Johnny Näsman

Two human α2-adrenoceptor subtypes α2A-C10 and α2B-C2 expressed in Sf9 cells couple to transduction pathway resulting in opposite effects on cAMP production

Two human alpha-2 adrenoceptor subtypes alpha-2A-C10 and alpha-2B-C2 expressed in Sf9 cells couple to transduction pathways resulting in opposite effects on cAMP production

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.

Autocrine Endocannabinoid Signaling through CB1 Receptors Potentiates OX1 Orexin Receptor Signaling

It has been proposed that OX1 orexin receptors and CB1 cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX1 receptors and potentiate OX1 receptor signaling to extracellular signal–regulated kinase (ERK). We have recently shown that OX1 receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX1-CB1 receptor interaction in signaling, for instance, in retrograde synaptic transmission. In the current study, we set out to investigate this possibility utilizing recombinant Chinese hamster ovary K1 cells. 2-AG released from OX1 receptor–expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB1 receptor–expressing cells. When OX1 and CB1 receptors were expressed in the same cells, OX1 stimulation–induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX1 response as such was fully abolished by specific inhibition of CB1 receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX1-CB1 heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX1-CB1 synergism and thus suggest a functional rather than a molecular interaction of OX1 and CB1 receptors.

Selective interference with TRPC3/6 channels disrupts OX1 receptor signalling via NCX and reveals a distinct calcium influx pathway.

TRPC channels play significant roles in the regulation of neuronal plasticity and development. The mechanism by which these nonselective cation channels exert their trophic actions appears to involve entry of Ca(2+) into the cells. Using a neuronal cell model (differentiated human IMR32 neuroblastoma cells), we demonstrate a central role for sodium entry via TRPC3/6 channels in receptor-mediated increases in intracellular calcium. These Na(+)-dependent Ca(2+) influxes, which were observed in a subpopulation of cells, were efficiently blocked by protein kinase C activation, by the Na(+)/Ca(2+) exchanger inhibitors, and by molecular disruption of TRPC3/6 channel function. On the other hand, another subpopulation of cells showed a Na(+)-independent Ca(2+) entry upon stimulation of the same receptors, orexin/hypocretin and bradykinin receptors. This second type of response was not affected by the above mentioned treatments, but it was sensitive to polyvalent cations, such as ruthenium red, spermine and Gd(3+). The data suggest that a NCX-TRPC channel interaction constitutes an important functional unit in receptor-mediated Ca(2+) influx in neuronal cells.

Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR-32 human neuroblastoma cells

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non‐selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR‐32 undergoes a remarkable differentiation in response to treatment with 5‐bromo‐2‐deoxyuridine. The cells acquire a neuronal morphology with increased expression of N‐type voltage gated calcium channels and neurotransmitters. Here we show using RT‐PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR‐32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl‐isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC‐030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR‐32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR‐32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression. J. Cell. Physiol. 221: 67–74, 2009.

Extracellular superoxide dismutase is a thyroid differentiation marker down-regulated in cancer

Reactive oxygen species, specifically hydrogen peroxide (H(2)O(2)), have a significant role in hormone production in thyroid tissue. Although recent studies have demonstrated that dual oxidases are responsible for the H(2)O(2) synthesis needed in thyroid hormone production, our data suggest a pivotal role for superoxide dismutase 3 (SOD3) as a major H(2)O(2)-producing enzyme. According to our results, Sod3 is highly expressed in normal thyroid, and becomes even more abundant in rat goiter models. We showed TSH-stimulated expression of Sod3 via phospholipase C-Ca(2+) and cAMP-protein kinase A, a pathway that might be disrupted in thyroid cancer. In line with this finding, we demonstrated an oncogene-dependent decrease in Sod3 mRNA expression synthesis in thyroid cancer cell models that corresponded to a similar decrease in clinical patient samples, suggesting that SOD3 could be used as a differentiation marker in thyroid cancer. Finally, the functional analysis in thyroid models indicated a moderate role for SOD3 in regulating normal thyroid cell proliferation being in line with our previous observations.

The three-finger toxin MTα is a selective α2B-adrenoceptor antagonist

Muscarinic toxins (MTs) are three-finger folded peptides isolated from mamba snake venoms. In this report we describe a selective antagonistic interaction of MTalpha with the human alpha(2B)-adrenoceptor. In a functional assay, measuring the alpha(2B)-adrenoceptor-induced increase in intracellular [Ca(2+)], we found that both venomous MTalpha and synthetic MTalpha inhibited the response in a concentration-dependent way. MTalpha did not affect the responses of alpha(2A)-, alpha(2C)-, alpha(1A)- or alpha(1B)-adrenoceptors. To further explore the binding of MTalpha to the alpha(2B)-adrenoceptor, we performed ligand binding experiments on Sf9 cell homogenates with [(3)H]RX821002 as reporter ligand. MTalpha bound to the receptor rather slowly requiring about 60 min to reach equilibrium. In equilibrium binding experiments, MTalpha displaced the radioligand with an IC(50) of 3.2 nM, but was not able to displace all bound radioligand. Using a saturation binding protocol, we found that MTalpha suppressed the maximum binding without any greater impact on the affinity of the radioligand, indicating a non-competitive mode of inhibition. The toxin bound reversibly to alpha(2B)-adrenoceptor, but extensive washing was needed for full recovery of binding sites at high toxin concentrations. Surprisingly, MTalpha did not affect [(3)H]-N-methylscopolamine binding to the muscarinic receptor subtypes at concentrations found to fully block alpha(2B)-adrenoceptors, showing that the toxin is a more potent antagonist for the alpha(2B)-adrenoceptor than for muscarinic receptors. These findings should open up new views in terms of selective adrenoceptor drug design as well as in elucidation of alpha(2)-adrenoceptor physiology.

Orexin/hypocretin receptor chimaeras reveal structural features important for orexin peptide distinction

We wanted to analyze the basis for the distinction between OX1 and OX2 orexin receptors by the known agonists, orexin‐A, orexin‐B and Ala11, d‐Leu15‐orexin‐B, of which the latter two show some selectivity for OX2. For this, chimaeric OX1/OX2 and OX2/OX1 orexin receptors were generated. The receptors were transiently expressed in HEK‐293 cells, and potencies of the agonists to elicit cytosolic Ca2+ elevation were measured. The results show that the N‐terminal regions of the receptor are most important, and the exchange of the area from the C‐terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptors ligand profile.

Adrenoceptor Activity of Muscarinic Toxins identified from Mamba Venoms

BACKGROUND AND PURPOSE Muscarinic toxins (MTs) are snake venom peptides named for their ability to interfere with ligand binding to muscarinic acetylcholine receptors (mAChRs). Recent data infer that these toxins may have other G‐protein‐coupled receptor targets than the mAChRs. The purpose of this study was to systematically investigate the interactions of MTs with the adrenoceptor family members.

Canonical Transient Receptor Potential Channel 2 (TRPC2) as a Major Regulator of Calcium Homeostasis in Rat Thyroid FRTL-5 Cells: IMPORTANCE OF PROTEIN KINASE C δ (PKCδ) AND STROMAL INTERACTION MOLECULE 2 (STIM2)*

Background: The identity of calcium channels in thyroid is not defined. Results: TRPC2 functions as a major regulator of calcium homeostasis in rat thyroid cells. TRPC2 appears to be receptor-regulated and participates in regulating ER calcium content. Conclusion: We have defined a novel physiological role for TRPC2 channels. Significance: TRPC2, by regulating STIM2, PKC expression, and SERCA activity, regulates calcium homeostasis in thyroid cells. Mammalian non-selective transient receptor potential cation channels (TRPCs) are important in the regulation of cellular calcium homeostasis. In thyroid cells, including rat thyroid FRTL-5 cells, calcium regulates a multitude of processes. RT-PCR screening of FRTL-5 cells revealed the presence of TRPC2 channels only. Knockdown of TRPC2 using shRNA (shTRPC2) resulted in decreased ATP-evoked calcium peak amplitude and inward current. In calcium-free buffer, there was no difference in the ATP-evoked calcium peak amplitude between control cells and shTRPC2 cells. Store-operated calcium entry was indistinguishable between the two cell lines. Basal calcium entry was enhanced in shTRPC2 cells, whereas the level of PKCβ1 and PKCδ, the activity of sarco/endoplasmic reticulum Ca2+-ATPase, and the calcium content in the endoplasmic reticulum were decreased. Stromal interaction molecule (STIM) 2, but not STIM1, was arranged in puncta in resting shTRPC2 cells but not in control cells. Phosphorylation site Orai1 S27A/S30A mutant and non-functional Orai1 R91W attenuated basal calcium entry in shTRPC2 cells. Knockdown of PKCδ with siRNA increased STIM2 punctum formation and enhanced basal calcium entry but decreased sarco/endoplasmic reticulum Ca2+-ATPase activity in wild-type cells. Transfection of a truncated, non-conducting mutant of TRPC2 evoked similar results. Thus, TRPC2 functions as a major regulator of calcium homeostasis in rat thyroid cells.