Differential stimulation of second messenger pathways by distinct classes of odorants

Using ciliary preparations from rat olfactory epithelia, representative compounds of different odorant classes were assayed for the capability of stimulating the formation of second messengers in a subsecond time range. Fruity, floral and minty odors were found to induce a transient accumulation of cyclic adenosine monophosphate, whereas the herbaceous and putrid compounds under test caused a rise in inositol trisphosphate concentration.     

Cytochemical localization of adenylate cyclase activity in rat olfactory cells

Summary Adenylate cyclase activity was demonstrated in the cilia, dendritic knob and axon of rat olfactory cells by using a strontium-based cytochemical method. The activity in the cilia and the dendritic knob was enhanced by non-hydrolyzable GTP (guanosine triphosphate) analogues and forskolin, and inhibited by Ca²⁺, all in agreement with biochemical reports of the odorant-sensitive adenylate cyclase. The results support the hypothesis of cyclic AMP working as a second messenger in olfactory transduction and imply that the transduction sites exist not only in the olfactory cilia but also in the dendritic knob. Enzymatic activity was also observed in the olfactory dendritic shaft by treating the tissue with 0.0002% Triton X-100, although the properties and role of the enzyme in this region are uncertain. The detergent inhibited the enzymatic activity in the cilia and the dendritic knob.     

Facilitation of adenylate cyclase stimulation in macrophages by lectins.

Preincubation of guinea pig peritoneal macrophages with concanavalin A (Con A) markedly enhanced the accumulation of 3′,5′-cyclic-adenosine monophosphate (cAMP) in response to the adenylate cyclase (AC) stimulators prostaglandin E₁ (PGE₁) and isoproterenol (IP). Basal cAMP levels were not altered. Maximal enhancement of cAMP accumulation was induced by preincubation with 50–100 μg/ml Con A for 10 min at 37 °C. Con A-induced facilitation of macrophage responsiveness was prevented by α-methyl-d-mannoside (αMM). No facilitation was induced by the divalent derivative, succinyl-Con A or by Con A immobilized on Sepharose beads. Con A-induced facilitation developed normally in macrophages treated with the microfilament blocking agent, cytochalasin B. The responsiveness of macrophages to PGE₁ and IP was also augmented by phytohemagglutinin (PHA) but wheat germ agglutinin (WGA), soy bean agglutinin (SBA), pokeweed mitogen (PWM), and Lotus tetragonolobus lectin (LL) showed no enhancing effect. The effect of Con A on cAMP levels was the result of augmented cAMP synthesis and not of reduced degradation or a block in cAMP egress from the cells. Lectin-induced facilitation of AC stimulation could be mediated via one of the following mechanisms: (i) induction of receptor clustering; (ii) causing a conformational change in the receptors; (iii) inhibition of negative cooperativity; (iv) causing an increase in membrane fluidity; (v) disruption of microtubules by acting as a Ca²⁺ ionophore; or (vi) inactivation of a sugar-containing inhibitor of AC.     

Olfactory adenylate cyclase of the rat. Stimulation by odorants and inhibition by Ca2+.

The Ca2+-dependent regulation of the activation of myosin MgATPase by vascular-smooth-muscle thin filaments involves caldesmon. This effect may be due to the direct interaction of caldesmon with a Ca2+-binding protein such as calmodulin or phosphorylation of caldesmon by a Ca2+-dependent kinase. I have found that Ca2+ switches on aorta thin filaments in less than 10 s, whereas the caldesmon in the thin filaments is phosphorylated only slowly (half-time greater than 10 min) and the maximum phosphorylation is very low (1 molecule per 7 molecules of caldesmon). I conclude that the phosphorylation of caldesmon hypothesis is untenable.     

The stimulation of hepatic adenylate cyclase by prostaglandin E1

Adenylate cyclase activity associated with particulate preparations from rat, mouse, rabbit, and dog liver is stimulated 2-to 5-fold by prostaglandin E₁ (PGE₁). This stimulation is dependent upon the presence of guanosine-5′-triphosphate (GTP). Prostaglandins F_1a and F_2a do not alter the enzymatic activity under these same conditions. Optimal concentrations of PGE₁ + GTP stimulate rat liver adenylate cyclase more than glucagon alone, but less than glucagon + GTP. Activity measured with glucagon + GTP is not affected by addition of PGE₁. Stimulation from PGE₁ + GTP is increased by glucagon to the same level measured with glucagon + GTP.     

Differential ion dependence of frog olfactory responses to various odorants.

1. Dependence of the fron olfactory bulbar responses on NaCl concentration greatly varied from odorant to odorant. The responses to odorants such as 1-carvone and isoamyl acetate were essentially unchanged by removal of NaCl, while those to odorant such as citral and beta-ionone were greatly decreased by removal of NaCl. 2. The NaCl requirement for the responses to certain odorants was greatly decreased by an increase in pH or temperature of the stimulating solution. 3. It was concluded that changes in ion permeability at the apical membranes of olfactory cells including olfactory ciliary membranes are not involved in generation of the in vivo olfactory responses to certain odorants.     

Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects

The olfactory receptors of terrestrial animals exist in an aqueous environment, yet detect odorants that are primarily hydrophobic. The aqueous solubility of hydrophobic odorants is thought to be greatly enhanced via odorant binding proteins (OBP) which exist in the extracellular fluid surrounding the odorant receptors. We have isolated and partially sequenced 14 candidate OBPs from six insect (moth) species. All 14 represent a single homologous family based on conserved sequence domains. The 14 proteins can be divided into three subfamilies based on differences in tissue specific expression and similarities in amino acid sequences. All 14 proteins are specifically expressed in antennal olfactory tissue. Subfamily I represents previously described pheromone binding proteins (PBP), which are male-specific, associate with pheromone-sensitive neurons, and are highly variable in their sequences when compared among species. Subfamilies II and III are expressed in both male and female antennae, appear to associate with general-odorant-sensitive neurons, and are highly conserved when compared among species. The properties of the subfamily II and III proteins suggest these are general-odorant binding proteins (GOBP). The properties of the respective insect OBP subfamilies suggest that they have different odorant binding specificities. The association of different insect OBP subfamilies with distinct classes of olfactory neurons having different odorant specificities suggests that OBPs can act as selective signal filters, peripheral to the actual receptor proteins.     

Representation of odorants by receptor neuron input to the mouse olfactory bulb.

To visualize odorant representations by receptor neuron input to the mouse olfactory bulb, we loaded receptor neurons with calcium-sensitive dye and imaged odorant-evoked responses from their axon terminals. Fluorescence increases reflected activation of receptor neuron populations converging onto individual glomeruli. We report several findings. First, five glomeruli were identifiable across animals based on their location and odorant responsiveness; all five showed complex response specificities. Second, maps of input were chemotopically organized at near-threshold concentrations but, at moderate concentrations, involved many widely distributed glomeruli. Third, the dynamic range of input to a glomerulus was greater than that reported for individual receptor neurons. Finally, odorant activation slopes could differ across glomeruli, and for different odorants activating the same glomerulus. These results imply a high degree of complexity in odorant representations at the level of olfactory bulb input.     

Brevibacterium liquefaciens adenylate cyclase and its in vivo stimulation by pyruvate.

Adenylate cyclase of Brevibacterium liquefaciens depends on pyruvate for activity. Growing in a simple medium containing glucose and DL-alanine, the microorganism excreted pyruvate, which reached 20 mM in the medium at stationary phase. Using [3H]adenosine to label the adenosine 5'-triphosphate pool, we showed that pyruvate in the medium stimulated adenylate cyclase of B. liquefaciens in vivo, in a manner similar to the stimulation observed in vitro. Adenylate cyclase in cells harvested at different phases of growth was equally responsive to exogenous pyruvate, indicating that the allosteric site for pyruvate was present in the enzyme throughout the various phases of cell growth. The specific activity of adenylate cyclase was highest in cells harvested at early log phase; thereafter it declined and was substantially lower at stationary phase. Although adenylate cyclase appears to be activated by pyruvate throughout the life span of the cell, the activity appears not to be critical to cell growth, which was comparable whether the medium contained high or low pyruvate.     

The detergent Solulan C-24 reveals properties of the olfactory adenylate cyclase system.

We have been developing the use of plasma-membrane-bound fluorescent probes to measure the pH values at the surfaces of living chondrocytes. For this purpose, three lipophilic pH indicators were made by covalently binding the xanthene dyes fluorescein, eosin or dichlorofluorescein to the amino group of phosphatidylethanolamine. The probes were incorporated into phospholipid vesicles and the effect of pH on the fluorescence was characterized. Fluorescence was measured at a single emission wavelength during excitation at two wavelengths, and the ratio of the intensities was calculated. The experimentally observed pKobs. values were determined by fitting the fluorescence ratios to the Henderson-Hasselbalch equation. All three probes acted as pH indicators, and the eosinyl-, dichlorofluoresceinyl- and fluoresceinylphosphatidylethanolamines had pKobs. values of 3.5, 6.3 and 7.5 respectively. At physiological salt concentrations, changes in the composition of the vesicle membrane had little effect on these values. We concluded that these probes were promising candidates for the measurement of pH values at cell surfaces.     

The effect of alpha and beta adrenergic receptor stimulation on the adenylate cyclase activity of human adipocytes.

The effects of the mixed agonist epinephrine and the beta agonist isoproterenol, each alone and in combination with the alpha adrenergic blocker phentolamine and the beta blocker propranolol on the adenylate cyclase activity of human adipocyte membrane fragments were determined in a calcium free buffer. Neither phentolamine (10 muM) nor propranolol (32 muM) affected basal adenylate cyclase activity. Epinephrine (10 muM) stimulated adenylate cyclase activity and this effect was slightly enhanced by phentolamine. The combination of epinephrine plus propranolol depressed adenylate cyclase below the basal level. Isoproterenol (10 muM) markedly stimulated adenylate cyclase; the addition of phentolamine caused an equivocal further increase while the addition of propranolol depressed adenylate cyclase activity to, but not below, the basal level. These findings are consistent with the hypothesis that human adipocytes have both alpha and beta adrenergic receptors and that these receptors are associated with the cell membrane adenylate cyclase system.     

Refractoriness of ovarian adenylate cyclase to continued hormonal stimulation.

Culture of preovulatory rat follicles with luteinizing hormone, follicle-stimulating hormone or prostaglandin E2 for 24 h reduced the subsequent response of adenylate cyclase to the homologous by 80, 50 and 90%, respectively; yet follicles refractory to luteinizing hormone fully responded to follicle-stimulating hormone responded to luteinizing hormone and prostaglandin E2, and those refractory to prostaglandin E2 could be stimulated by either gonadotropin. Desensitization of the adenylate cyclase system by luteinizing hormone was achieved by hormone concentrations of 0.8--2.0 mug/ml in the medium; a lower dose of luteinizing hormone (0.4 mug/ml), though effective in stimulating adenylate cyclase, did not induce refractoriness. Prostaglandin E2 caused partial refractoriness at dose levels of 0.1--0.25 mug/ml; higher dose levels were more effective. These findings suggest that continued exposure to the preovulatory follicle to elevated levels of hormones may cause perturbations in either the interaction between the hormone and its specific receptor or in a subsequent step essential for activation of adenylate cyclase.     

Enhancement of hormonal stimulation in intact cells. Potentiation of GTP-dependent activation of adenylate cyclase.

The ability of prostaglandin E1 (PGE1) and cholera toxin to increase cyclic AMP levels is potentiated 6-fold when normal rat kidney (NRK) cells are treated with picolinic acid or histidinol, or grown in isoleucine-deficient medium. The response to (-)-isoproterenol is increased 2-fold in NRK cells treated with picolinic acid but not in cells subjected to isoleucine deprivation. The increase in agonist responsiveness is time-dependent, reaches its maximum at 40 h, and is quickly reversed following removal of picolinic acid or addition of medium with normal amounts of isoleucine. The cholera toxin response is also increased about 7-fold in simian virus 40-transformed NRK cells and Moloney sarcoma virus-transformed NRK cells treated with picolinic acid. GTP-stimulated, but not fluoride-stimulated, adenylate cyclase activities are increased in membranes from NRK cells treated with picolinic acid or starved for isoleucine, indicating that the increased response is due, at least in part, to a specific potentiation of GTP-dependent functions of the adenylate cyclase system. The results demonstrate that GTP-dependent events in hormonal stimulation of adenylate cyclase can be altered in intact cells to modulate hormonal enhancement of cyclic AMP production.     

Sensitized guinea-pig lung: altered adenylate cyclase stimulation by epinephrine

Adenylate cyclase (AC) was measured in healthy and sensitized quinea-pig lungs. Basal activities were 24.49 ± 2.50 and 26.73 ± 3.03 pmols cyclic AMP mg protein/minute, respectively. NaF produced about threefold activity increase in both groups. Low concentrations of epinephrine (EPI) 10^?9 ? 10^?6M, maximally stimulated the enzyme in sensitized lungs. In contrast, these concentrations had no effect in healthy lungs. Higher EPI concentrations, 10^?5 ? 10^?2M, while progressively stimulating less the AC in sensitized lungs, increased the response in the healthy lungs. The maximal increase in AC activity, about 200%, was achieved with 10^?6 and 10^?3M EPI in sensitized and healthy lungs, respectively. Propranolol blocked the effect of EPI in both groups. The results indicate that sensitization altered the AC system in guinea-pig lungs.     

Direct stimulation of adenylate cyclase by mechanical forces in S49 mouse lymphoma cells during hyposmotic swelling.

S49 mouse lymphoma cells respond to swelling deformation with rapid increases in intracellular calcium and cAMP. Experiments demonstrate that these increases in calcium and cAMP concentrations are not coupled in a regulatory manner. Direct inhibition of adenylate cyclase in wild type cells with miconazole prevented swelling-induced accumulation of cAMP. No effect of swelling was observed on the activity of cAMP phosphodiesterase. Additionally, complete inhibition of cAMP phosphodiesterase did not prevent swelling-induced cAMP accumulation. Experiments involving cyc- mutants (lacking the Gs-alpha protein) and 2',5'-dideoxyadenosine indicate that increased adenylate cyclase activity with swelling is not mediated by Gs. No evidence was found for attenuation of Gi-mediated inhibition of adenylate cyclase activity following swelling. In addition, exposure to pertussis toxin or phorbol ester, which disrupts Gi inhibition of adenylate cyclase did not prevent cAMP accumulation following swelling. Disruption of the actin membrane skeleton resulted in a significant accumulation of cAMP which was not further increased by swelling. Disruption of the microtubular cytoskeleton also increased cAMP content in S49 cells which could be further increased by swelling. It is concluded that S49 cell-adenylate cyclase responds directly to mechanical forces transmitted through the actin membrane skeleton.     

The lipid environment of the glucagon receptor regulates adenylate cyclase activity.

1. The lipids composition of rat liver plasma membranes was substantially altered by introducing synthetic phosphatidylcholines into the membrane by the techniques of lipid substitution or lipid fusion. 40-60% of the total lipid pool in the modified membranes consisted of a synthetic phosphatidylcholine. 2. Lipid substitution, using cholate to equilibrate the lipid pools, resulted in the irreversible loss of a major part of the adenylate cyclase activity stimulated by F-, GMP-P(NH)P or glucagon. However, fusion with presonicated vesicles of the synethic phosphatidylcholines causes only small losses in adenylate cyclase activity stimulated by the same ligands. 3. The linear form of the Arrhenius plots of adenylate cyclase activity stimulated by F- or GMP-(NH)P was unaltered in all of the membrane preparations modified by substitution or fusion, with very similar activation energies to those observed with the native membrane. The activity of the enzyme therefore appears to be very insensitive to its lipid environment when stimulated by F- or gmp-p(nh)p. 4. in contrast, the break at 28.5 degrees C in the Arrhenius plot of adenylate cyclase activity stimulated by glucagon in the native membrane, was shifted upwards by dipalmitoyl phosphatidylcholine, downwards by dimyristoyl phosphatidylcholine, and was abolished by dioleoyl phosphatidylcholine. Very similar shifts in the break point were observed for stimulation by glucagon or des-His-glucagon in combination with F- or GMP-P(NH)P. The break temperatures and activation energies for adenylate cyclase activity were the same in complexes prepared with a phosphatidylcholine by fusion or substitution. 5. The breaks in the Arrhenius plots of adenylate cyclase activity are attributed to lipid phase separations which are shifted in the modified membranes according to the transition temperature of the synthetic phosphatidylcholine. Coupling the receptor to the enzyme by glucagon or des-His-glucagon renders the enzyme sensitive to the lipid environment of the receptor. Spin-label experiments support this interpretation and suggest that the lipid phase separation at 28.5 degrees C in the native membrane may only occur in one half of the bilayer.     

Calmodulin stimulation of adenylate cyclase of intestinal epithelium

The effect of dicyclohexylcarbodiimide (DCCD) on the proton pumping two-subunit cytochrome c oxidase from Paracoccus denitrificans was investigated. Purified Paracoccus oxidase was reconstituted into phospholipid vesicles by cholate dialysis. Following incubation with increasing amounts of DCCD, proton ejection was recorded in response to reductant pulses with reduced cytochrome c. Concentrations of DCCD which greatly reduced proton pumping by bovine cytochrome c oxidase used as a control were found to exert only a minor effect on proton translocation by Paracoccus oxidase. Similarly, incubation of the bacterial enzyme with [14C]DCCD failed to reveal the specific covalent interaction previously demonstrated to occur with bovine cytochrome c oxidase, and here also shown for the oxidase of yeast. Thus, Paracoccus oxidase differs in its interaction with DCCD from the functionally analogous eukaryotic enzymes.