Regulation of insulin secretion by α2-adrenergic agonists

Regulation of insulin secretion by aa-adrenergic agonists Catecholamine-induced inhibition of insulin secretion is mediated by cur-adrenoceptors’ although controversy surrounds the pharmacological characteristics of these receptors and their biochemical coupling mechanisms. Initial studies suggested that islet cw-receptorsare of high affinity but are present in very low abundance (-55 fmol mg-i protein?,“). However, this work was performed using a tissue preparation which was not well characterized, and more recent evidence obtained with hand-picked islets has suggested that the receptors may be of lower affinity but are present in much greater numbers (645 fmol mg-i protein)*. Further studies using islet membrane preparations will be required to resolve these discrepancies. It has been well documented that activation of ara-receptors causes inhibition of adenylate cyclase in islet homogenates5e6, and that era-agonists can lower CAMP levels in intact islet cells’. It is not surprising, therefore, that the inhibition of insulin secretion has been attributed to a reduction in cAMP~,~.However, a number of problems have arisen with this inte~retation. Firstly, it is clear that while being a positive modulator of insulin secretion, CAMP cannot itself directly initiate an increase in secretion rateg. It seems unlikely, therefore, that the apparently ubiquitous inhibitory effect of ara-agonists (which are effective against many different stimuli, not ail of which alter CAMP levels) would be mediated entirely via this system. In support of this idea, Ullrich and Wollheim have reported a paradoxical augmenta~on of &Ml? levels in islets treated with both ara-agonists and forskolin, under conditions was when insulin secretion abolishedlo. Treatment of isolated rat islets with dibutyryl CAMP also fails to prevent o+adrener$c inhibition of insulin secretior& , a result which cannot be readily

explained if these agents exert their influence only at the level of adenylate cyclase. A complicating factor in this story, however, derives from conflicting evidence relating to the extent to which or-agonists inhibit insulin secretion when celI CAMP levels are elevated. Some studies have demonstrated total abolition under these conditionsi’, while others have shown only partial inhibitio#. In an attempt to address this issue more directly, experiments have recently been performed with permeabilized islets, which offer the advantage that intracellular messengers can be introduced directly into the cells at defined concentrations. These studies have revealed that islets permeabilized with either detergentsi or by high voltage electric discharger3 remain responsive to ara-agonists. Unfortunately, however, the two systems have not provided unequivocal data relating to the effects of oz-agonists in the presence of CAMP. In detergentpermeabilized islets, oz-agonists totally inhibited insulin secretion induced by cAMP1* whereas in electrically permeabilized cells, secretion was still enhanced by CAMP despite the presence of noradrenaliner3. The reasons for this discrepancy remain unresolved. The crucial point raised by all of these studies is whether araagonists can directly regulate SCretion by interaction at a site which lies distal to CAMP generation {thereby desensitizing the B cell to the effect of an increase in CAMP) or whether they simply reduce the effectiveness of CAMP as a potentiator of secretion by lowering its concentration. This latter proposition is consistent with much of the data and seems the more attractive one, since it requires the postulation of fewer control points to explain the observed effects of csa-agonists in islet cells. Thus, their primary effect is to decrease the rate of

secretion induced by initiators of the response (by an unknown mechanism which is independent of CAMP)but, in parallel, they also minimize the extent of any potentiation of secretion by inhibiting adenylate cyclase activity. This model would also explain the observation that in dissociated B cells stimulated with glucagon (where the rate of secretion is directly controlled by the CAMP level) the extent of inhibition mediated by ala-agonists exact1 parallels the reduction in CANIP’P. Despite the uncertainties outlined above, there is a developing concensus that ruz-recePtors must exert an influence at (at least) two separate sites in the B cell. One of these is at the level of adenylate cyclase but the second is likely to be at a more fundamental point in the stimulus-secretion coupling pathway. It remains to be established whether this dual control is effected by two distinct subtypes of or-receptor, or whether it is achieved by the coupling of a single class of receptors to separate signalling systems. Effects of aa-agonists on Set ceII Ca2+ handling With respect to the CAMPeffects of arzindependent agonists, it has been proposed that control of cell Ca” handling might represent the major site of adrenergic regulation. Indeed, it has been known for a number of years that catecholamines can reduce the rate of Ca’+ influx into glucose-stimulated islets15. Sowever, more recent data has shown that this effect can be dissociated insulin from inhibition of secretion1,16, and may be secondary to changes in CAMP’. In addition, direct measurements made in the insulin-secreting, catechol~ine-sensitive, RINm5F cell, have revealed that ailrenergic inhibition of secretion is not accompanied b a decrease in cytosolic free Ca X concentratioiG7. Furthermore, in islets permeabilized with either detergents or electric fields, Ca2’-dependent secretion can be abolished by noradrenalinel’*‘*, and in intact islets, changes in Ca’+ efflux do not correlate with adrener ic inhibition of insulin secretion’ 8. q

El

cl

Taken iogether, these data pro-

0 1987, Elsevier Publietimr,

Cambridge

0165 - 6147/67/soZ.W

370 tide a body Of evidence which suggests that cu,-receptors do not contr01 the rate of insulin Secretion by directly modulating islet Cell Ca2* handlinp. It is also unlikely that they exert a direct influence On the inOsitO1lipid signafiing system in i~let$~ or, by inference, On the activatirin Ofpr0tein kinase C. The data point, therefore, to a primary interaction at a crucial step in ihe pathway of stimulus-secretion coupling which lies at, or beyond, the point at &&&

these

signalling

systems

converge. The challenge t0 identify and &aracterize this site still remains. Success will provide new insights into the molecular events which control insulin secretion and will afford a clearer under&ES!fzg of the wq5 in which f+= fecep&rs regulate cell activation. NOEL G. MORGAN

Department of Biological Sciences, University of Keele, Keek, Staffordshire ST5 5BG, UK.

1 @agarb N. G- andBdmtagm, W. (2985) Bio+m* f. 226,571-5% 2 Chherksey, B., Altszuler, N. and Zadunia~ky, J. (1981) Diabetes 30,172-174 3 Cherkuey, B., Mendelsohn, S., Zaduniasky, J*and Altszuler, N. (X9@) PharnlaroloPv 27.95-102 4 F$es,“$ tk, Cawfharne,M. A. a& How&# s. L. (19857 B&CL Rep. 7, n-22 5 Howe& S, L. and Montague, W. (1973) Biochim. Biapkys. Acta 320,4442 ii Gazcia&Rmxles, P., Dufrane, S. .P.. Sener. A., Valverde. I. and Malaisse. W. J. il9&Q) Biosci. Rep. 4,511-521 . 7 Yama&& S., Katada, T. and Ui, M. (19fQ Mal. ~~~~u~~~~ 21,643-653 8 F;iin, J_N. (19%) M~~~~ 33,6X2-679 9 Henquin, 3. C (19351 Arck. I& Physiol Biochem. 93,374 10 Zurich. S. and Wollheim, C. B. (1984) 1. Biol. C&em. 259.4111-4115 11 -Nakaki, T, Nakahate, T., Yamamoto, S. and Kate, IL (1983) Life Sci. 32,X91-195 12 Tamagawa, T.. Niti,* L, N&i, H. and Niki, M. (%&LB3iomed. Res. 6, -32 I3 Jones, P. M., Fykes. 1. M. and Hawe& S. L. (1986) DiabetorOgia 29,554A 14 Schuit,E C. and Pipeleers, D. G. (1986) Science 2X&875-877 15 WoUheim, C. B., Kikuchi, M., Renold, A. E. and Sharp, G. W_ G. (1977) 1. cb’rz. bznoest_60,1165-1173

17 LUlrich, i. and Wollheim, C. B. (1985) Mol. Pknrmacof. 28,100-106 18 Fyies, J. M I, pries, .P. M. and HoweX, S. L. (19%) Diubetnfoggia 29, J99A 19 hlorgan, N. G., Rumford, GM. and Mmtague, W. (19S5) Biosci. Rep. 5, l&53-%%I 20 Morgan, N. G. and Montague, W. (19%) Biuehem. Sue. Trans. 14,101~1020 21 Montage, W., Morgan, N. G., Rumford, G. M. and Prince, C. A. (1985) Bii;&rn. ,‘. 227,4&3-$89

The renin-angiotensin system (IUS) is a multiregulated proteolytic cascade that produces two potent pressor and aldosteranogenit peptides, angiOter&n II (AID and angiotensin III @III). Pharmacologic interruption of this system at the angiotensin converting enzyme (ACE) reaction, which produces the Octapeptide AII from its decapeptide precursor AI, has been shown to be effective therapy far a majority of hypertensive patienrts and is a majOr advance in the treatment of hypertension and congestive heart failure”“. The first, and rate-limiting, pryte&y&z step in the RAS is the renin reaction, in which AI is cieaved by the enzyme renin from the N-terminus of a protein substmtle, angiotensinogen. Inhibition of the RAS by blockade at the renin reaction could be an extraordinarily specific alternative t0 inhibition of the relatively nonspecific dipeptidyl carboxypeptidase, ACE, and attempts to design medicinally-useful r&n inhibitors have been underway for more than 20 years, predating the Merest in ACE. Despite this early interest in renin and the intense research efforts underway at most of the major pharmaceutical laboratories, orally bioavailable, longacting zenin inhibitors have not yet been de&bed which are candidates for widespread clinical useM. The extraordinary specificity of renin has long been an attraction for the pharmacologist and medicinal chemist, yet this same specificity presents prcrblems in designing a medicinally-use~l renin inhibitor. While ACE, for example, will accept substrates as small as acylated tripeptides, the minimum known substrate for renin is au oetapeptide, 1 (see Table 1): spanning the &avasp site. Thus, while there have been a wealth Of potent ACE inhibitors described which mimic two or three amino acids of its substrate, inhibitors of renin have tended to be rather larger, and this seemingiy necessary size has brOught along larger problems Of metabolism, oral bioavailability, and duraticm of action. Early efforts at design of renin

inhibitors focused On analogs af the known minimum substrate sequence I, in which the Leu residues of the scissile dipeptide (Pl-E’l’) were substituted with Phe, giving non-cleaved analogs af micromOlar potency. These inhibitors were shown to lower blood pressure in animals and one was studied acutely in humans, where sOme precipitOus drops in blood pressun: and heart rate were noted6. More potent inhibitors brave been designed which replace the scissile dipeptide unit within substrate sequences with transitionstate Or intermediate analogs (see Fig. 1). Boger and cOI!eagues designed a family of inhibitors based upon the natural product pepstatin A, 3, which is a subnanomolar inhibitOr Of most aspartic proteinases. Renti is an aspartic proteinase, in the pepsin family of enzymes, hill it is inhibited by pepstatin only weakly. The central statine residue (Sta) of pepstatin is thought to be rt?sponsible for its inhibition of aspar& proteinases. This residue is a noncteavable transition-state or intermediate analOg of the scissile dipeptide. Incorporation of Sta as a dipeptide mimic into an an&lag Of the renin substrate octapeptide sequence gave SCRIP, 4, which has been shOwn to be an effective, though short-lived, renin administered inhibitor when intravenously7. Experiments in which SCRIP was infused for up to 48 hours in Na4-deficient, conscious dogs were the first to demonstrate that a small mofecule renin enzyme inhibitor could maintain a stable blood-pressure decrement for extended period@. A nonapeptide analog, 5, incorporates Sta into the human substrate sequence. An increase in duration Of action upon i.v. administratiOn Of 5 was attributed to the additional amino acids blocking the Nand C-tenninae. Some oral activity was detected with a heraic dase (lC0 mg kg-‘), although the bioava~a~lity was clearly very low? It has been widely assumed that the primary drawback to peptides as drugs is their susceptibility to proteolytic degradation, While