Ca2+-Independent Stimulation of Adenylate Cyclase Activity by Muscarinic Receptors in Rat Olfactory Bulb

In rat olfactory bulb homogenate, carbachol stimulated adenylate cyclase activity in a concentration-dependent manner (EC50 = 1.1 microM). The carbachol stimulation occurred fully in membranes that had been prepared in the presence of 1 mM EGTA and incubated in a Ca2(+)-free enzyme reaction medium. Under these conditions, exogenous calmodulin (1 microM) failed to stimulate adenylate cyclase activity. In miniprisms of olfactory bulb, carbachol (1 mM) increased accumulation of inositol phosphates, but this response was markedly reduced in a Ca2(+)-free medium. Moreover, the carbachol stimulation of adenylate cyclase activity was not affected by staurosporine at a concentration (1 microM) that completely blocked the stimulatory effect of phorbol 12-myristate 13-acetate, an activator of Ca2+/phospholipid-dependent protein kinase. Quinacrine, a nonselective phospholipase A2 inhibitor, reduced the carbachol stimulation of adenylate cyclase activity, but this inhibition appeared to be competitive with a Ki of 0.2 microM. Nordihydroguaiaretic acid and indomethacin, two inhibitors of arachidonic acid metabolism, failed to affect the carbachol response. These results indicate that in rat olfactory bulb, muscarinic receptors stimulate adenylate cyclase activity through a mechanism that is independent of Ca2+ and phospholipid hydrolysis.     

Olfactory transduction: cross-talk between second-messenger systems

Chemosensory cilia of olfactory receptor neurons contain an adenylate cyclase which is stimulated by high concentrations of odorants. Cyclic AMP produced by this enzyme has been proposed to act as second messenger in olfactory transduction. Here we report that olfactory cilia contain calmodulin and that calmodulin potently activates olfactory adenylate cyclase by a mechanism additive to and independent from direct stimulation by odorants. Activation by calmodulin is calcium dependent and enhanced by GTP. Thus, olfactory transduction may involve a second-messenger cascade in which an odorant-induced increase in intracellular calcium concentration leads to activation of adenylate cyclase by calmodulin.     

Information from e.x.a.f.s. spectroscopy on the structures of different forms of molybdenum in xanthine oxidase and the catalytic mechanism of the enzyme.

In isolated perfused rat hearts, epidermal growth factor (EGF; 15 nM) increased cellular cyclic AMP (cAMP) content by 9.5-fold. In rat cardiac membranes, EGF also stimulated adenylate cyclase activity in a dose-dependent manner, with maximal stimulation (35% above control) being observed at 10 nM-EGF. Half-maximal stimulation of adenylate cyclase was observed at 40 pM-EGF. Although the beta-adrenergic-receptor antagonist propranolol markedly attenuated the isoprenaline-mediated increase in cAMP content of perfused hearts and stimulation of adenylate cyclase activity, it did not alter the ability of EGF to elevate tissue cAMP content and stimulate adenylate cyclase. The involvement of a guanine-nucleotide-binding protein (G-protein) in the activation of adenylate cyclase by EGF was indicated by the following evidence. First, the EGF-mediated stimulation of adenylate cyclase required the presence of the non-hydrolysable GTP analogue, guanyl-5'-yl-imidodiphosphate (p[NH]ppG). Maximal stimulation was observed in the presence of 10 microM-p[NH]ppG. Secondly, in the presence of 10 microM-p[NH]ppG, the stable GDP analogue guanosine 5'-[beta-thio]diphosphate at a concentration of 10 microM blocked the stimulation of the adenylate cyclase by 1 nM- and 10 nM-EGF. Third, NaF + AlCl3-stimulated adenylate cyclase activity was not altered by EGF. The ability of EGF to stimulate adenylate cyclase was not affected by pertussis-toxin treatment of cardiac membranes. However, in cholera-toxin-treated cardiac membranes, when the adenylate cyclase activity was stimulated by 2-fold, EGF was ineffective. Finally, PMA by itself did not alter the activity of cardiac adenylate cyclase, but abolished the EGF-mediated stimulation of this enzyme activity. The experimental evidence in the present paper demonstrates, for the first time, that EGF stimulates adenylate cyclase in rat cardiac membranes through a stimulatory GTP-binding regulatory protein, and this effect is manifested in elevated cellular cAMP levels in perfused hearts exposed to EGF.     

Functional reconstitution of the olfactory membrane: incorporation of the olfactory adenylate cyclase in liposomes

Chemosensory cilia isolated from the olfactory epithelium of Rana catesbeiana were solubilized with Lubrol PX in the presence of supplementary lipid, forskolin, and sodium fluoride. Subsequent removal of the detergent by adsorption onto Biobeads SM2 results in the formation of proteoliposomes that display forskolin- and GTP gamma S-sensitive adenylate cyclase activity. Sucrose gradient centrifugation of liposomes formed in the presence of fluorescently labeled phosphatidylcholine demonstrates association between the olfactory adenylate cyclase and the exogenously added lipid. Forskolin stimulates the enzyme in reconstituted membranes with the same potency as in native membranes (EC50 = 1-2 microM). However, GTP gamma S is 350-fold more potent in native membranes (EC50 = 4.0 +/- 0.5 nM) than in reconstituted membranes (EC50 = 1.4 +/- 0.3 microM). These studies represent a first step toward the functional reconstitution and molecular dissection of the olfactory membrane.     

Gossypol modulation of nucleotide metabolizing enzymes in the reproductive tract of male rats

The activities of several pivotal nucleotide metabolizing enzymes from the testis and vasal sperm of rats treated for 7 wk with 0, 20 or 30 mg X kg X day gossypol acetic acid were examined. Total testicular lactate dehydrogenase (LDH) activity increased 40% above control in the highest treatment group examined. However, the specific activity of the testis-specific isozyme of LDH, LDH-C4, decreased to 50 and 20% of control in the 20 and 30 mg X kg X day treatment groups, respectively. Basal soluble adenylate cyclase from a 100,000 X g supernatant of testis homogenate exhibited a 25% decrease in activity only in the 30-mg treatment group. Basal adenylate cyclase activity in the testicular membrane fraction increased 20 to 30% above control in response to gossypol administration. Testis membranes from the 20- and 30-mg treatment group exhibited a 2- and 4-fold greater activation of adenylate cyclase by guanine nucleotides. In vitro dose-response curves showed a half-maximal inhibitory concentration (IC50) for inhibition of soluble testicular adenylate cyclase by gossypol of 400 microM in each treatment group. Caudal epididymal sperm adenylate cyclase activity decreased to 25% of control levels in gossypol-treated animals, and the in vitro sensitivity of the enzyme to the inhibitory effects of gossypol increased 4-fold. IC50 values for gossypol inhibition of sperm adenylate cyclase decreased from 200 microM in control animals to 75 and 50 microM in the 20 and 30 mg X kg X day treatment groups, respectively. Cyclic adenosine 3':5' monophosphate phosphodiesterase activity in caudal sperm increased 6-fold in the 20- and 30-mg treatment groups. These results demonstrate that nucleotide metabolizing enzymes in sperm are major targets for the actions of gossypol and provide a possible mechanism for the inhibition of normal sperm function by this compound.     

Distinct interactions between Ca2+/calmodulin and neurotransmitter stimulation of adenylate cyclase in striatum and hippocampus

1. Ca2+ and cAMP both act as intracellular second messengers of receptor activation. In neuronal tissue, Ca2+ acting via calmodulin can elevate cAMP levels. This regulation by Ca2+ provides a means whereby the elevation of intracellular [Ca2+] might modulate cAMP generation. 2. In the present studies, the impact of the Ca2+/calmodulin regulation on receptor-mediated stimulation of activity is compared in striatum and hippocampus--regions of differing sensitivity to Ca2+/camodulin. Ca2+/calmodulin stimulated striatal and hippocampal adenylate cyclase activity by 1.4- and 2.7-fold respectively, while dopamine and vasoactive intestinal peptide (VIP) stimulated the enzyme activity of these respective regions by 1.3- and 2-fold. 3. In the presence of Ca2+/calmodulin, the dopamine dose-response curve in the striatum was shifted upward, without alteration of the slope of the curve or of the maximal stimulation of activity elicited by dopamine. In the hippocampus, the ability of VIP to stimulate adenylate cyclase activity was reduced by the presence of calmodulin. 4. The dose dependence of these actions of calmodulin was examined. In the striatum, the stimulation of adenylate cyclase activity by 0.1 to 0.3 microM calmodulin obscured dopamine stimulation, while 1 to 10 microM was additive with the dopamine stimulation. In the hippocampus, all concentrations of calmodulin (0.1 to 10 microM) reduced VIP-mediated stimulation of enzyme activity. 5. These data suggest that the ratio of calmodulin-sensitive to calmodulin-insensitive adenylate cyclase activity varies in different rat brain regions and that, in those regions in which this ratio is low (e.g., rat striatum and most peripheral systems), calmodulin- and receptor-mediated activation of adenylate cyclase activity will be additive, while in those systems in which this ratio is high (e.g., most of the central nervous system), calmodulin will reduce receptor-mediated stimulation of enzyme activity.     

Calcium-independent activation of adenylate cyclase by calmodulin

Adenylate cyclase of Bordetella pertussis is stimulated by calmodulin by two distinct interactions. At low activator concentrations (approximately equal to 1 nM) the process is Ca2+-dependent (i.e. inhibited by EGTA added before calmodulin). High activator concentrations (approximately equal to 0.1-10 microM) stimulate adenylate cyclase also in the presence of EGTA, an effect not accounted for by residual Ca2+ or low concentrations of Ca X calmodulin, which thus appears to be due to calcium-free calmodulin. Some calmodulin dose-response curves show both phases of stimulation, separated by a plateau of activity, and half-maximal activating concentrations differ by 100-300-fold. Both effects are on the V and not the Km for ATP and are not mimicked by 10(5)-fold greater concentrations of parvalbumin or by various polyanions. In addition, adenylate cyclase stimulation at high calmodulin concentrations is greater in the presence of EGTA than in its absence. This enhancement is also produced by 1,10-phenanthroline and 8-hydroxyquinoline but not by non-chelating isomers. These compounds are poor Ca2+ chelators, stimulate at any calmodulin concentration (unlike EGTA), and suggest regulation of this adenylate cyclase by a second metal ion.     

Calmodulin-mediated adenylate cyclase from mammalian sperm

Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by the CaM antagonists chlorpromazine and calmidazolium (IC50 = 400 and 50 microM, respectively). The inhibition of enzyme activity by these agents correlated with their known potency and specificity as anti-CaM agents. The activity of the enzyme in the presence of 200 microM calmidazolium was restored by the addition of authentic CaM (EC50 = 15 microM); full activity was restored by the addition of 50 microM CaM. La3+, an ion that dissociates CaM from tightly bound CaM-enzyme systems, inhibited equine sperm adenylate cyclase (IC50 = 1 mM). Incubation of equine sperm adenylate cyclase with La3+ dissociated endogenous CaM from the enzyme so that most of the enzyme bound to a CaM-Sepharose column equilibrated with Ca2+. Specific elution of CaM-binding proteins from the CaM-Sepharose column with EGTA yielded a CaM-depleted adenylate cyclase fraction that was stimulated 2-fold by the addition of exogenous CaM.     

Involvement of calmodulin in the regulation of adenylate cyclase activity in guinea-pig enterocytes

The involvement of calmodulin as an activator of adenylate cyclase activity was examined in isolated guinea-pig enterocytes and in a membrane preparation. In enterocytes, which responded to prostaglandin E1, vasoactive intestinal peptide and cholera toxin with a significant increase in the rate of cAMP formation trifluoperazine, a calmodulin antagonist, completely inhibited cAMP formation. In a membrane preparation adenylate cyclase activity was stimulated 10-20-fold by the GTP analog, guanosine 5'-[beta-imido]5'-triphosphate (Gpp[NH]p). Prostaglandin E1 and vasoactive intestinal peptide enhanced cAMP formation in this system by 2-3- and 1.2-1.6-fold. respectively. Addition of 200 nM calmodulin to membranes, in which endogenous calmodulin was decreased from 1.4 microgram/mg protein to 0.5 microgram/mg protein by washing with buffer containing EGTA and EDTA, resulted in a 3-4-fold increase of adenylate cyclase activity. The absolute increment in adenylate cyclase activity caused by calmodulin (10-15 pmol cAMP/min per mg protein) was approximately the same in the absence or presence of Gpp[NH]p. The apparent Ka for Gpp[NH]p (6 . 10-7 M) was not significantly changed by the addition of calmodulin. Although endogenous calcium (approx. 10 microM) in the enzyme assay was adequate to affect stimulation by calmodulin, a maximal effect was observed at a calcium concentration of 100 microM. These findings indicate that a calmodulin-sensitive form of adenylate cyclase is present in guinea-pig enterocytes, and that stimulation of cAMP formation in the intestinal mucosa may involve a calmodulin-mediated mechanism.     

Biphasic Ca2+ response of adenylate cyclase. The role of calmodulin in its activation by Ca2+ ions

The Ca2+-dependent regulation of human platelet membrane adenylate cyclase has been studied. This enzyme exhibited a biphasic response to Ca2+ within a narrow range of Ca2+ concentrations (0.1-1.0 microM). At low Ca2+ (0.08-0.3 microM) adenylate cyclase was stimulated (Ka = 0.10 microM), whereas at higher Ca2+ (greater than 0.3 microM) the enzyme was inhibited to 70-80% control (Ki = 0.8 microM). Membrane fractions, prepared by washing in the presence of LaCl3 to remove endogenous calmodulin (approximately equal to 70-80% depletion), exhibited no stimulation of adenylate cyclase by Ca2+ but did show the inhibitory phase (Ki = 0.4 microM). The activation phase could be restored to La3+-washed membranes by addition of calmodulin (Ka = 3.0 nM). Under these conditions it was apparent that calmodulin reduced the sensitivity of adenylate cyclase to Ca2+ (Ki = 0.8 microM). Prostaglandin E1 (PGE1) did not alter Ki or Ka values for Ca2+. Calmodulin did not alter the EC50 for PGE1 stimulation of adenylate cyclase but increased the Vmax (1.5-fold). The calmodulin antagonist trifluoperazine potently inhibited adenylate cyclase in native membranes (80%) and to a much lesser extent in La3+-washed membranes (15%). This inhibition was due to interaction of trifluoperazine with endogenous calmodulin since trifluoperazine competitively antagonized the stimulatory effect of calmodulin on adenylate cyclase in La3+-washed membranes. We propose that biphasic Ca2+ regulation of platelet adenylate cyclase functions to both dampen (low Ca2+) and facilitate (high Ca2+) the haemostatic function of platelets.     

Guanylate cyclase in olfactory cilia from rat and pig

A guanylate cyclase was identified in cilia from rat and pig olfactory epithelia. Enzyme activities were 200-250 and 90-100 pmol/min.mg-1, respectively. Activity required the presence of non-ionic detergents, e.g., 0.1% Lubrol PX. MnGTP, not MgGTP was used as a substrate. Furthermore, 0.9 mM free Mn2+ was necessary for optimal activity indicating a regulatory site for a divalent cation. The guanylate cyclase displayed sigmoidal Michaelis-Menten kinetics suggesting cooperativity between MnGTP and enzyme. S0.5 was 160 microM MnGTP. The Hill coefficient of 1.7 indicates that more than one class of substrate-binding sites interact in a positive cooperative manner. ATP inhibited the enzyme and linearized plots of substrate kinetics with MnGTP. SH-Blocking agents reversibly inhibited enzyme activity. Sodium azide and nitroprusside were without effect as were several odorants. A guanylate cyclase activity in cilia from tracheal tissue had properties similar to the olfactory enzyme.     

Guanine nucleotide-independent inhibition of adenylate cyclase by a stimulatory hormone.

Stimulation of basal adenylate cyclase activity in membranes of neuroblastoma x glioma hybrid cells by prostaglandin E1 (PGE1) is half-maximal and maximal (about 8-fold) at 0.1 and 10 microM respectively. This hormonal effect requires GTP, being maximally effective at 10 microM. However, at the same concentrations that stimulate adenylate cyclase in the presence of GTP, PGE1 inhibited basal adenylate cyclase activity when studied in the absence of GTP, by maximally 60%. A similar dual action of PGE1 was observed with the forskolin-stimulated adenylate cyclase, although the potency of PGE1 in both stimulating and inhibiting adenylate cyclase was increased and the extent of stimulation and inhibition of the enzyme by PGE1 was decreased by the presence of forskolin. The inhibition of forskolin-stimulated adenylate cyclase by PGE1 occurred without apparent lag phase and was reversed by GTP and its analogue guanosine 5'-[gamma-thio]triphosphate at low concentrations. Treatment of neuroblastoma x glioma hybrid cells or membranes with agents known to eliminate the function of the inhibitory GTP-binding protein were without effect on PGE1-induced inhibition of adenylate cyclase. The data suggest that stimulatory hormone agonist, apparently by activating one receptor type, can cause both stimulation and inhibition of adenylate cyclase, and that the final result depends only on the activity state of the stimulatory GTP-binding protein, Gs. Possible mechanisms responsible for the observed adenylate cyclase inhibition by the stimulatory hormone PGE1 are discussed.     

Potent and cooperative feedback inhibition of adenylate cyclase activity by calcium in pituitary-derived GH3 cells

Calcium (Ca2+) ion concentrations that are achieved intracellularly upon membrane depolarization or activation of phospholipase C stimulate adenylate cyclase via calmodulin (CaM) in brain tissue. In the present study, this range of Ca2+ concentrations produced unanticipated inhibitory effects on the plasma membrane adenylate cyclase activity of GH3 cells. Ca2+ concentrations ranging from 0.1 to 0.8 microM exerted an increasing inhibition on enzyme activity, which reached a plateau (35-45% inhibition) at around 1 microM. This inhibitory effect was highly cooperative for Ca2+ ions, but was neither enhanced nor dependent upon the addition of CaM (1 microM) to EGTA-washed membranes. The inhibition was greatly enhanced upon stimulation of the enzyme by vasoactive intestinal peptide (VIP) and/or GTP. Prior exposure of cultured cells to pertussis toxin did not affect the inhibition of plasma membrane adenylate cyclase activity by Ca2+, although in these membranes, hormonal (somatostatin) inhibition was significantly attenuated. Maximally effective concentrations of Ca2+ and somatostatin produced additive inhibitory effects on adenylate cyclase. The addition of phosphodiesterase inhibitors demonstrated that inhibitory effects of Ca2+ were not mediated by Ca2(+)-dependent stimulation of a phosphodiesterase activity. These observations provide a mechanism for the feedback inhibition by elevated intracellular Ca2+ levels on cAMP-facilitated Ca2+ entry into GH3 cells, as well as inhibitory crosstalk between Ca2(+)-mobilizing signals and adenylate cyclase activity.     

Calcium-dependent adenylate cyclase of pituitary tumor cells

Effects of Ca2+ and calmodulin on the adenylate cyclase activity of a prolactin and growth hormone-producing pituitary tumor cell strain (GH3) were examined. The adenylate cyclase activity of homogenates was stimulated approx. 60% by submicromolar free Ca2+ concentrations and inhibited by higher (microM range) concentrations of the cation. A 2-3-fold stimulation of the activity in response to Ca2+ was observed at physiologic concentrations of KCl, with both the stimulatory and inhibitory responses occurring at respectively higher free Ca2+ concentrations. Calmodulin in incubations at low KCl concentrations increased the enzyme activity at all Ca2+ concentrations tested. In incubations conducted at physiologic KCl concentrations, both the inhibitory and stimulatory responses to Ca2+ were shifted by calmodulin to lower respective concentrations of the cation, without significant change occurring in the maximal rate of enzymic activity at optimal free Ca2+ X Mg2+ concentrations in the incubation also influenced the Ca2+ concentration dependence of adenylate cyclase; at high Mg2+ more Ca2+ was required to obtain maximal activity. Trifluoperazine inhibited adenylate cyclase of GH3 cells only in the presence of Ca2+; as Ca2+ concentrations in the assay were increased, higher drug concentrations were required to inhibit the enzyme. Ca2+ was also observed to reduce the extent of enzyme destabilization which occurred during pretreatments at warm temperatures. Vasoactive intestinal polypeptide and phorbol myristate acetate, which stimulate prolactin secretion in intact GH3 cells, enhanced enzyme activity 4- and 2.5-fold, respectively, without added Ca2+. Increasing free Ca2+ concentrations reduced the enhancement by VIP and eliminated the stimulation by PMA.     

Cross talk between stimulatory and inhibitory guanosine 5'-triphosphate binding proteins: role in activation and desensitization of the adenylate cyclase response to vasopressin

The inhibitory GTP-binding protein (Gi) is known to mediate the effects of a number of hormones that act through specific receptors to inhibit adenylate cyclase. In this study we examined the mechanism whereby Gi modulates the response of adenylate cyclase to a stimulatory hormone and its role in desensitization. In membranes prepared from the cultured renal epithelial cell line LLCPK1, adenylate cyclase activity was stimulated 16-fold by 1-2 microM lysine vasopressin. Addition of GTP (1-100 microM) resulted in stimulation of basal activity but inhibition of hormone-stimulated activity (approximately 40% inhibition at 100 microM GTP). This contrasts with the usual effect of GTP to support or augment activation by stimulatory receptors. The inhibitory effect was abolished by pertussis toxin, which had little effect on basal activity in the absence or presence of added GTP or on vasopressin-stimulated activity in the absence of added GTP. GTP-mediated inhibition was vasopressin concentration dependent. At concentrations of vasopressin below the K1/2 for enzyme activation (approximately 0.6 nM), GTP was stimulatory, and at higher concentrations, GTP was inhibitory. The inhibitory effect of GTP was also observed for a V2-receptor agonist and was not abolished by a V1-receptor antagonist, indicating that a distinct V1 receptor did not mediate inhibition of adenylate cyclase. Using the known subunit structure of adenylate cyclase, we developed the minimal mechanism that would incorporate a modulatory role for Gi in determining net activation of adenylate cyclase by a stimulatory hormone. The predicted enzyme activities for basal and maximal hormone stimulation in the presence and absence of GTP were generated, and model parameters were chosen to match the experimental observations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium.

In the ciliated protozoan Paramecium, Ca2+ and cyclic nucleotides are believed to act as second messengers in the regulation of the ciliary beat. Ciliary adenylate cyclase was activated 20-30-fold (half-maximal at 0.8 microM) and inhibited by higher concentrations (10-20 microM) of free Ca2+ ion. Ca2+ activation was the result of an increase in Vmax., not a change in Km for ATP. The activation by Ca2+ was seen only with Mg2+ATP as substrate; with Mn2+ATP the basal adenylate cyclase activity was 10-20-fold above that with Mg2+ATP, and there was no further activation by Ca2+. The stimulation by Ca2+ of the enzyme in cilia and ciliary membranes was blocked by the calmodulin antagonists calmidazolium (half-inhibition at 5 microM), trifluoperazine (70 microM) and W-7 (50-100 microM). When ciliary membranes (which contained most of the ciliary adenylate cyclase) were prepared in the presence of Ca2+, their adenylate cyclase was insensitive to Ca2+ in the assay. However, the inclusion of EGTA in buffers used for fractionation of cilia resulted in full retention of Ca2+-sensitivity by the ciliary membrane adenylate cyclase. The membrane-active agent saponin specifically suppressed the Ca2+-dependent adenylate cyclase without inhibiting basal activity with Mg2+ATP or Mn2+ATP. The ciliary adenylate cyclase was shown to be distinct from the Ca2+-dependent guanylate cyclase; the two activities had different kinetic parameters and different responses to added calmodulin and calmodulin antagonists. Our results suggest that Ca2+ influx through the voltage-sensitive Ca2+ channels in the ciliary membrane may influence intraciliary cyclic AMP concentrations by regulating adenylate cyclase.     

Olfactory transduction mechanisms in sheep

The enzyme adenylyl cyclase from sheep olfactory epithelium is dually regulated by GTP and is highly sensitive to the nucleotide analogues GTPS and GppNHp, as well as to fluoride ions and forskolin. Many, but not all, odorants tested are able to stimulate adenylyl cyclase in a dose-dependent manner and with different potencies. Such an effect is detectable only in the presence of GTP. The odorants belonging to the putrid class are the least effective in stimulating adenylyl cyclase activity, and only furfuryl mercaptan significantly increases cAMP biosynthesis. Mixtures of two odorants, chosen among those able to activate adenylyl cyclase, induce additive or supra-additive effects, suggesting the presence of many different receptor types. The presence of an alternative olfactory signal transduction process, i.e. the inositol phospholipid second messenger system, has been evaluated. Triethylamine, a putrid odorant completely ineffective on cAMP levels, is able to significantly increase inositol phosphate accumulation, indicating the coexistence of both cAMP- and InsP₃-mediated signalling pathways in sheep olfactory epithelium.     

Properties of Muscarinic-Stimulated Adenylate Cyclase Activity in Rat Olfactory Bulb

The muscarinic stimulation of adenylate cyclase activity in rat olfactory bulb was characterized, with the aim of elucidating the nature of the molecular mechanism involved. Carbachol (CCh) stimulated the enzyme activity in either crude or purified cell membrane preparations and increased cyclic AMP accumulation in miniprisms of olfactory bulb. The CCh stimulation of adenylate cyclase activity displayed a fast onset and was rapidly reversed by addition of atropine. The stimulation was associated with an increase in the apparent Vmax of the enzyme, with no change in the Km for Mg-ATP. The affinity of the enzyme for Mg2+ was enhanced by CCh. The muscarinic effect required GTP at concentrations higher than those needed for enzyme stimulation with either l-isoproterenol or vasoactive intestinal peptide. Moreover, contrary to the beta-adrenergic stimulation, the muscarinic effect disappeared when guanosine 5'-O-(3'-thiotriphosphate) was substituted for GTP. In vivo treatment of olfactory bulbs with pertussis toxin completely prevented the muscarinic stimulation of adenylate cyclase, whereas cholera toxin was without effect. These results indicate that in rat olfactory bulb muscarinic receptors increase adenylate cyclase activity by interacting with a pertussis toxin-sensitive GTP-binding protein different from the stimulatory GTP-binding protein.     

Calmodulin activation of adenylate cyclase in the mouse B16 melanoma.

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.     

Effects of Odorants on Pigment Aggregation and cAMP in Fish Melanophores

Odor perception within olfactory neuroepithelium and pigment translocation within melanophores both seem to rely on a cAMP-based second messenger system. From studies on cultured frog melanophores, Lerner et al. (Proc. Natl. Acad. Sci. USA 85:261-264, 1988) suggested that some aspect of odor perception may be mediated by a nonspecific mechanism whose signal is transduced by a cAMP-based second messenger system. In the present study, odorants (β-ionone, benzylaldehyde, cineole, cinnamaldehyde, and octanol), which previously have been shown to stimulate formation of cAMP in the olfactory neuroepithelium, were investigated for possible pigment dispersing and cAMP-increasing effects. Pretreatment of fish melanophores with the adenylate cyclase activator forskolin (1 μM) resulted in an approximately 300% increase in cAMP and an almost complete blockage of noradrenaline-induced pigment aggregation. However, none of the tested odorants were able to increase the cAMP level and only cinnaldehyde and β-ionone were found to have any pigment dispersing activity.