Repolarization of the membrane potential of blood platelets after complement damage: evidence for a Ca++ -dependent exocytotic elimination of C5b-9 pores

Gel-filtered blood platelets exposed to complement proteins C5b-9 have previously been shown to undergo a reversible depolarization of membrane potential (Em) in the absence of lytic plasma membrane rupture. In this paper, we examine the mechanism by which C5b-9 damaged platelets restore their basal electrochemical state, despite increased ion conductance due to membrane insertion of these cytolytic serum proteins. Repolarization of Em after formation of the C5b-9 membrane pore is shown to be accompanied by a Ca++-dependent vesiculation of the platelet surface, which results in the release of these proteins from the plasma membrane and a restoration of the membrane's functional integrity. This exocytotic elimination of C5b-9 complexes from the plasma membrane is accompanied by a ouabain-inhibitable repolarization of Em, which presumably reflects restoration of transmembrane cation gradients by the plasma membrane Na/K ATPase. The role of external Ca++ in the platelet's response to membrane-insertion of the C5b-9 proteins is discussed both in the context of the known cellular effects of this ion and in the context of recent observations suggesting sublytic changes in platelet function after complement-mediated plasma membrane damage.     

Effect of agents that produce membrane disorder on lysis of erythrocytes by complement.

To evaluate the effect of membrane lipid acyl-chain packing on the efficiency of cell lysis by complement, we have studied membrane modulation by 2-(2-methoxy)-ethoxyethyl-8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) and by myristoleyl alcohol, the cis isomer of a C14:1 aliphatic alcohol. These substances are known to increase the membrane lipid disorder by virtue of the bend in their acyl chains, which is believed to loosen the phospholipid acyl-chain packing. We have found that both of these compounds markedly enhance the lysis of erythrocytes by the terminal complement proteins C5b-9. The enhancing effect by A2C is operative in the formation of erythrocytes carrying complement components C5b, C6, and C7, as well as in the subsequent reactions with complement components C8 and C9. We have also found that A2C-treated erythrocytes bind C5b6 to a measurable extent, whereas untreated erythrocytes do not. We attribute this to a shift in the partition equilibrium of C5b6 toward membrane association, which would improve lytic efficiency. The increase of membrane lipid disorder by these agents would also be expected to increase insertion of hydrophobic peptides from C7, C8, and C9, with consequent gain in lytic efficiency. Treatment of erythrocytes with sublytic doses of NaDodSO4, or Triton X-100 did not enhance lysis by C5b-9 appreciably, suggesting that enhancement of lysis by C5b-9 is not a general property of amphiphiles.     

Steady-state analysis of tracer exchange across the C5b-9 complement lesion in a biological membrane.

Resealed erythrocyte ghosts have been used to define the kinetics of tracer exchange across the membrane-bound terminal complex of the complement cascade (C5b-9). Under steady-state conditions and at net chemical equilibrium, C5b-9 ghosts showed no significant lysis above control levels as measured by hemoglobin efflux. In 1 mM sucrose at 37 degrees C, [14C]sucrose isotopic exchange diffusion into C5b-9 ghosts occurred at 4.8 (+/- 0.5, SEM) X 10(-20) mol sec-1 per functional lesion, equivalent to an apparent permeability coefficient of 4.8 X 10(-14) cm3 sec-1 for the single C5b-9 lesion. No significant uptake of [14C]sucrose above control levels was observed in C5b67 ghosts. The apparent rate of tracer permeation through the complement lesion is one to two orders of magnitude slower than predicted by a model of a transmembrane channel of dimensions permitting free diffusion of sucrose. The data support earlier assertions from this laboratory that diffusion of small molecules across the complement lesion in biological membranes is significantly restricted.     

Permeability characteristics of complement-damaged membranes: evaluation of the membrane leak generated by the complement proteins C5b-9.

Permeability characteristics of the membrane lesion generated by the terminal complement proteins are considered in light of recent observations that the measured diffusion of solute across complement-damaged membranes does not conform to the "doughnut hole" model of a discrete transmembrane pore formed by the inserted C5b-9 complex. By using the measured kinetics of steady-state tracer isotope diffusion of nonelectrolytes across resealed erythrocyte ghost membranes treated with C5b-9, a new transport model is developed. This model considers the apparent membrane lesion strictly in terms of the operational criteria of a functional conducting pathway for the observed diffusing solute, independent of a priori assumptions about the geometry or molecular properties of the membrane lesion. With this definition of the unit membrane lesion and the assumption that the exclusion size of the conducting pathway varies directly with the multiplicity of bound C5b-9 (as suggested by previous measurements under conditions of varying input of C5b-9), numerical estimates of te apparent permeability of the complement-damaged membrane to four diffusing nonelectrolytes are derived. These results suggest that the pathway for a particle diffusing across the complement lesion cannot be a pore and is functionally equivalent to an aqueous leak pathway, free of pore constraints. Implications of these results are discussed in terms of current molecular models for the mechanism of membrane damage by the complement proteins.     

Cation transport and adenosine triphosphatase activity in rat erythrocytes: a comparison of spontaneously hypertensive rats with the normotensive Brown-Norway strain.

The activity of transport adenosine triphosphatases (ATPases) in saponin-treated erythrocytes as well as the passive membrane permeability for 86Rb+ (K+), 45Ca2+ uptake (in the presence of orthovanadate) and the rate of Na(+)-H+ exchange in intact erythrocytes were studied in spontaneously hypertensive rats (SHR), Wistar-Kyoto (WKY) and Brown-Norway (BN.lx) rats. Higher Na+,K(+)-ATPase activity, lower Ca(2+)-ATPase activity, increased passive K+ permeability and greater 45Ca2+ uptake were observed in erythrocytes from SHR compared with BN.lx rats. Similar differences in the last two parameters were also disclosed by a comparison of SHR and WKY rats. The rate of Na(+)-H+ exchange in SHR erythrocytes was greater than in WKY rats but equal to that of BN.lx rats. A genetic analysis did not reveal a significant correlation between Na(+)-H+ exchange rate and blood pressure in F2 SHR x WKY hybrids.     

Charge movements during the Na+-Ca2+ exchange in heart sarcolemmal vesicles.

The Na+-Ca2+ exchange was studied in a highly purified vesicular preparation derived from heart sarcolemma. The initial velocity of the Na+-driven Ca2+ influx, which was monitored continuously with a specific electrode, was 15 nmol/mg of protein per sec; the total Ca2+-accumulation capacity was 80 nmol/mg of protein. The Na+-Ca2+ exchange generated a current that was compensated for by the uptake of tetraphenylphosphonium in (Ph4P+) (when the latter was present in the medium), the influx of K+, and the efflux of Cl-. The movements of Ph4P were followed with a specific electrode. Ca2+ in the concentration range 3-50 microM induced an increase in the permeability of the sarcolemmal membrane to K+. Under conditions of optimal charge neutralization by K+ (i.e., in the presence of valinomycin), the Km (Ca2+) of the exchanger was 1.5 microM. The Na+-Ca2+ exchange was inhibited by chlorpromazine and was not inhibited by vanadate.     

Inhibition of deoxygenation-induced membrane protein dephosphorylation and cell dehydration by phorbol esters and okadaic acid in sickle cells

Deoxygenation (DO) of sickle cell anemia red blood cells (SS cells) induces membrane permeabilization to Ca2+, Na+, and K+ and cell dehydration mostly through the activation of the Ca(2+)-dependent K+ channels. We show that DO of both SS cells and normal red blood cells was accompanied by a nonspecific dephosphorylation of membrane proteins. After treatment with a protein kinase C activator (phorbol myristate acetate) or a phosphoprotein phosphatase inhibitor (okadaic acid), the level of membrane protein phosphorylation in deoxygenated cells was maintained higher or equal, respectively, to that of the oxygenated controls. We found that these drugs in SS cells (1) inhibited by 40% the DO-stimulated net Ca2+ uptake, without affecting the DO-stimulated Ca2+ influx, suggesting that they activated the Ca2+ efflux; (2) slightly increased the DO-induced Na+ uptake and decreased the DO-induced K+ loss; and (3) prevented the DO-induced cell dehydration. Both drugs are known to stimulate both phosphorylation and activity of the Ca pump and of the Na/H antiport. Inhibition of SS cell dehydration might be due to an activation of the Ca pump preventing [Ca2+]i elevation responsible for the stimulation of the K+ channels and/or to an activation of the Na/H exchange resulting in cell water gain.     

Participation of protein kinases in complement C5b-9-induced shedding of platelet plasma membrane vesicles.

The formation of membrane microparticles through vesiculation of the platelet plasma membrane is known to provide catalytic surface for several enzyme complexes of the coagulation system, and to underlie the procoagulant responses elicited with platelet activation. This induced shedding of vesicles from the plasma membrane is most prominent when platelets are activated by the terminal complement proteins, C5b-9, by a Ca2+ ionophore, or by the combination of thrombin plus collagen. Although shown to require elevated [Ca2+], the cellular events that initiate plasma membrane evagination and fusion to form the shed vesicles remain unresolved. To gain additional insight into the cellular events that regulate membrane microparticle formation, we have examined how this process is influenced by the activity of cellular protein kinases. Cytoplasmic [Ca2+] of gel-filtered platelets was increased by membrane assembly of the terminal complement proteins C5b-9 in the presence of selective inhibitors of protein kinase or phosphatase reactions, and resulting microparticle formation was quantitated by fluorescence-gated flow cytometry. Pre-equilibration of the phosphatase inhibitor vanadate into the platelet cytosol increased microparticle formation by as much as 40%, suggesting that vesiculation of the platelet plasma membrane is influenced by the state of phosphorylation of a cellular constituent. By contrast to the stimulatory effects of vanadate, microparticle formation was partially inhibited in platelets treated with the protein kinase inhibitor sphingosine, the myosin light chain kinase inhibitor ML-7, the calmodulin-antagonist W-7, and under conditions of elevated cytosolic concentration of cyclic adenosine monophosphate. These results indicate that complement-induced platelet microparticle formation is influenced by one or more protein kinase(s) as well as by calmodulin, and suggest a role for the platelet myosin light chain kinase or another Ca(2+)-pluscalmodulin-regulated membrane component.     

Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement.

The ion permeability of planar lipid bilayers, as measured electrically, was found to increase modestly upon treatment with purified complement complex C5b,6 and complement components C7 and C8. The subsequent addition C9 greatly amplified this change. No permeability changes occurred when components were added individually to the membrane, or when they were used in paired combinations, or when C5b, C7, C8, and C9 were admixed prior to addition. Thus, there is a significant parallel between the permeability changes induced in the model membrane and damage produced in biological membranes by the C5b-9 complement attack sequence. The efficiency of membrane action by C5b-9 was critically dependent on the order in whcih components were added to the membrane. There were also differences in the electrical properties of membranes treated with C5b-8 and C5b-9, though in both cases the enhanced bilayer permeability is best attributed to the formation of trans-membrane channels. Collectively, the data are consistent with the hypothesis that the mechanism of membrane action by complement involves the production of a stable channel across the lipid bilayer, resulting in cell death by colloid-osmotic lysis.     

Elevated levels of parathyroid hormone in essential hypertensive patients with increased erythrocyte potassium efflux

Previous observations suggest that Ca(2+)-dependent K+ efflux is increased in erythrocytes from spontaneously hypertensive rats. On the other hand, it has been reported that hyperparathyroidism induces an increase in Ca(2+)-dependent K+ efflux of human erythrocytes. To investigate whether Ca(2+)-dependent K+ efflux is altered in essential hypertension quinine-sensitive K+ efflux was measured in erythrocytes from 20 normotensive controls and 30 nontreated essential hypertensives. The quinine-sensitive K+ efflux was similar for hypertensive patients (593 +/- 20 mmol/L cells/h) as compared with normotensive controls (532 +/- 34 mmol/L cells/h). Ten hypertensives exhibited values of quinine-sensitive K+ efflux above an upper normal limit of 650 mmol/L cells/h. As compared with controls those patients presented elevated plasma levels of parathyroid hormone (P less than .05). In addition, a positive correlation was found between parathyroid hormone and quinine-sensitive K+ efflux in the above ten hypertensives (R = 0.85, P less than .001). These results suggest that an excess of parathyroid hormone may be involved in the increase of Ca(2+)-dependent K+ efflux present in some essential hypertensive patients.     

Erythrocyte membrane permeability of monovalent cations in rats with spontaneous arterial hypertension

Intraerythrocyte Na and K concentrations were not different in spontaneously hypertensive rats (SHR) from those of normotensive rats (WKY). During incubation of the cells in LiCl solution for 24 h at 2-4 degrees C Li accumulation and K efflux in erythrocytes of SHR were higher as compared to WKY. A positive linear correlation was found between influx of Li and efflux of K in the erythrocytes. Li efflux from the erythrocytes at 37 degrees C was independent of the presence of a Na medium that indicated the absence of Na-Li countertransport in rat erythrocytes. There was no difference in Li efflux in both rat groups. The results provide further evidence in favor of the hypothesis of increased passive permeability for monovalent cations in erythrocytes of SHR.     

Platelet and erythrocyte Mg2+, Ca2+, Na+, K+ and cell membrane adenosine triphosphatase activity in essential hypertension in blacks.

OBJECTIVE: To assess the relationship between intracellular Mg2+, Ca2+, Na+ and K+ and cell membrane adenosine triphosphatase (ATPase) activity in normotensive and hypertensive blacks. DESIGN: Intracellular cations and cell membrane ATPase activity were studied in black patients with untreated essential hypertension and age-, weight- and height-matched normotensive controls. Platelet, erythrocyte and serum Mg2+, Ca2+, Na+ and K+ levels as well as platelet and erythrocyte membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities were measured in all subjects. METHODS: Intracellular Na+ and K+ were measured by flame photometry and Mg+ and Ca+ by atomic absorption spectrophotometry. Cell membrane ATPase activity was determined by a colorimetric method. RESULTS: The hypertensive group consistently demonstrated depressed activity of each ATPase studied, with significantly lower serum Mg2+, serum K+, erythrocyte Mg2+ and platelet Mg2+ levels compared with the normotensive group. Platelet Na+ and Ca2+ and erythrocyte Ca2+ were significantly elevated in the hypertensive group. In the hypertensive group, mean arterial pressure (MAP) was inversely correlated with platelet and erythrocyte membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase. Serum Mg2+, serum Ca2+ and platelet Mg2+ were negatively correlated with MAP in the hypertensive group whilst erythrocyte and platelet Ca2+ were positively correlated. In the normotensive group, platelet Mg2+ and MAP were negatively, and erythrocyte Ca2+ and MAP, positively correlated. CONCLUSIONS: Black patients with essential hypertension have widespread depression of cell membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities with serum and intracellular Mg2+ depletion and cytosolic Na+ and Ca2+ overload, which may reflect an underlying membrane abnormality in essential hypertension. These cellular abnormalities may be related to the defective transport mechanisms that in turn may be aggravated by Mg2+ depletion.     

Sodium-calcium ion exchange in cardiac membrane vesicles.

Membrane vesicles isolated from rabbit ventricular tissue rapidly accumulated Ca2+ when an outwardly directed Na+ gradient was formed across the vesicle membrane. Vesicles loaded internally with K+ showed only 10% of the Ca2+ uptake activity observed with Na+-loaded vesicles. Dissipation of the Na+ gradient with the monovalent cation exchange ionophores nigericin or narasin caused a rapid decline in Ca2+ uptake activity. The Ca2+-ionophore A23187 inhibited Ca2+ uptake by Na+-loaded vesicles and enhanced the rate of Ca2+ loss from the vesicles after uptake. Efflux of preaccumulated Ca2+ from the vesicles was stimulated 30-fold by the presence of 50 mM Na+ in the external medium. Na+-dependent uptake and efflux of Ca2+ were both inhibited by La3+. The results indicate that cardiac membrane vesicles exhibit Na+-Ca2+ exchange activity. Fractionation of the vesicles by density gradient centrifugation revealed a close correspondence between Na+-Ca2+ exchange activity and specific ouabain-binding activity among the various fractions. This relationship suggests that the observed Na+-Ca2+ exchange activity derives from the sarcolemmal membranes within the vesicle preparation.     

Altered calcium homeostasis and membrane destabilization in erythrocytes of hamsters infected with Leishmania donovani

Homeostatic mechanisms regulating intracellular concentrations of Ca2+ at a low level are prerequisites for maintaining the integral and cytoskeletal structure of erythrocytes under normal physiological conditions. The present study was undertaken to assess the contribution of Ca2+ homeostasis in modifying red-cell stability in hamsters, during the anaemia caused by Leishmania donovani. Erythrocytes from the infected animals became increasingly fragile as infection progressed. This fragility may be the result of a gradual change in membrane permeability, as indicated by enhanced uptake of 45Ca2+. The increase in cytosolic Ca2+ and decrease in membrane-bound Ca2+ observed indicate the release of Ca2+ from the membrane store, leading to [Ca2+]i accumulation in the later stages of the post-infection period. Decline in the efficacy of Ca(2+)-effluxing enzyme may also contribute to the enhanced [Ca2+]i level, with subsequent degradation of membrane proteins in the erythrocytes of the infected animals. Marked inhibition of proteolytic degradation by the Ca(2+)-dependent thiol protease inhibitor leupeptin, with concomitant thiol depletion, indicates the involvement of Ca(2+)-induced thiol protease in the observed degradation of membrane proteins. The results indicate that an altered Ca2+ homeostasis in erythrocytes following leishmanial infection causes enhanced cellular accumulation of Ca2+, which in turn may lead to haemolysis in experimental visceral leishmaniasis.     

Effect of protein kinase C activation on cytoskeleton and cation transport in human erythrocytes. Reproduction of some membrane abnormalities revealed in essential hypertension

Certain manifestations of alterations of membrane cytoskeleton, protein kinase C activity, and ion transport were revealed in erythrocytes of patients with essential hypertension: 1) the average volume of erythrocytes is reduced by 4%; 2) about 7% of the total number of erythrocytes is represented by cup-shaped forms compared with 1.5 to 3.0% in the control group; 3) basal phosphorylation of Band 4.9 protein is increased 1.6-fold to 1.8-fold; 4) activity of protein kinase C is increased by 60 to 70%; 5) the rate of proton electrochemical gradient (delta mu H+)-induced Na+-H+ exchange is increased twofold. Treatment of erythrocytes of healthy donors with protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate) leads to similar but more marked changes in cell shape (17% of cup-shaped forms), volume reduction (by 7%), an increase of Band 4.9 protein phosphorylation (threefold), and an increase in the rate of Na+-H+ exchange (fourfold). Protein kinase activation does not modify Na+-Li+ exchange and slightly increases (by 20-50%) Na+-K+ pump activity, Na+-K+ cotransport, and the rate of 45Ca influx. It may be assumed that the increase of protein kinase C activity is one of the most probable molecular mechanisms conditioning abnormalities of the membrane skeleton and Na+-H+ exchange in primary hypertension.     

Increased potassium permeability by calcium in hypochromic red blood cells.

The red blood cell (RBC) content of Na+ and K+ were measured both on fresh cells from normal, heterozygous beta-thalassaemic and iron-deficiency-anaemic subjects, and on the same cells incubated for 24 h, at 37 degrees C, either in presence or in absence of Calcium (Ca2+). Ca2+ did not increase membrane permeability to Na+, but increased the K+ loss, both from normal cells and to a greater degree much more from hypochromic cells. Glucose largely prevented the K+ loss from hypochromic cells incubated either in absence or in presence of Ca2+, probably maintaining an adequate level of ATP during the incubation. EDTA only partially decreased the permeability to K+ in hypochromic cells incubated for 24 h at 37 degrees C, possibly removing Ca2+ bound to the cell membrane. The results suggest that Ca2+ does not represent the primary cause of K+ leak in hypochromic cells, but it is able to enhance a pre-existing peculiar abnormality of the cell membrane when the ATP level slows down.     

23Na and 39K NMR studies of ion transport in human erythrocytes.

Ion transport in human erythrocytes was studied by 23Na and 39K NMR with an anionic paramagnetic shift reagent, Dy(P3O10)2(7-). The intra- and extracellular 23Na and 39K NMR signals were well separated (over 10 ppm) at 5 mM concentration of the shift reagent. The NMR visibility of the intracellular Na+ and K+ was determined to be 100% in human and duck erythrocytes. The intracellular ion concentrations were 8.1 +/- 0.8 mM Na+ (n = 7) and 110 +/- 12 mM K+ (n = 4) for fresh human erythrocytes. The ouabain-sensitive net Na+ efflux was 1.75 +/- 0.08 mmol/hr per liter of cells at 37 degrees C (n = 3). The gramicidin-induced ion transport in human erythrocytes was also studied by 23Na and 39K NMR or by simultaneous measurements of 23Na NMR and a K+-selective electrode. The time courses of the Na+ and K+ transport induced by the ionophore were biphasic. The initial rapid fluxes were due to an exchange of Na+ for K+, which were found to occur with a 1:1 stoichiometry. The subsequent slow components were the net Na+ and K+ effluxes rate-limited by the Cl- permeability and accompanied by a reduction in cell volume. The Cl- permeability determined from the NMR measurements of these slow fluxes was 3.2 +/- 0.5 X 10(-8) cm/sec at 25 degrees C (n = 4).     

Isolation of a human erythrocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels.

Erythrocytes are poorly lysed by homologous complement, whereas they are readily lysed by heterologous complement. This phenomenon had been attributed to an interference by the cell surface with the action of complement components C8 and C9. To isolate the responsible membrane constituent, detergent-solubilized human erythrocyte (EH) membranes were subjected to affinity chromatography by using human C9-Sepharose. The isolated protein had a mass of 38 kDa and, incorporated into liposomes, was highly effective in inhibiting complement-mediated channel expression, including the C5b-8, membrane attack complex, and tubular polymer of C9 channels. Antibody produced to the 38-kDa protein caused a 20-fold increase in reactive lysis of EH by isolated C5b6, C7, C8, and C9. The antibody did not enhance C5b-7 uptake, but it affected C9 binding to the target cell membrane. Antibody to human decay-accelerating factor, used as a control, had no effect on reactive lysis of EH. Anti-38-kDa protein did not enhance the action on EH of C8 and C9 from other species, indicating that the action of this regulatory protein is species specific. It was therefore termed homologous restriction factor (HRF). Blood cells other than erythrocytes, such as polymorphonuclear leukocytes, also exhibited cell-surface HRF activity. In immunoblots of freshly isolated EH membranes, anti-38-kDa HRF detected primarily a 65-kDa protein, suggesting that the 38-kDa protein constitutes an active fragment of membrane HRF. Because of the specific binding reaction observed between HRF and C8 or C9, HRF was tested with anti-human C8 and anti-human C9. A limited immunochemical relationship of HRF to C8 and C9 could be established and solid-phase anti-C9 proved an efficient tool for the isolation of HRF from solubilized EH membranes.     

A p-nitrophenylphosphatase activity associated with the human erythrocyte membrane.

A p-nitrophenylphosphatase activity has been identified as a component of the human erythrocyte membrane. This activity is distinct from that associated with the cell's Na(+)+K(+)-dependent ATPase, Ca(2+)-dependent ATPase, or spectrin phosphatase. The activity described here is stimulated by Mn2+ but not by Ca2+ with or without calmodulin. A potential erythrocyte membrane substrate for this activity is a 95 kDa phosphoprotein that can be shown to undergo Mn(2+)-stimulated but not Mg(2+)-stimulated dephosphorylation.     

Galanin and epinephrine act on distinct receptors to inhibit insulin release by the same mechanisms including an increase in K+ permeability of the B-cell membrane.

The mechanisms by which galanin and epinephrine affect pancreatic B-cell function were studied in normal mouse islets. In the presence of 15 mM glucose and 2.5 mM Ca2+, galanin (50 nM) and epinephrine (100 nM) hyperpolarized the B-cell membrane and suppressed electrical activity only transiently. These changes were accompanied by a decrease in 86Rb+ efflux from islet cells and nearly complete inhibition of insulin release. Both agents also decreased 86Rb+ efflux in the absence of Ca2+. Low concentrations (10-15 microM) of diazoxide, an activator of ATP-sensitive K+ channels, mimicked some effects of galanin and epinephrine. However, insulin release was more markedly inhibited by galanin or epinephrine than by diazoxide when electrical activity was similarly decreased, and diazoxide had no effect on 86Rb+ efflux in the absence of Ca2+. When the permeability to K+ was increased by 100 microM diazoxide and the hyperpolarization reversed by high extracellular K+, galanin and epinephrine still inhibited insulin release, but did not affect the membrane potential or 86Rb+ efflux. Galanin and epinephrine decreased glucose utilization and oxidation in islet cells by about 10%, whereas diazoxide had no effect. Blockade of alpha 2-adrenoceptors by yohimbine suppressed the effects of epinephrine, but not those of galanin. It is concluded that activation of galanin and alpha2-adrenergic receptors inhibits insulin release by the same mechanisms. These may involve an increase in K+ permeability of the B-cell membrane by opening ATP-sensitive K+ channels and an additional effect independent of the membrane potential.