation with and removal of angiotensin II. Carbachol also elicited changes in the radiolabeled levels of both myristate- and arachidonate-containing diacylglycerol. However, the rapid decline in diacylglycerol content following carbachol removal resembled the rapid fall in arachidonate-diacylglycerol; we therefore proposed that the diacylglycerol species generated with carbachol stimulation contains predominantly arachidonic acid. In summary, our results suggest that prolonged elevations in diacylglycerol content following removal of hormones such as angiotensin II, as well as the identity of the diacylglycerol species itself, may be important in the regulation of cellular responses.

Information Flow in the Calcium Messenger System

Work in the past 20 years has led to an ever-expanding appreciation of the intracellular messenger function of calcium ion [Ca2+]. What began as an interest in the role of Ca2+ in stimulus-response coupling in skeletal muscle has expanded to the point where Ca2+ is recognized as one of the few universal intracellular messengers involved in coupling a wide variety of stimuli to an equally diverse range of responses: Ca2+ participates in the control of neuro-, exocrine, and endocrine secretion, the contraction of all forms of muscle, fluid and electrolyte transport, energy and fuel metabolism, and growth and development. The control of these diverse tissue responses requires a considerably more elaborate system than simply a rise and fall in intracellular free Ca2+ concentration—the original model of the way Ca2+ was thought to serve its messenger function. Hence, it is more appropriate to introduce the concept of the calcium messenger system. Encompassed within this term are the cellular components involved in generating, terminating, transmitting, and receiving the Ca2+ message.

The Cellular Basis for Short-Term Memory in Endocrine Systems

A common property of many endocrine systems is an anamnestic response. For instance, when pancreatic islets are reexposed to a standard glucose stimulus after a prior period of exposure to the same glucose challenge, their insulin secretory response is greater to the second than to the first stimulus (Gold et al., 1982; Grill et al., 1978, 1979; Grill and Rundfeldt, 1979; Grill, 1981; Grodsky et al., 1969). A similar pattern is seen on repeated exposure of adrenal glomerulosa cells to the peptide hormone angiotensin II. Recent work in both of these systems indicates that protein kinase C plays a central role in mediating the sustained phase of each of these endocrine responses (Kojima et al., 1984; Tanigawa et al., 1982; Zawalich et al., 1983, 1984). Work in our laboratories over the past 18 months has focused on the possible role of protein kinase C in this type of short-term cellular memory in bovine adrenal glomerulosa cells and isolated rat pancreatic islets.

Chapter 11 Mechanism of action of angiotensin II

Publisher Summary This chapter discusses the mechanism of action of angiotensin II (AII). The octapeptide AII is the primary effector hormone of the rennin-angiotensin-aldosterone system, regulating not only the release of aldosterone from the adrenal but also that of renin from the kidney. By inhibiting the secretion of renin—the enzyme which catalyses the proteolysis of angiotensinogen to the AII-precursor angiotensin I (AI)—AII effectively regulates its own formation as well via a negative feedback loop. AII receptors have also been identified in the kidney, brain, uterus, placenta, pituitary, and reticuloendothelium. Saturable binding of radiolabeled AII is demonstrable in adrenal glomerulosa, vascular smooth muscle, and hepatic plasma membranes. In some smooth-muscle cell types, AII alters the activity of both phospholipase C and adenylate cyclase, presumably via a single-receptor type. The biological effect of AII on its target tissues can be altered by changing either receptor affinity or receptor number. In the adrenal, liver, and vasculature the affinity of the receptor for AII is modulated by cations, such as sodium, calcium or magnesium, and by reducing agents.

Temporal and Spatial Events in the Calcium Messenger System

The great complexity of the central nervous system makes it a difficult object of biochemical study. Yet, some of the most important biochemical discoveries having implications for the field of cell regulation have been made in CNS tissue. A case in point is the discovery by Nishizuka and coworkers of a new kind of protein kinase, the so-called phospholipid-dependent, calcium-activated protein kinase or C-kinase, in brain tissue (Takai et al.). This kinase was found to be distinct from either the classic cAMPdependent or Ca++-CaM-dependent protein kinases. It was, however, found to be activated by Ca++, phospholipids, and diacylglycerols (Takai et al., 1977; Kishimoto et al., 1980). After its discovery in the brain, where it exists in very large amounts, it was found to be widely distributed in animal tissues (Nishizuka and Takai, 1981). Its discovery coincided in time with a significant breakthrough in our understanding of the role of inositol polyphosphatase in the transducing events which occur in the calcium messenger system (Michell, 1975; Berridge, 1984) (see chapters by Agranoff and by Lapetina in this volume).

Chapter 4 Mechanism of action of Ca2+-dependent hormones

Publisher Summary This chapter describes mechanism of action of Ca 2+ -dependent hormones. Ca 2+ is a major and nearly universal intracellular messenger throughout the animal kingdom. An important aspect of Ca 2+ messenger function is its complexity. There are multiple mechanisms by which a Ca 2+ messenger can be generated or terminated. Messenger generation can result from the turnover of polyphosphoinositides, the activation of the cyclic AMP (cAMP)-messenger system, and the activation of receptor-operated or potential-dependent Ca 2+ channels in the plasma membrane. The most common mechanism by which Ca 2+ -dependent hormones act is via a receptor-linked phospholipase C which catalyses the hydrolysis of polyphosphatidylinositol, resulting in the production of two intracellular messengers: inositol 1,4,5- trisphosphate and diacylglycerol. The mechanisms by which the Ca 2+ message leads to a change in cell function are also complex. The action of Ca 2+ can be achieved via either amplitude or sensitivity modulation. The actions of Ca 2+ can also be expressed in different spatial domains within the cell, and these domains may change during a sustained cellular response.

Control of Plasma Membrane Transducing Systems by Second Messengers and by Cellular Metabolism

The focus of this meeting on receptor-receptor interactions is not so much on the direct interaction of one receptor protein molecule with another different protein receptor molecule, but rather on the immediate products of the transducing events mediated by one class of receptors acting as modulators of the functions of a second class of receptors. This is a special case of a much larger phenomenon, that of the interactions between components of one messenger system, e.g., the Ca2+ messenger system, with those in another, e.g., the cAMP messenger system (Rasmussen, 1981). It is an intramembrane integrative mechanism. The special feature of the receptor-receptor theme is that the interactions are those which specifically influence other transducing events at the level of the plasma membrane. Even within this category of interactions, receptor-receptor interactions are only one example of this class of phenomena. We will first discuss examples of receptor-receptor interaction in endocrine systems, and then focus attention on two other types of control processes in which a coupling occurs between a transducing event and either a change in ion transport or a change in cellular metabolism. We do so for two reasons: first, to emphasize the fact that signal transducing events other than those directly linked to receptor-transducer interactions are an important feature of plasma membrane function; and second, to emphasize the importance of these membrane-linked events in the regulation of sustained cellular responses.