Panel discussion VI: Receptor-transmembrane signaling, August 8, 1989

Panel Discussion

VI: Receptor-Transmembrane

Signaling,

August 8, 1989

Y.C. Patel, MD, PhD, Moderator

Y.C. Pate1 (Montreal, Canada): In the morning session, I believe there was reasonable agreement for the existence of pharmacological receptor subtypes. Evidence was presented that these subtypes may be due to multiple receptor proteins. In the subsequent session, the one that just took place, we learned that there are different receptor-linked signalingpathways, operating either proximally at the cell membrane or distally within the cell. Dr Dichter presented data showing opposite effects of SS- 14 and SS-28 on K’ conductance, which appears to be the first demonstration of differential postreceptor effects of these two peptides. There are some areas of dispute. You heard Dr Schonbrunn not finding evidence for the distal effect-the post-CAMP, postcalcium effect of somatostatin in GH cells. Dr Wollheim, on the other hand, does find such an effect in that cell line as well as in HIT cells. We should address this issue. Is the distal effect cellspecific? Secondly, are the proximal and distal effects operating in concert, are they parallel or alternative signaling pathways, and what are their relative significances with respect to the final biological response, the inhibition of secretion. Perhaps we could start off with comments from the panel concerning the distal effects of somatostatin. A. Schonbrunn (Houston, TX): I would like to ask Dr Wollheim if he found an effect in GH cells. I thought I only saw data on the HIT cells, in which case the resolution of the dispute could be simple. C.B. Wollheim (Geneva, Switzerland): We have actually not carried out experiments in permeabilized GH cells. But Luini in Italy has in press experiments carried out in AtT20 cells, a related cell line, where he showed that calciumstimulated ACTH release is inhibited by somatostatin in digitonin-permeabilized AtT-20 cells. This ties in with earlier experiments in GH3 cells showing that ionophore-stimulated secretion of growth hormone can be inhibited by somatostatin, which, for all intents and purposes, corresponds to the experiments carried out in permeabilized cells, since the ionophore effect should not be modified by changes in membrane potential. Some 10 years ago, we showed that A23 187 calcium ionophore-induced secretion was indeed inhibited by somatostatin in insulin-secreting cells. A. Schonbrunn: Okay. I can address those kinds of experiments, although I do not know whether this will resolve all the discrepant results. When a lot of those experiments were done looking at the ability of somatostatin to inhibit ionophore-stimulated secretion, phorbol ester-stimulated secretion or for that matter, a variety of secretogogues, there weren’t careful controls done, to try to make sure that somatostatin was not inhibiting ionophore-stimulated secretion by having an effect on membrane potential, or by having an effect on CAMP. This is because the mechanisms of somatostatin action were not well understood at that time. So in those kinds of studies CAMP levels could and probably were changing, because calcium does have effects on CAMP levels. It is possible through calcium-dependent effects on potassium channels that there were simultaneous membrane effects going on. In Mefabohm, Vol39,

No 9, Suppl 2 (September),

1990: pp 105-107

hindsight, had those experiments been done with rigorous controls they may have provided suggestive evidence for second mechanisms, but I do not think they come anywhere close to proving it. I think the permeabilized studies that you are doing are very very elegant and convincing. So, on the basis of those, I would say there might will be cell-specific differences to explain. Indeed, if there are subtypes of somatostatin receptors, then it would not be surprising to see different kinds of mechanisms going on where those subtypes exist. Y.C. Patek There is a fundamental difference between the cells that you (Dr Schonbmnn) have used all along in your studies, the GH& cell line and AtT-20 cells or normal pituitary cells. Binding studies show that GH& cells are SS14 selective, which is opposite to AtT-20 cells, normal pituitary cells, and probably also HIT cells, all of which bind SS-28 more than SS-14. So there may be these variations to account for the kinds of differences that you see in GH cells and what you (Dr Wollheim) are referring to in AtT-20 and perhaps HIT cells. T.D. Reisine (Philadelphia, PA): Dr Schonbrunn, those studies you are talking about were controlled for in the AtT20 cells. Phorbol esters do not affect CAMP levels; calcium ionophore does not affect CAMP levels. In addition, they are most likely independent of effects on membrane potential. Whether they occur through some other nonspecific effect, nobody knows. But, then again, nobody really knows what hormone secretion really involves. But definitely, the studies that Luini is talking about, as well as the old work showing that 8-bromo-CAMP-evoked release is blocked by somatostatin, tend to argue against your statements about AtT-20 cells at least. A. Schonbrunn: Well, in AtT-20 cells there are the electropermeabilization results indicating that there is another mechanism, and there may well be one. C.B. Wollheim: I just wanted to correct something that our chairman said. The HIT cell does not show a preference for somatostatin-14, at least for nutrient-stimulated insulin secretion. The dose-responses I showed were identical for somatostatin-28 and 14. Y.C. Patek Which is then different from the normal /3 cell I presume. The normal j3 cell has been shown in a variety of secretion studies to be more sensitive to somatostatin-28 than 14. By autoradiography there appear to be a higher density of somatostatin-28 compared to somatostatin- 14 binding sites on /3 cells. G. Makhlouf (Richmond, VA): Dr Schonbrunn, could you perhaps tell us something more about potassium channels that you have looked at? In particular, I am interested to find out whether they are ATP-sensitive potassium channels, whether glibenclamide or tolbutamide blocks these channels, and whether specific potassium agonists will open them. A. Schonbrunn: Those are very interesting questions, and I wish I had an answer for you. I do not know the answers to those questions, but perhaps Dr Yatani does. 105

106 A. Yatani (Houston, TX): In our experiment, somatostatin activated one set of potassium channels, which is different from ATP sensitive channels. So my answer is no. Somatostatin is not coupled to ATP-regulated channels. G. Makhlouf: In that case may I readdress the question to Dr Wollheim? You have used galanin, and galanin is known at least in two cell systems to hyperpolarize the cells, and in one cell system the hyperpolarization has been linked to the opening of potassium channels. Have you had occasion to characterize those potassium channels? C. Wollheim: In collaboration with Dr Peterson’s group in Liverpool, we have actually recently published the characterization of the galanin sensitive potassium channel in the insulin-secreting RlNMSf cell and carried insulin secretion experiments in parallel. And, yes, in this cell line, galanin opens the ATP-sensitive potassium channel, the same channel that is closed by sulfonylurea drugs such as tolbutamide and glibenclamide. But, in the same cell line, galanin inhibits up to 50% of the calcium-stimulated insulin secretion in electrically stabilized cells. So we are faced again with the two actions in the same cell system, hyperpolarization and the transient lowering of calcium, and a good inhibition in permeabilized cells. So I think that we should come to an agreement that there can be different potencies of the various effects, and they all lead to hyperpolatize the membrane, lower calcium influx, and, finally, inhibit secretion by a direct effect. Probably, there can be various degrees of these three effects expressed in various cell types. I wanted to just add to your former question to Dr Schonbrunn. In the HIT cell line, the insulin-secreting cell line, I have personal communication from Dr Ciani in Los Angeles that somatostatin does open ATP-sensitive potassium channel in these cells. Unfortunately, I have not seen the data, but he told me so when I met him a couple of weeks ago. It has been published by Lazdunski’s group for RIN cells. that in this cell line somatostatin actually opens the ATP-sensitive potassium channel. But there is a strange permutation which may be in favor of my argument about the the distal effect, that the RIN cell, despite displaying this mechanism of hyperpolarization and opening of the potassium channel by somatostatin, is not sensitive to somatostatin in terms of inhibition of insulin secretion. We have carried out experiments in various types of RIN cells, and they never show an inhibition of insulin secretion by somatostatin, in marked contradistinction to the HIT cell line. G. Makhloti One last question. Does apamine block the potassium channels in any system? A. Schonbrunn: I have not tested that. C. Wollheim: It does not usually block the ATP-sensitive one. G. Makhlouf: Yes, I know, but in the case of galaninapamine, the potassium channels that are opened in smooth muscle cells for example, in our hands are blocked by low concentrations of apamine. C. Wollheim: I am not sure that that has actually been tested, but I must ask my friends in Liverpool. J.E. GerIch (Pittsburgh, PA): Increases in intracellular calcium which might trigger secretion could come about by two m~hanisms: increased uptake from the outside or dis-

PANEL DISCUSSION VI

charge from intracellular calcium stores. Is there any evidence that ~matostatin could work by eff~ting release from intracellular calcium stores? A. Schonbrunn: I would say there is no evidence at all, despite the fact that lots of people have looked. All of the studies that we have done have shown that the effect of somatostatin on intracellular calcium can be entirely accounted for by an inhibitory effect on calcium influx. Furthermore, somatostatin does not seem to have any acute effects on inositol phosphate formation. Y.C. Patel: Dr Wollheim, seeing that you are the one who finds this distal effect of somatostatin action, can you elaborate further on my first question namely, how do you see the proximal and distal effects working together in a given cell where they both exist? C. Wollheim: I already on that some extent said that could be We have from different lines that may be expressions of effects. We unfortunately never the same of somatostatin inhibit secretion ~~~biIiz~ cells in intact This was same with or with We do know what is due It could that we some component the system our permeaprocedure, since maximum inhibition can get 50% rather 80% or So it very hard actually answer question directly, one devises periments to at that, we have done. Y.C. Patek I have a question for Dr Yatani which is quite topical and concerns the role of subunits. There are some groups now that have shown direct effects of the &-subunits ofG proteins on atrial potassium channels. Can you comment on that? You have shown very nicely with your electrophysiological studies, direct linkage of Gk to a subset of potassium channels. Can you comment on the presumptive roles of ar and & subunits? A. Yatani: Two years ago we published additional data on G protein, because human erythrocyte G, and Gk share the same j3, component. Gt activated the channels, G, did not which indicated that the a-subunit was responsible for channel activation. Then we purified the CYsubunit and showed the same potency for G1, and (Yk.At the same time, other laboratories showed that 8, directly activated potassium channels at nanomolar concentrations which we have shown at picomolar concentrations. Then there was a dispute. Right now, the other group has confirmed that LYactivates the channel. But they are saying that & also activates the channel at very very high concentrations. So at least G protein-mediated channel activation is due to the at-subunit. On that both groups are agreed. Now the question is, what biological role does & play? In our hands, 0, blocks channel activation, which is the same as the adenylate cyclase system. It reduces background noise. When you add more &, it binds to the asubunit, so cyclase is reduced. Y.C. Patek You have not carried out the kinds of studies that these other groups have done to look at direct coupling of &-subunit or direct activation by 8, subunits of phospholipase A2 and the leucotriene system? A. Yatani: Yes. We found channel activation with & only when & is contaminate by the a-subunit. You can ADP

PANEL DISCUSSION VI

ribosylate with pertussis toxin and see how much a-subunit contamination of p, there is. When we used 10 nmol/L 0, which contaminates with about 10 pmol/L a-subunit, we can see activation. When we used uncontaminated ~3, we never saw activation. As for phospholi~ AZ, we have no evidence to support the idea that &-mediated effects are due to phospholipase A*. E.A. Woltering (Portland, OR): We have made some clinical observations that I would like the basic scientists to address. We have taken a group of patients with functional endocrine tumors through a variety of provocative tests: tolbu~mide for insulinom~, meals, and calcium infusions. What we have observed is a unique phenomenon, that is, the calcium seems to neutralize or inhibit the inhibitory effect of somatostatin and the analogs. We have observed the same thing also in cells from those identical patients. Can you close the loop for us? You have shown us that somatostatin reduces intm~lluiar calcium. My question is, what happens when you bring us to reality? That is, you take us from 10 pmol/ L into mmol/L concentrations of 1, 2, and 3 that patients with hypercalcemia would exhibit. What happens then? A. Schonbrunn: Well, the experiments that I showed you, except where we were specifically manipulating calcium ion ~ncen~tions, the extracellular calcium concentration is 1 mmol/L. We can bring that up to 2 or 3 mmol/L and somatostatin still inhibits, because there is a good driving force than for calcium influx. So at least at the cellular level, I cannot explain your observations with raising of the calcium, Maybe them are some indirect effects. Maybe the high calcium is stimulating the secretion of something which interferes with ~matos~tin action. I don’t know. C.B. Woilheim: I have the same comment. We published data some 10 years ago where we looked at the calcium concentration range between 1 and 10 mmol/L, and somatostatin was absolutely capable at each instance of having the same inhibitory effect on glucose-induced insulin secretion. This was in vitro. In the case of your in vivo studies, I agree with Dr Schonbrunn, that when you increase the calcium, you may secrete other agonists, which may disturb the balance between agonists and inhibitors. E.A. Woltering: Dr Bhathena who is in the audience, observed 10 years ago very different things that calcium could inhibit ~matos~tin action on both insulin and glucagon, and Bennett and Curry have shown the opposite effect with insulin, that calcium could overcome somatostatin inhibition. I wonder what could be the potential cellular mechanism of that. C.B. Wollheim: We have become more direct. I think when you raise the calcium concentration outside an intact cell, you do not really do very much to the cytosohc calcium, as long as the cell stays alive, because it has the tremendous capacity, as I showed in my initial scheme, to get rid of the overload of calcium. So I really think that there is no explanation for the old controversy that you have alluded to. When we permeabilize cells and control the calcium con~ntration down to the micromolar range, there we have the effect. You know, we always verify our extracellular calcium concentration in the experiments on permeabilized cells with a calcium-

sensitive electrode, and we know exactly what our calcium concentration is. Since the cells are permeabilized, they actua.lly see this concentration. So this is a calcium-independent effect as long as calcium levels are concerned. I am sorry if I sound arrogant, but I think we should now look forward and not 10 years back. R. Couper (Toronto, Canada): I was wondering how Dr Wollheim controlled in his solubilized cell model the intracellular pH and whether this may actually explain some of the things that he has seen. Secondly, has anyone looked at the levels of calmodulin and calcitonin-like peptides after they have chronically treated cells with ~matos~tin? C.B. WoBheinx If I may answer the first question, we clamp the pH inside the cell, because it is the same pH as outside the cell, and we usually carry out our experiments at the pH concentration that we have measured in these cell lines which is around 7.0 to 7.05, but we usually work at 7.0. So this cannot explain the effects. Unidentified Speaker: This is for Dr Dichter. You showed that somatostatin-28 and - 14 essentially affected the same microscopic current. But you alluded to the fact that pertussis toxin affected both. If my understanding of the mechanism is correct, pertussis toxin works basically at Gi, I think. I stand to be corrected. In light of that, have you done single channel recordings to make sure that you are dealing with one and the same channel in the case of the response of somatostatin-28 versus - 14? M.A. Dichter (Philadelphia, PA): Let me be clear about that and also correct a slight oversight. I should have mentioned that Hung-Li Wang in my laboratory in collaboration with Terry Reisine and I did those experiments. I meant to mention that during the talk. We did not look at single-channel data about those potassium currents. I think it is very likely that pertussis toxin will be acting on a number of G proteins, not all of them, and not just one specific one. In terms of the voltage-dependent potassium current, which now is generated by different channels than the channels that were discussed by other speakers, this is the potassium channel that opens when you depolarize the cell but is closed at rest and is not affected directly by somatostatin. So somatostatin did not open the channel itself. The channel had to be opened by depolarization and then somatostatin affected its gating. In that ~rcums~nce, ~matostatin-14 and -28 had opposite effects on the same current. Now, we did not take it to the same channel level, and it is possible theoretically at least, that the same exact looking current was mediated by two different channels. I think that is highly unlikely, because everything else that we know about the current indicates that it is a uniform channel~e~v~ current. Now, when you treat the cells with pertussis toxin, it blocked both the effects of 14 and 28. So our scheme is that I4 and 28 for that response have two different receptors, which are coupled to two different G proteins, both of which are affected by pertussis toxin. Those two G proteins would have opposite effects on the final common pathway, which is that particular voltagedependent potassium channel. Now, we have a little way to go before we put ail those pieces together, but that is the hypothesis at present.