Karl E. O. Åkerman

The Orexin OX1 Receptor Activates a Novel Ca2+ Influx Pathway Necessary for Coupling to Phospholipase C

Ca2+ elevations in Chinese hamster ovary cells stably expressing OX1 receptors were measured using fluorescent Ca2+ indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca2+elevations with an EC50 around 1 nm. When the extracellular [Ca2+] was reduced to a submicromolar concentration, the EC50 was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mm external Ca2+ was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca2+. The shift in the potency was not caused by depletion of intracellular Ca2+ but by a requirement of extracellular Ca2+ for production of inositol 1,4,5-trisphosphate. Fura-2 experiments with the “Mn2+-quench technique” indicated a direct activation of a cation influx pathway by OX1 receptor independent of Ca2+ release or pool depletion. Furthermore, depolarization of the cells to +60 mV, which almost nullifies the driving force for Ca2+ entry, abolished the Ca2+ response to low concentrations of orexin-A. The results thus suggest that OX1 receptor activation leads to two responses, (i) a Ca2+ influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.

OX1 orexin receptors couple to adenylyl cyclase regulation via multiple mechanisms

In this study, the mechanism of OX1 orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC50 ≈ 1nm) inhibition and a strong (4-fold), low potency (EC50 = ≈300 nm) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of Gi/o proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC50 = ≈500 nm). cAMP elevation was not caused by Ca2+ elevation or by Gβγ. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCδ, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of Gs activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC50 of orexin-A = ≈10 nm and EC50 of orexin-B = ≈100 nm), and the response was fully dependent on PKCδ. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Gαs antibodies. We conclude that OX1 receptors stimulate adenylyl cyclase via a low potency Gs coupling and a high potency phospholipase C → PKC coupling. The former or some exogenous Gs activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX1 receptors couple to Gs almost as efficiently as the orexin-A-activated receptors, in contrast to Ca2+ elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.

*Orexin signaling in recombinant neuron-like cells

To assess the role of orexin receptor signaling in neuron‐like cells, Neuro‐2a murine neuroblastoma and PC12 human pheochromocytoma cells were stably transfected with human OX1 or OX2 receptors. Activation of both receptors strongly elevated cellular inositol phosphates and Ca2+. A difference in the potency between orexin‐A and ‐B was seen for OX1, but not OX2 receptors. Dependence of the orexin‐mediated Ca2+ response on extracellular Ca2+ and the observed Ba2+ influx indicate that in addition to phospholipase C, orexin receptors also may couple to similar non‐voltage‐gated Ca2+ channels in neuronal cells as previously characterized in non‐neuronal cells.

Contribution of glycolytic and mitochondrial pathways in glucose-induced changes in islet respiration and insulin secretion

Abstract. The different roles of glycolytic and mitochondrial pathways in glucose-induced metabolic activation and insulin secretion were studied in islets of Langerhans. Single islets were perifused with 3xa0mM glucose together with agents affecting the production or consumption of ATP. Subsequently, glucose was raised to 11xa0mM and the effects of the agents on metabolic and secretory responses were evaluated. Metabolism was monitored continuously with an oxygen-sensitive microelectrode inserted into the islet. Insulin secretion was determined by assaying insulin in perifusate with ELISA. Inhibitors of mitochondrial ATP production reduced the metabolic and secretory response to glucose. When glycolytic ATP production was reduced, initial but not sustained glucose-stimulated insulin release was observed. Inhibition of mitochondrial pyruvate transport reduced the glucose-induced decline in pO2. Although mitochondrial metabolism was eventually similar to normal, insulin release was only 20% of normal. Increased energy expenditure also changed the kinetics of the glucose-induced decline in pO2 and decreased the insulin release by 50%. In conclusion, glucose-induced enhancement of insulin release was only seen when the rise of the sugar concentration triggered a rapid and sustained increase of mitochondrial metabolism. This activation of mitochondrial metabolism required a good metabolic state prior to the glucose challenge.

Agonist trafficking of Gi/o-mediated α2A-adrenoceptor responses in HEL 92.1.7 cells

The ability of 19 agonists to elevate Ca2+ and inhibit forskolin‐induced cyclic AMP elevation through α2A‐adrenoceptors in HEL 92.1.7 cells was investigated. Ligands of catecholamine‐like‐ (five), imidazoline‐ (nine) and non‐catecholamine‐non‐imidazoline‐type (five) were included. The relative maximum responses were similar in both assays. Five ligands were full or nearly full agonists, six produced 20u2003–u200370% of the response to a full agonist and the remaining eight gave lower responses (<20%) so that their potencies were difficult to evaluate. Marked differences in the potencies of the agonists with respect to the two measured responses were seen. The catecholamines were several times less potent in decreasing cyclic AMP than in increasing Ca2+, whereas the other, both imidazoline and ox‐/thiazoloazepine ligands, were several times more potent with respect to the former than the latter response. For instance, UK14,304 was more potent than adrenaline with respect to the cyclic AMP response but less potent than adrenaline with respect to the Ca2+ response. All the responses were sensitive to pertussis toxin‐pretreatment. Also the possible role of PLA2, β‐adrenoceptors or ligand transport or metabolism as a source of error could be excluded. The results suggest that the active receptor states produced by catecholamines and the other agonists are markedly different and therefore have different abilities to activate different signalling pathways.

Oscillations in oxygen tension and insulin release of individual pancreatic ob/ob mouse islets.

Aims/hypothesis. The role of beta-cell metabolism for generation of oscillatory insulin release was investigated by simultaneous measurements of oxygen tension (pO2) and insulin release from individual islets of Langerhans.¶Methods. Individual islets isolated from the ob/ob-mice were perifused. Insulin in the perifusate was measured with a sensitive ELISA and pO2 with a modified Clark-type electrode inserted into the islets.¶Results. In the presence of 3 mmol/l d-glucose, pO2 was 102 ± 9 mmHg and oscillatory (0.26 ± 0.04 oscillations/min). Corresponding insulin measurements showed oscillatory release with similar periodicity (0.25 ± 0.02 oscillations/min). When the d-glucose concentration was increased to 11 mmol/l, pO2 decreased by 30 % to 72 ± 10 mmHg with maintained frequency of the oscillations. Corresponding insulin secretory rate rose from 5 ± 2 to 131 ± 16 pmol · g–1· s–1 leaving the frequency of the insulin pulses unaffected. The magnitude of glucose-induced change in pO2 varied between islets but was positively correlated to the amount of insulin released (r2 = 0.85). When 1 mmol/l tolbutamide was added to the perifusion medium containing 11 mmol/l glucose no change in average oscillatory pO2 was observed despite a doubling in the secretory rate. When 8 mmol/l 3-oxymethyl glucose was added to perifusion medium containing 3 mmol/l d-glucose, neither pO2 nor insulin release of the islets were changed. Temporal analysis of oscillations in pO2 and insulin release revealed that maximum respiration correlated to maximum or close to maximum insulin release.¶Conclusion/interpretation. The temporal relation between oscillations in pO2 and insulin release supports a role for metabolic oscillations in the generation of pulsatile insulin release. [Diabetologia (2000) 43: 1313–1318]

The STC-1 cells express functional orexin-A receptors coupled to CCK release.

Orexins are newly discovered neuropeptides regulating feeding and vigilance and have been detected in neuroendocrine cells of the gut. Potential neuroendocrine functions of orexin are unknown. Therefore, the effects of orexin-A on the intestinal neuroendocrine cell line, STC-1, were investigated as a model system. RT-PCR demonstrated the presence of both OX(1) and OX(2) receptors. Stimulation with orexin-A produced a dose-dependent release of cholecystokinin (CCK), which was abolished by removal of extracellular Ca(2+) or the presence of the voltage-gated L-type Ca(2+)-channel blocker diltiazem (10 microM). Orexin-A (Ox-A) elevated intracellular Ca(2+), which was dependent on extracellular Ca(2+). Furthermore, orexin-A caused a membrane depolarization in the STC-1 cells. Ox-A neither elevated cAMP levels nor stimulated phosphoinositide turnover in these cells. These data demonstrate a functional orexin receptor in the STC-1 cell line. Ox-A produces CCK release in these cells, by a mechanism involving membrane depolarization and subsequently activation of L-type voltage-gated Ca(2+)-channels.

Modelling of promiscuous receptor–Gi/Gs-protein coupling and effector response

A single G-protein-coupled receptor might activate multiple G-protein species. This multiplex coupling ability can be used by tissues to regulate signalling; for the pharmacologist, such multiplex coupling might cause difficulties in the interpretation of experimental data. In this article, we present mathematical models for the activation of two separate G-protein species by a single receptor. Issues addressed concern mutual antagonism between the G proteins and the availability of an already activated receptor for interaction with a new G protein (receptor-G-protein-effector complexing versus free diffusion of G proteins) in addition to receptor-G-protein precoupling at different G-protein and receptor expression levels. The output from the receptor models uses, as readout, a new model for adenylyl cyclase regulation by two allosteric regulators (i.e. G(s) and G(i)).

Phospholipase C activator m-3M3FBS affects Ca2+ homeostasis independently of phospholipase C activation

In this study, we have investigated responses to the phospholipase C (PLC) activator m‐3M3FBS in SH‐SY5Y human neuroblastoma cells. As measured using fura‐2, m‐3M3FBS caused a slowly developing − full response was obtained within 4–6 min − Ca2+ elevation both in the presence and absence of extracellular Ca2+, indicating Ca2+ release from intracellular stores, putatively from endoplasmic reticulum and mitochondria. PLC activity was also measured using two methods, the classical ion‐exchange separation and the more novel fluorescent real‐time method. In the time frame in which m‐3M3FBS caused Ca2+ elevation (up to 7 min), no PLC activation was detected. Instead, more than 20 min were required to see any inositol phosphate generation in response to m‐3M3FBS. m‐3M3FBS also interfered with store‐operated Ca2+ influx and Ca2+ extrusion. In conclusion, m‐3M3FBS cannot be considered either potent or specific PLC activator.

Role of G-protein availability in differential signaling by alpha 2-adrenoceptors

The impact of G-protein expression on the coupling specificity of the human alpha(2B)-adrenergic receptor (alpha(2B)-AR) was studied in Sf9 cells. The alpha(2B)-AR was shown to activate both coexpressed G(s)- and G(i)-proteins in a [(35)S]GTPgammaS binding assay. Noradrenaline and the synthetic agonist UK14,304 were equally potent and efficacious in stimulating G(i) activation. At the effector level (adenylyl cyclase), both ligands stimulated cAMP production. In the presence of forskolin, the effects of the agonists were more complex. Noradrenaline stimulated cAMP production, while UK14,304 showed a biphasic concentration-response curve with inhibition of stimulated cAMP production at low agonist concentrations and further stimulation at high agonist concentrations. G(s) coexpression caused a monophasic stimulatory response with both ligands. Coexpression with G(i) resulted in a biphasic concentration-response curve for noradrenaline and a monophasic inhibition with UK14,304. Experiments with a panel of agonists demonstrated that the more efficacious an agonist is in stimulating cAMP production, the weaker is its ability to couple to inhibition of cAMP accumulation via exogenous G(i). To be able to explain the mechanistic consequences of dual G-protein coupling described above, we developed a mathematical model based on the hypothesis that an agonist induces different conformations of the receptor having different affinity for different G-proteins. The model reproduced the profiles seen in the concentration-response curves with G(s) and G(i) coexpression. The model predicts that the affinity of the receptor conformation for G-proteins as well as the availability of G-proteins will determine the ultimate response of the receptor.