DISCUSSION Summarized by Ronald P. Rubin Department of Pharmacology, Medical College of Virginia Richmond, Virginia 23298 Discussion of the papers in this session focused on the breakdown of phosphoinositides and the resulting formation of inositol phosphates. While the papers demonstrated that receptor agonists induce breakdown of PI P 2 in a variety of cell types, employing diverse stimuli, the mechanism by which the inositol phosphates are formed was a matter of some debate. I n response to Dr. I rene Litosch's (Brown University) comment that one really cannot exclude the possibility that PI breakdown occurs in parallel with PIP 2 hydrolysis, Dr. Michell acknowledged that I P may be derived from PI breakdown directly. However, he felt that the law of parsimony militates against a situation where three lipids are being broken down in parallel. Dr. Michell felt that the simplest explanation and the one consistent with the presently available data - but certainly not the only one - is that PI P 2 is broken down to I P 3, which is then sequentially degraded by phosphatases to inositol. Another point raised by Drs. Nic~olas Bazan and Michael Berridge related to the high K concentrations and the prolonged duration of stimulation employed by Dr. Michell in neural tissue. This prompted Dr. Michell to express the opinion that his findings probably reflected responses not of a rapid regulatory pathway, but rather a more gentle, modulatory one. Both Drs. Abde!;Latif and Michell agreed that the PI effect produced by K depolarization had nothing to do with activation of voltage-sensiti e calcium channels, but was merely a reflection of K -induced neurotransmitter release. 329
Rubin The findings reported by Dr. John Exton indicating that phosphoinositide breakdown was not involved in the effects of vasopressin and alpha-agonists on calcium mobilization in the liver was challenged by Dr. James Putney, who noted that, in liver, I P 3 has been shown to have biological activity and is formed during hormonal stimulation. However, Dr. Putney acknowledged the existence of the problem of precisely correlating tissue levels of IP3 with the functional response. In response to Dr. Thomas Martin's (University of Wisconsin) question as to whether the effects of hormones on calcium pumping were mimicked either by agents that translocate calcium or alter diacylglycerol-dependent protein phosphorylation, Dr. Exton noted that ionophores abolish the ability of membrane vesicles to accumulate calcium, although he had not tested whether mitochondrial inhibitors, such as FCCP, would produce hormone-like effects on Ca-ATPase. In response to a question raised by Dr. Pedro Cuatrecasas (Wellcome Research Laboratories), Dr. Exton indicated that IP2 and IP 3 do not influence calcium metabolism of isolated mitochondria, although he further stated that negative results might be accounted for by the absence of critical co-factors in the media. In relation to Dr. John Fain's paper, Dr. Michell raised the question as to how, after steady state labeling of inositol phospholipids, stimulation could bring about a decreased labeling of PI with an accompanying increase in total concentration. Dr. Fain suggested that 32 P labeling may not be equilibrating with all pools. Dr. Downes later reaffirmed the possibility that there may be multiple pools of phosphoinositide labeling at different rates. Dr. Michell countered with the proposal that the 32 P labeling may be comprised of some compound in addition to [32P]_ PI P 2' The findings of Dr. Fain that phorbol esters stimulate glycogenolysis raised some questions, although Dr. Fain agreed that the action of phorbol ester is not certain to be on protein kinase C. Dr. Putney suggested that protein kinase C may not be involved in the stimulus-response pathway in hepatocytes, since an increase in [Ca.] can fully activate phosphorylase kinase, which is app~rently not the case in platelets. With regard to the phorbol esters, Dr. John Williamson supported the statement of Dr. Exton by noting that phorbol esters have no effect on free calcium levels in liver cells as monitored by 330
Discussion Quin-2 fluorescence. However, he also warned that one must be certain that the vehicle e. g. ethanol or DMSO, is not responsible for any effects observed. In response to the question raised by Dr. Mark Seyfred (Michigan State University), Dr. Fain offered the suggestion that the enzymes for PI breakdown and resynthesis may be localized in the plasma membrane, as well as in the endoplasmic reticulum. He further hypothesized that resynthesis of PI is the result of calcium release from cellular membranes, which relieves the inhibitory constraint on the enzymes involved in PI synthesis. Following Dr. Abdel-Latif's paper, Dr. Berridge acknowledged the pioneer work of Dr. Abdel-Latif on the agonist-dependent breakdown of polyphosphoinositides in 1977. He also reiterated the important fact that the observed calcium-dependency merely reflected inhibition of release of an endogenous neurotransmitter. Dr. Berridge speculated that in smooth muscle the primary phase of contraction, which is independent of extracellular calcium, may be caused by internal calcium release induced by IP 3 The secondary, tonic phase could be explained on the basis of IP 3 short-circuiting internal calcium pools, thereby placing the emphasis for calcium signalling on the plasma membrane, whereby the calcium sequestering system is toned down to allow the accumulation of cellular calcium. This hypothesis would be consistent with the findings of Drs. Fain and Exton, who demonstrated some relationship between phosphoinositide metabolism and calcium pumping activity. Dr. Abdel-Latif agreed that the calcium pump, as well as the sodium pump, was involved in phosphoinositide breakdown. The ability of light to stimulate phosphoinositide metabolism in the retina at earlier response times was questioned by Dr. Mark Dibner (DuPont); it was suggested by Dr. Robert Anderson that this event could reflect adaptation to light, rather than a more rapid functional response of the retina. Dr. Anderson acknowledged that his system has a high signal: noise ratio and that only a small percentage of cells responded. Dr. Michell addressed the issue of Mn stimulation of radiolabeled inositol incorporation into PI, but Dr. Anderson indicated that Mn, while stimulating the exchange reaction, caused the total disruption of photoreceptor cells 331
Rubin after 4 h. Dr:... Michell offered the suggestion that the actions of Mg 2 were likely to be exerted throug h some inhibitory mechanism, even tho+ugh it mimics the effects of light. He proposed that Mg 2, like light, decreases the inhibitory input of a neurotransmitter whose function is to decrease PI turnover. This would imply that retinal stimuli may function in a manner opposite from that observed in other model systems. Dr. Anderson supported this proposal by noting that light hyperpolarizes retinal cells. The short papers by Drs. Peter Downes and Peggy Zelenka also prompted fruitful discussion. Dr. Abdel Latif addressed the question as to whether lower concentrations of agonist exhibit differential effects on the formation of one or another of the inositol phosphates. Dr. Abdel-Latif indicated that when a lesser carbachol concentration is employed in smooth muscle, only IP 3 formation is observed. Dr. Downes speculated that the enzymes involved in PIP 2 breakdown are probably carefully controlled in perhaps the same way that nucleotide phosphodiesterase is regulated. Discussion then focused on the possibility that more than one mechanism may exist for phosphoinositide breakdown. Dr. Marvin Gershengorn (NYU) stated that in GH 3 pituitary cells, calcium ionophores stimulate the hydrolysis of PI P, but not PI P 2. He suggested that the stimulation of these cells by TRH may cause the breakdown of PIP 2 causing calcium release, which in turn brings about the hydrolysis of PIP. However, Dr. Putney responded to this proposal by noting that in parotid cells the dose-response curves for formation of each of the inositol phosphates were superimposable and all were calcium-independent. Dr. Yoram Oron (Tel Aviv University) postulated the existence of a second mechanism in cell-free systems either because the polyphosphoinositides had disappeared from such a preparation and/or because this system enables the enzyme to come into contact with PI. To add to the complexities, Dr. Downes noted that the present techniques do not separate or distinguish different isomers of inositol phosphates. More sophisticated procedures are needed to ascertain the true chemical identity of the inositol phosphates that are being measured. Dr. Downes further suggested that the 332
Discussion energy-dependent steps (kinases) in the synthesis of phosphoinositides would allow a greater degree of control of the levels of these potent putative mediators. So, by simply altering the activity of a single kinase or phosphatase, inositol phosphate levels can be drastically modified. Dr. Robert Farese supported the notion of the separate mechanisms for the breakdown of PIP 2 and PI from data derived from his studies on exocrine pancreas. I n response to the statement that people had been considering PI metabolism in lectin-treated lymphocytes and calcium mobilization since the 1960's and 70's, Dr. Peggy Zelenka stated that her system enabled her to study PI breakdown in cells which are in the complete range of division states. In response to a question regarding the effect of serum, she noted that in epithelial cells taken from explants after 15 days, stimulation with serum increases the rate of cell division, while PI metabolism becomes comparable to that of other phospholipids. These data fortify the hypothesis that PI metabolism may playa regulatory role in the cell cycle. In conclusion, while this session established that inositol phosphate formation is enhanced by receptor agonists in several different systems, the following questions were still left unanswered: (a) What is the true chemical identity of the inositol phosphates being measured? (b) What is the biochemical mechanism of inositol phosphate formation? (c) Are the levels of inositol phosphates produced temporally and quantitatively appropriate for playing a second messenger role in the mobilization of cellular calcium? In other words, as stated by Dr. Fain, it is still not clear what the "Hokin phenomenon" truly represents. 333