Isolation of a photosystem II reaction center consisting of D-1 and D-2 polypeptides and cytochrome b-559

A photosystem II reaction center complex consisting of D-1 and D-2 polypeptides and cytochrome b-559 was isolated from spinach grana thylakoids, treated with 4% (wt/vol) Triton X-100, by ion-exchange chromatography using DEAE-Toyopearl 650S. The isolated complex appears to contain five chlorophyll a, two pheophytin a, one β-carotene, and one or two cytochrome b-559 heme(s) (molar ratio) and exhibits a reversible absorbance change attributable to the photochemical accumulation of reduced pheophytin typical for the intermediary electron acceptor of photosystem II reaction center. These results strongly suggest that the site of primary charge separation in photosystem II is located on the heterodimer composed of D-1 and D-2 subunits.     

Evidence for a biological role in photosynthesis for cytochrome b-559--a component of photosystem II reaction center

Absorbance changes in untreated intact leaf discs, produced upon excitation with high-intensity red light, were shown to be due to the photooxidation of cytochrome b-559. At low intensities (<100 W/m2), photooxidation was almost undetectable. Photooxidation occurred with a half-time of 4.3 sec and an extent of 0.64 mol of cytochrome per 320 mol of chlorophyll. Upon transition to darkness, an additional oxidation occurred that exhibited faster kinetics (t/12 < 100 msec) and 0.32 mol of cytochrome was oxidized per 320 mol of chlorophyll. Photooxidation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and was specifically induced by red light since far-red light did not cause any absorbance decrease. These results suggest that the redox changes of cytochrome b-559 are driven by photosystem II. Photooxidation was increased by 67% and its initial rate was doubled upon incubation of the leaf in carbonylcyanide p-trifluoromethoxy-phenylhydrazone. Exposure of the leaf to mild water stress or mild heat stress resulted in an increase in the extent of photooxidation and in a 6-fold decrease in the rate constant. Mild heat stress also induced a large increase of the rate constant for the dark reduction of the cytochrome. The dependence of photooxidation on high-intensity red light, its inhibition criteria, the fast transient dark oxidation, and enhancement of both photooxidation and dark transient oxidation by treatments that affect Z, the primary donor to P680, indicate that cytochrome b-559 in vivo is involved in cyclic electron flow around photosystem II. Its primary role in photosynthesis is to divert excess photons from a linear to a cyclic electron flow at high light intensities for protection of the D1 and D2 proteins against photodamage. Dark oxidation of the cytochrome is suggested to reflect a second role, that of deactivation of the powerful oxidant Z+ in the dark.     

New transients in the electron-transfer dynamics of photolyzed mixed-valence CO-cytochrome c oxidase.

Electron transfer following photolysis of CO from mixed-valence (cytochrome a3+ Cu2+A cytochrome a2+3-CO Cu+B) cytochrome oxidase (ferrocytochrome-c; oxygen oxidoreductase, EC 1.9.3.1) was studied on time scales of nanoseconds to milliseconds by multichannel time-resolved optical absorption spectroscopy. In this method, the optical absorption was measured at many wavelengths simultaneously by using an optical spectrometric multichannel analyzer system. The high-quality time-resolved difference spectra showed a large increase on a microsecond time scale in the visible region centered at approximately 520 nm and in the UV region centered at approximately 390 nm. These absorbance changes were not observed after photodissociation of CO from the fully reduced enzyme and therefore are attributed to intramolecular electron transfer. Simultaneously, there was a blue shift and a small increase in the alpha band, which is attributed to the reduction of cytochrome alpha. Approximately one-third of the absorbance change at 520 nm can be attributed to reduction of cytochrome a. The absorbance changes associated with the 520- and the 390-nm bands are on the same time scale (t1/2 approximately 2 microseconds) as the dissociation of CO from Cu+B reported previously by time-resolved infrared spectroscopy. The position and shape of these bands are reasonable for charge-transfer transitions involving copper(II). We suggest that the absorbance increase at 520 nm, which cannot be attributed to a reduction of cytochrome a, may represent a charge transfer involving Cu2+B accompanying the oxidation of Cu+B to Cu2+B. The absorbance increase at 390 nm is also partially attributed to this transition. These results suggest that Cu2+B may be observed spectrophotometrically in the electron-transfer dynamics of cytochrome oxidase.     

A functional model for the role of cytochrome b559 in the protection against donor and acceptor side photoinhibition.

A quinone-independent photoreduction of the low potential form of cytochrome b559 has been studied using isolated reaction centers of photosystem II. Under anaerobic conditions, the cytochrome can be fully reduced by exposure to strong illumination without the addition of any redox mediators. Under high light conditions, the extent and rate of the reduction is unaffected by addition of the exogenous electron donor Mn2+ and, during this process, no irreversible damage occurs to the reaction center. However, prolonged illumination in strong light brings about irreversible bleaching of chlorophyll, indicative of photoinhibitory damage. When the cytochrome is fully reduced and excess Mn2+ is present, the effect of moderate light is to facilitate the photoaccumulation of reduced pheophytin. The dark reoxidation of the reduced cytochrome is very slow under anaerobic conditions but significantly speeded up on addition of oxidized 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. From these results it is suggested that the low potential form of cytochrome b559 can accept electrons directly from reduced pheophytin and in so doing help to protect the reaction center against acceptor side photoinhibition as suggested by Nedbal et al. [Nedbal, J., Samson, G. & Whitmarsh, J. (1992) Proc. Natl. Acad. Sci. USA 89, 7929-7933]. This conclusion has been incorporated into a model that further suggests that in its high potential form the cytochrome primarily acts to protect against donor side photoinhibition due to increased lifetime of highly oxidized species as previously proposed by Thompson and Brudvig [Thompson, L. & Brudvig, G. W. (1988) Biochemistry 27, 6653-6658]. The particular feature of our scheme is that it incorporates reversible interconversion between the two redox forms so as to protect against either type of photoinhibition.     

Femtosecond photodichroism studies of isolated photosystem II reaction centers.

Photosynthetic conversion of light energy into chemical potential begins in reaction center protein complexes, where rapid charge separation occurs with nearly unit quantum efficiency. Primary charge separation was studied in isolated photosystem II reaction centers from spinach containing 6 chlorophyll a, 2 pheophytin a (Pheo), 1 cytochrome b559, and 2 beta-carotene molecules. Time-resolved pump-probe kinetic spectroscopy was carried out with 105-fs time resolution and with the pump laser polarized parallel, perpendicular, and at the magic angle (54.7 degrees) relative to the polarized probe beam. The time evolution of the transient absorption changes due to the formation of the oxidized primary electron donor P680+ and the reduced primary electron acceptor Pheo- were measured at 820 nm and 545 nm, respectively. In addition, kinetics were obtained at 680 nm, the wavelength ascribed to the Qy transition of the primary electron donor P680 in the reaction center. At each measured probe wavelength the kinetics of the transient absorption changes can be fit to two major kinetic components. The relative amplitudes of these components are strongly dependent on the polarization of the pump beam relative to that of the probe. At the magic angle, where no photoselection occurs, the amplitude of the 3-ps component, which is indicative of the charge separation, dominates. When the primary electron acceptor Pheo is reduced prior to P680 excitation, the 3-ps component is eliminated.     

Cytochrome b-559 and proton conductance in oxygenic photosynthesis

Although cytochrome b-559 has long been known as a membrane-bound redox component closely linked to the reaction center of the oxygen-generating photosystem (PSII), its role in photosynthesis has remained obscure. This paper reports evidence and outlines a hypothesis in support of a “b-559 cycle”—i.e., a light-induced, cytochrome b-559-dependent, cyclic electron transport pathway around PSII that promotes translocation of protons from plastoquinol into the aqueous domain (lumen) of photosynthetic membranes (thylakoids). Light-induced proton transport coupled to light-induced electron transport is an essential aspect of energy transduction in photosynthesis because it generates an electrochemical proton gradient that drives ATP synthesis by the process of photosynthetic phosphorylation. The principal carrier of electrons and protons in thylakoids is the plastoquinone/plastoquinol couple. We propose that the b-559 cycle functions as a redox-linked proton pump that may operate jointly with the Rieske iron-sulfur pathway in oxidizing plastoquinol. The overall effect of such concerted oxidation of plastoquinol would be the translocation into the thylakoid lumen of two protons for each electron transferred from water to plastocyanin via plastoquinone.     

Three Forms of Cytochrome b559 and Their Relation to the Photosynthetic Activity of Chloroplasts

Spinach chloroplasts were found to contain three forms of cytochrome b(559) that have the same alpha-peak at 559 nm, but are distinguished from one another by their oxidation-reduction potentials. The high-potential (H) form (E(m) about 330-350 mV) is reducible by hydroquinone, the middle-potential (M) form (E(m) about 50-80 mV) is reducible by ascorbate but not by hydroquinone, and the low-potential (L) form is reducible by dithionite but not by ascorbate. The H form was the predominant one in freshly prepared chloroplasts and was strongly correlated with Photosystem II activity. Chloroplast treatments such as aging, sonication, and mild heating, singly or in combination, brought about a shift of the H to the M form. More drastic treatments resulted in shifts of the H and M forms to the L form.The role of cytochrome b(559) as an electron carrier in System II of plant photosynthesis is discussed.     

Structural model of cytochrome b559 in photosystem II based on a mutant with genetically fused subunits

Photosystem II is a reaction center protein complex located in photosynthetic membranes of plants, algae, and cyanobacteria. Using light energy, photosystem II catalyzes the oxidation of water and the reduction of plastoquinone, resulting in the release of molecular oxygen. A key component of photosystem II is cytochrome b₅₅₉, a membrane-embedded heme protein with an unknown function. The cytochrome is unusual in that a heme links two separate polypeptide subunits, α and β, either as a heterodimer (αβ) or as two homodimers (α₂ and β₂). To determine the structural organization of cytochrome b₅₅₉ in the membrane, we used site-directed mutagenesis to fuse the coding regions of the two respective genes in the cyanobacterium Synechocystis sp. PCC 6803. In this construction, the C terminus of the α subunit (9 kDa) is attached to the N terminus of the β subunit (5 kDa) to form a 14-kDa αβ fusion protein that is predicted to have two membrane-spanning α-helices with antiparallel orientations. Cells containing the αβ fusion protein grow photoautotrophically and assemble functional photosystem II complexes. Optical spectroscopy shows that the αβ fusion protein binds heme and is incorporated into photosystem II. These data support a structural model of cytochrome b₅₅₉ in which one heme is coordinated to an α₂ homodimer and a second heme is coordinated to a β₂ homodimer. In this model, each photosystem II complex contains two cytochrome b₅₅₉ hemes, with the α₂ heme located near the stromal side of the membrane and the β₂ heme located near the lumenal side.     

Reconstitution of active transport in proteoliposomes containing cytochrome o oxidase and lac carrier protein purified from Escherichia coli.

Most active transport across the bacterial cell membrane is driven by a proton electrochemical gradient (delta-muH+, interior negative and alkaline) generated via electron transfer through a membrane-bound respiratory chain. This phenomenon is now reproduced in vitro with proteoliposomes containing only two proteins purified from the membrane of Escherichia coli. An o-type cytochrome oxidase was extracted from membranes of a cytochrome d terminal oxidase mutant with octyl beta-D-glucopyranoside after sequential treatment with urea and cholate and was purified to homogeneity by ion-exchange chromatography. The purified oxidase contains four polypeptides (MrS 66,000, 35,000, 22,000, and 17,000), two b-type cytochromes (b558 and b563), and 16-17 nmol of heme b per mg of protein, and it catalyzes the oxidation of ubiquinol and other electron donors with specific activities 20- to 30-fold higher than crude membranes. The lac carrier protein was purified as described. Proteoliposomes were formed in the presence of the oxidase and lac carrier protein by detergent dilution, followed by freeze-thaw/sonication. The system generates a delta-muH+ (interior negative and alkaline) with ubiquinol as electron donor and the magnitude of delta-muH+ is dependent on the concentration of cytochrome o in the proteoliposomes. Furthermore, the proteoliposomes transport lactose against a concentration gradient to an extent that is commensurate with the magnitude of delta-muH+ generated. The results provide powerful additional support for the "chemiosmotic hypothesis" and demonstrate that purified lac carrier protein retains the ability to function in a physiological manner.     

LIGHT-INDUCED OXIDATION OF A CHLOROPLAST B-TYPE CYTOCHROME AT -189 degrees C

The b-type cytochromes of chloroplasts have heretofore been viewed as photosynthetic electron carriers that probably occupy an intermediate position in a light-induced electron flow. The oxidation-reduction of such intermediate electron carriers, being removed from the primary photochemical reaction linked to photon capture by chlorophyll, would be expected to show a temperature dependence. Evidence has now been obtained that cytochrome b₅₅₉ is photooxidized at -189°C and that this photooxidation can be induced only by “short-wavelength” monochromatic light which activates the oxygen-evolving system in chloroplasts (photosystem II). In appears, therefore, that photooxidation of cytochrome b₅₅₉ is closely linked with photon capture by the chlorophyll pigments characteristic of photosystem II.     

Photoinduced degradation of the D1 polypeptide in isolated reaction centers of photosystem II: evidence for an autoproteolytic process triggered by the oxidizing side of the photosystem.

When the isolated D1/D2/cytochrome b559 complex was exposed to bright light, a distinctive pattern of D1 polypeptide fragments was observed under both aerobic and anaerobic conditions. The major degradation product had an apparent molecular mass of 24 kDa, while other fragments were detected at 17, 14, and 10 kDa by immunoblotting. This pattern was observed when the electron acceptors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone or silicomolybdate were present during illumination. It is known that these conditions stabilize P680+ chlorophyll and bring about the photooxidation and destruction of pigments in the reaction center, particularly chlorophyll absorbing at 670 nm and beta-carotene. When P680+ was not allowed to accumulate, either by omission of an electron acceptor or by addition of both an electron donor (Mn2+) and an acceptor, no breakdown fragments were observed. In the former case, however, some degradation of the D1 and D2 polypeptides did occur. Under conditions that gave rise to the characteristic D1 breakdown pattern, the D2 polypeptide was also degraded to specific fragments detected at about 29 and 21 kDa by immunoblotting. The results indicate that the photoinduced degradation of D1 (and D2) does not involve exogenous proteases but is most likely an autoproteolytic process. Moreover, our data indicate that the photochemical damage giving rise to D1 and D2 degradation occurs on the oxidizing rather than the reducing side of photosystem II and involves photooxidation of the accessory pigments. The results are discussed in terms of D1 and D2 turnover and photoinhibition.     

Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.

The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC 1.9.3.1) of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 in the presence of a protonophore, and (c) a fast-responding membraneless oxygen electrode. The reliability of the rate measurements was first established against the known stoichiometry of the scalar reaction of cytochrome oxidase (2ferrocytochrome c + 2H+ + 1/2O2 leads to 2ferricytochrome c + H2O) in the presence of excess protonophore. With all three methods the directly observed vectorial H+/O ejection ratios in the presence of K+ + valinomycin significantly exceeded 3.0. However, because the rate of backflow of the ejected H+ into the mitoplasts is very high and increases with the increasing delta pH generated across the membrane, there is a very rapid decline in the observed H+/O ratio from the beginning of the reaction. Kinetic analysis of ferrocytochrome c oxidation by the mitoplasts, carried out with a fast-responding membraneless oxygen electrode, showed the reaction to be first order in O2 and allowed accurate extrapolation of the rates of O2 uptake and H+ ejection to zero time. At this point, at which there is zero delta pH across the membrane, the H+/O ejection ratio of the cytochrome oxidase reaction, obtained from the rates at zero time, is close to 4.0.     

On the primary nature of fluorescence yield changes associated with photosynthesis

Absorbance changes of C-550 and cytochrome b₅₅₉, and fluorescence-yield changes were measured during irradiation of chloroplasts at -196°. The photo-reduction of C-550 proceeded more rapidly than the photo-oxidation of cytochrome b₅₅₉, and the fluorescence-yield change had similar kinetics to the cytochrome b₅₅₉ change. The fluorescence yield of chloroplasts exposed to a 16-μsec flash at -196° did not increase during the flash, but increased in the dark after the flash. Both of these experiments indicate that the fluorescence yield follows the dark reduction of the primary electron donor of Photosystem II, not the photoreduction of the acceptor. This explanation would also account for the recent results of Mauzerall [Proc. Nat. Acad. Sci. USA (1972) 69, 1358-1362] showing that the fluorescence yield of chloroplasts at room temperature requires about 20 μsec to reach a maximum after a very brief flash.     

Release of oxidized plastocyanin from photosystem I limits electron transfer between photosystem I and cytochrome b6f complex in vivo

We used fast absorbance spectroscopy to investigate in vivo binding dynamics and electron transfer between plastocyanin (pc) and photosystem I (PSI), and cytochrome (cyt) f oxidation kinetics in Chlamydomonas reinhardtii mutants in which either the binding or the release of pc from PSI was diminished. Under single flash-excitation conditions, electron flow between PSI and the cyt complex was not affected by a 5-fold lowering of the binding affinity of pc to PSI, as induced by a mutation replacing the tryptophan-651 of the PsaA subunit by a serine residue (PsaA-W651S). On the other hand, electron flow from PSI to the cyt b(6)f complex was very sensitive to a 2- to 3-fold decrease in the rate of pc release from PSI, obtained by replacing the glutamic acid residue 613 of the PsaB subunit with glutamine (PsaB-E613N). Thus, our data indicate that under these experimental conditions the release of oxidized pc limits electron transfer between cyt b(6)f complex and PSI in vivo.     

Cytochrome b and FAD content in polymorphonuclear leucocytes in a family with X-linked chronic granulomatous disease

Chronic granulomatous disease (CGD), an immunodeficiency syndrome characterized by extreme susceptibility to bacterial infections, is due to a defect of the respiratory burst in human phagocytes. NADPH oxidase, the enzyme that catalyzes the reduction of oxygen and the release of oxidative radicals, was studied in polymorphonuclear leucocytes (PMNs) in a family affected by an x-linked inheritance form at high penetrance of the disease. The contents of cytochrome b, suggested as the terminal component of the oxidase electron transport chain, and FAD, the hypothetical proximal component of the chain, were determined in patients and in carriers. Cytochrome b showed the typical behaviour of x-linked CGD: total absence in patients, intermediate values in carriers. FAD content evaluated on plasma membranes was less decreased than cytochrome b. Carriers also showed a decrease of this flavoprotein. Cytochrome b and FAD contents were compared to NBT test and superoxide production: a clear correlation was observed for the cytochrome b, but FAD plasma membrane evaluation could also be an interesting tool for the metabolic characterization of the disease in patients and in carriers.     

ENERGY-COUPLING MECHANISMS IN MITOCHONDRIA: KINETIC, SPECTROSCOPIC, AND THERMODYNAMIC PROPERTIES OF AN ENERGY-TRANSDUCING FORM OF CYTOCHROME b

The primary event of coupled electron transfer at phosphorylation site II is identified with a modification in one of the two chemically distinct forms of cytochrome b, designated as the energy-transducing cytochrome b(T). This modification is expressed through a change in the redox midpoint potential and by an increase in its reaction half time with cytochrome c(1). In pigeon heart mitochondria cytochrome b(T) exhibits an absorption maximum at 564 nm and on this basis, it can be distinguished from Keilin's cytochrome b which exhibits an absorption maximum at 560 nm and serves as an electron carrier on the substrate side of cytochrome b(T). Kinetic capability of cytochrome b(T) is evidenced by its rapid electron transfer and energization time of less than 200 msec, its thermodynamic capability-by a 280 mV potential span suitable for providing one of the two electron transfer reactions required in ATP formation. Two secondary events of coupled electron flow may be identified with a charge separation across the lipid structure of the permeability barrier and a change in water structure; both events result in an increased 1-anilino-8-naphthalene-sulfonic acid (ANS) response to the altered environment.     

Resolution of the electronic transitions of cytochrome c oxidase: evidence for two conformational states of ferrous cytochrome alpha.

Second-derivative absorption spectra are reported for a variety of oxidation and ligation states of bovine cytochrome c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1). The high resolving power of the second-derivative method allows us to assign the individual electronic transitions of cytochrome alpha and cytochrome alpha 3 in many of these states. In the fully reduced enzyme, one observes a single electronic transition at 444 nm, corresponding to the Soret transition for both ferrous cytochrome alpha and ferrous cytochrome alpha 3. When the cytochrome alpha 3 site is occupied by an exogenous ligand (CN or CO), one observes two absorption bands assignable to the ferrous cytochrome alpha chromophore, one at ca, 443 nm and the other at ca, 450 nm. The appearance of the 450-nm band is dependent only on ligand occupancy at the cytochrome alpha 3 site and not on the oxidation state of the cytochrome alpha 3 iron. These results can be interpreted either in terms of a heterogeneous mixture of two ferrous cytochrome alpha conformers in the cytochrome alpha 3-ligated enzyme or in terms of a reduction in the effective molecular symmetry of the ferrous cytochrome alpha site that results in a lifting of the degeneracy of the lowest unoccupied molecular orbital associated with the Soret pi,pi* transition of cytochrome alpha. In either case, the present data indicate that ferrous cytochrome alpha can adopt two distinct conformations. One possible structural difference between these two states could be related to differences in the strength of hydrogen bonding between the ferrous cytochrome alpha formyl oxygen and a proton donor from an unidentified amino acid side chain of the enzyme. The implications of such modulation of hydrogen-bond strength are discussed in terms of possible mechanisms of proton translocation and electron transfer in the enzyme.     

Coevolution of exceptional longevity, exceptionally high metabolic rates, and mitochondrial DNA-coded proteins in mammals

Mammals' longevity is inversely related to mass-specific basal metabolic rate because the generation of reactive oxygen species constrains lifespan. Longevity increases with body mass because the latter is inversely related to mass-specific basal metabolic rates. In placental mammals the longevity residuals from the power laws that describe longevity as a function of mass-specific basal metabolic rates, or body mass, are positively correlated with the relative rates of evolution of cytochrome b, a generator of reactive oxygen species. Therefore, longevity is more accurately described as a function of both mass-specific basal metabolic rate and the relative rate of cytochrome b evolution. The longevity residuals from the power law that describe longevity as a function of body mass are positively correlated with the relative rate of evolution of most other mtDNA-coded proteins. In taxa with very high rate of cytochrome b evolution exceptional longevity is associated with an increase, rather than the predicted decrease, of basal metabolic rates. These finding are compatible with the hypothesis that, in placental mammals, the accelerated evolution of mtDNA-coded proteins, allowed the extension of lifespan by selecting mutations that reduce the generation of reactive oxygen species, mostly by increasing internal proton leak, that accelerates mitochondrial electron transport.     

Growth factor activation of an amiloride-sensitive Na+/H+ exchange system in quiescent fibroblasts: coupling to ribosomal protein S6 phosphorylation.

Chinese hamster lung fibroblast cells (CCl39) enter the G0/G1 nonproliferative state after serum deprivation. In this report, we show that reinitiation of DNA synthesis by serum or the combination of purified human thrombin and insulin (1-10 microgram/ml) is preceded by very early stimulation of ionic fluxes (Na+/Rb+) and protein phosphorylation (27,000 daltons, 62,000 daltons, and the ribosomal S6 proteins). The potentiating action of insulin on thrombin-stimulated DNA synthesis is also observed on thrombin-stimulated Na+ influx, Rb+ influx, and protein S6 phosphorylation. Moreover, we demonstrate that CCl39 cells possess a Na+/H+ exchange system sensitive to amiloride. Half-maximal inhibition of growth factor-activated Na+ influx and Na+-dependent H+ efflux is obtained with 3-10 microM amiloride. Two lines of evidence indicate that the extrusion of H+ via the activation of the Na+/H+ exchanger is coupled to protein S6 phosphorylation: serum-stimulated phosphorylation is blocked by (i) amiloride at a concentration that abolishes serum-stimulated Na+ influx and (ii) protonophores that acidify the cell interior. The present data support the idea that the regulation of intracellular pH is a key event in the mechanism of growth factor action.     

Effect of low temperatures on glucose-induced insulin secretion and ionic fluxes in rat pancreatic islets

The direct effect of cold on the inhibition of B cell secretion is well known in hibernating and experimentally hypothermic mammals. This temperature dependency may result from the inhibition of ion transport across the membranes. In order to verify this hypothesis, ionic effluxes and insulin secretion from rat islets loaded with 86Rb+ and 45Ca+ were measured during perifusion. At 37 degrees C, the rise in glucose concentration from zero to 16.7 mmol/l provoked a rapid decrease in 86Rb+ efflux, an early fall and subsequent rise in 45Ca2+ efflux and a typical biphasic pattern of insulin secretion. At 27 degrees C, glucose induced only a very slight increase in insulin secretion, while the fluxes of radioactive ions were not significantly modified in amplitude but were clearly delayed. At 17 degrees C, no insulin response to glucose was observed and the decrease in K+ conductance indicated by 86Rb+ flux decrease was less temperature-dependent than the movement of Ca2+. After supplementary stimulation with a high extracellular concentration of Ca2+, insulin secretion was enhanced at 27 degrees C and reached levels induced by glucose alone at 37 degrees C. An increase in hormone secretion occurred even at 17 degrees C, but only during a first phase of secretion. Regular increases in temperature potentiated insulin secretion and provoked changes in ionic fluxes which suggest that B cell depolarization (86Rb+ flux decrease) induced by glucose can occur at 15 degrees C but cannot induce the opening of voltage-dependent Ca2+ channels (increase in 45Ca2+ efflux) until temperatures higher than 27 degrees C are reached.(ABSTRACT TRUNCATED AT 250 WORDS)