RGS proteins reconstitute the rapid gating kinetics of gbetagamma-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Galpha(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20-40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of "regulators of G protein signaling" (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor --> GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent "basal" GIRK current was suppressed by approximately 40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of "slow" postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.     

Tissue-specific regulation of G-protein-coupled inwardly rectifying K+ channel expression by muscarinic receptor activation in ovo

We investigated the effects of muscarinic acetylcholine receptor stimulation on the expression levels of the G-protein-coupled inwardly rectifying K+ channel (GIRK) subunits using solution hybridization and immunoblot analyses. We report here that treatment of chick embryos in ovo with muscarinic agonist causes decreases in mRNA levels encoding GIRK1 and GIRK4 in atria but does not alter GIRK1 expression in ventricles. In addition, GIRK1 protein levels also demonstrate a decrease in atria upon muscarinic acetylcholine receptor stimulation. Numerous receptors couple to the activation of the GIRK family of inwardly rectifying K+ channels; thus, these decreases represent a novel mechanism for regulating physiological responses to chronic agonist exposure.     

Distribution of mRNA encoding the inwardly rectifying K+ channel, BIR1 in rat tissues

Chinese hamster P-glycoprotein ("multidrug-resistance protein") was purified and reconstituted in proteoliposomes by the procedure of I. L. Urbatsch, M. K. al-Shawi, and A. E. Senior (1994, Biochemistry 33, 7069-7076). The presence of lipid during the octylglucoside solubilization and Reactive Red 120 chromatography steps was found to be mandatory for retention of ATPase activity. Sheep brain or bovine liver lipid extracts could be substituted for the Escherichia coli lipids used previously. Stimulation of ATPase activity of purified, reconstituted P-glycoprotein by vinblastine, colchicine, and daunomycin was seen with sheep brain and bovine liver lipids, but not with E. coli lipids. Basal (i.e., not drug-stimulated) ATPase activity was different in the three lipids. Azidopine labeling of the drug binding sites in purified, reconstituted P-glycoprotein was carried out; vinblastine, colchicine, and daunomycin competed for labeling in all three lipids. It is therefore evident that the lipid environment can significantly influence the characteristics of purified, reconstituted P-glycoprotein ATPase activity and the apparent coupling between drug-binding and catalytic sites.     

Cloning of a Xenopus laevis inwardly rectifying K+ channel subunit that permits GIRK1 expression of IKACh currents in oocytes

Xenopus oocytes injected with GIRK1 mRNA express inwardly rectifying K+ channels resembling IKACh. Yet IKACh, the atrial G protein-regulated ion channel, is a heteromultimer of GIRK1 and CIR. Reasoning that an oocyte protein might be substituting for CIR, we cloned XIR, a CIR homolog endogenously expressed by Xenopus oocytes. Coinjecting XIR and GIRK1 mRNAs produced large, inwardly rectifying K+ currents responsive to m2-muscarinic receptor stimulation. The m2-stimulated currents of oocytes expressing GIRK1 alone decreased 80% after injecting antisense oligonucleotides specific to the 5' untranslated region of XIR, but GIRK1/CIR currents were unaffected. Thus, GIRK1 without XIR or CIR only ineffectively produces currents in oocytes. This result suggests that GIRK1 does not form native homomultimeric channels.     

Ceramide inhibits inwardly rectifying K+ currents via a Ras- and Raf-1-dependent pathway in cultured oligodendrocytes

Ceramide is a lipid mediator implicated in apoptosis induced by proinflammatory cytokines in many cell types, including oligodendrocytes (OLGs). To determine whether ceramide modulates transmembrane signaling events in OLGs, we studied its effect on intracellular Ca2+ (Cai), resting membrane potential and inwardly rectifying K+ currents (IKir) in cultured neonatal rat OLGs. We report here that (1) exposure to C2-ceramide (cer) rarely increases OLG Cai, whereas sphingosine elicits sustained increase in Cai; (2) cer causes OLG depolarization, an effect mimicked by sphingosine-1-phosphate but not by sphingosine; and (3) cer, but not its inactive analog dihydroceramide, inhibits OLG IKir. The cer effect is attenuated by Ras antibody Y13-259, by protein kinase C inhibitory peptide (19-36), and by suppression of c-Raf-1 expression with antisense raf-1 oligonucleotides. We conclude that cer-induced OLG depolarization is mediated via inhibition of IKir by a Ras- and raf-1-dependent pathway, which results in the phosphorylation of the inward rectifier K+ channel protein.     

Inhibition by taurine of the inwardly rectifying K+ current in guinea pig ventricular cardiomyocytes

Effects of taurine on the inwardly rectifying K+ current (IK1) in isolated guinea pig ventricular cardiomyocytes were examined using patch voltage-clamp methods. All experiments were performed at 36 degrees C. Taurine (10-20 mM) increased the action potential duration, but failed to affect the resting potential. Holding potential was maintained at -30 mV. The current was activated with an inwardly going rectification, and was completely blocked by Ba2+ (2 mM). Taurine inhibited IK1 at - 120 mV by 28.3+/-1.1% (n=6, P < 0.05) at 10 mM and by 36.0+/-2.1% (n=6, P < 0.01) at 20 mM. The reversal potential was shifted in the hyperpolarizing direction by 3.7+/-0.6 mV (n=6) at 20 mM. In inside-out patch-clamp experiments, the amplitude of unitary channels was -2.7+/-0.3 pA (n=21) at -90 mV. Symmetrical high-K+ (150 mM) solutions in both bath and pipette were used. The channel conductance was 32+/-2 pS (n=9). Taurine did not affect channel conductance, but markedly decreased the open probability at - 120 mV of channel by 21.5+/-2.4% (n=8, P < 0.01) at 10 mM, and by 56.7+/-3.8% (n=8, P < 0.001) at 20 mM. These responses were almost reversible. These results suggest that taurine directly modulates the open probability of the inwardly rectifying K+ current, resulting in regulation of the functions of heart cells.     

Pore mutation in a G-protein-gated inwardly rectifying K+ channel subunit causes loss of K+-dependent inhibition in weaver hippocampus

Weaver (wv) mice carry a point mutation in the pore region of a G-protein-gated inwardly rectifying K+ channel subunit (Kir3.2). wvKir3.2 conducts inward currents that may cause the loss of neurons in the cerebellum and substantia nigra. Although Kir3.2 is widely expressed in the CNS, significant morphological or physiological changes have not been reported for other brain areas. We studied the role of wvKir3.2 in hippocampal slices of young [postnatal day (P) 4-18] and adult wv/wv (>/=P24) mice, because protein levels of Kir 3. 1 and Kir3.2 appear to be normal in the first 3 postnatal weeks and only decrease thereafter. In disinhibited slices, the GABAB receptor agonist R-baclofen reduced burst activity in wv/wv mice but was much more potent in wild-type mice. Mean resting membrane potential, slope input resistance, and membrane time constant of CA3 neurons of adult wv/wv and wild-type mice were indistinguishable. However, R-baclofen or chloroadenosine did not induce K+ currents or any other conductance change in wv/wv mice. Moreover, electrical or chemical stimulation of inhibitory neurons did not evoke slow IPSPs in adult wv/wv mice. Only in a few cells of young wv/wv mice did GABAB receptor activation by R-baclofen or presynaptic stimulation induce small inward currents, which were likely caused by a Na+ ion influx through wvKir3.2 channels. The data show that the pore mutation in wvKir3.2 channels results in a hippocampal phenotype resembling Kir3.2-deficient mutants, although it is not associated with the occurrence of seizures.     

Human D2 and D4 dopamine receptors couple through betagamma G-protein subunits to inwardly rectifying K+ channels (GIRK1) in a Xenopus oocyte expression system: selective antagonism by L-741,626 and L-745,870 respectively

OBJECTIVE: To develop a prognostic model, based on clinical and pathological data, to estimate the probability of micrometastasis in the sentinel lymph node in patients with malignant melanoma. DESIGN: Retrospective analytical study. SETTING: University medical center. PATIENTS: Two hundred fifteen patients with American Joint Committee on Cancer stages I and II cutaneous malignant melanoma underwent sentinel lymph node biopsy. MEASUREMENTS: Presence of microscopic melanoma in the sentinel lymph node(s). Clinical attributes recorded included age, sex, and location of the primary melanoma. Pathological attributes recorded before lymph node evaluation included ulceration, microsatellites, angiolymphatic invasion, mitotic rate, tumor infiltrating lymphocytes, and regression. RESULTS: Forty-six patients (21.4%) overall had a positive sentinel lymph node. Patients with tumor thickness ranging from 3.0 to 3.9 mm had the highest incidence (50%) of nodal involvement, followed by those with tumors 4.0 to 4.9 mm thick (41%). Patients with melanomas measuring greater than 4.9 mm thick and those between 1.0 and 2.9 mm had a similar rate of nodal involvement (16%-17%). Clinical characteristics had minimal correlation with nodal status in multivariate analysis. The total number of histological high-risk features was significantly correlated with sentinel lymph node involvement. Important pathological risk factors included ulceration, high mitotic rate, angiolymphatic invasion, and microsatellites. Patients with tumor thickness greater than 1.0 mm but lacking these features had a 14% risk of occult metastases. CONCLUSION: Among patients with clinically node-negative primary melanoma, the presence of 1 or more high-risk histological features significantly increases the incidence of microscopic nodal involvement and can be used to predict the likelihood of a positive sentinel lymph node biopsy.     

Selective block by glyceryl nonivamide of inwardly rectifying K+ current in rat anterior pituitary GH3 cells

The effects of glyceryl nonivamide (GLNVA) on ionic currents were compared and examined in rat pituitary GH3 cells. Hyperpolarization-activated K+ currents in GH3 cells bathed in high-K+ Ca2+-free external solution were studied to assess effects of GLNVA on the an inwardly rectifying K+ current (I(K(IR))). GLNVA is very potent in blocking I(K(IR)) in a concentration-dependent manner, with a half maximal concentrations of 0.1 microM. The complete block of I(K(IR)) achieved with concentrations > or = 1 microM revealed the presence of a non-inactivating current. We also found that GLNVA at a concentration above 30 microM inhibited L-type voltage-dependent Ca2+ current and two components of K+ outward currents, while GLNVA (< or = 3 microM) did not have any effect on them. This study shows that GLNVA, in addition to retaining the capability of eliciting peptidergic neurons, is a selective block of I(K(IR)) in GH3 cells and will provide a useful tool for characterizing I(K(IR)) and understanding its physiological function. In addition, the carefulness should be taken about the interpretation of GLNVA-mediated responses in vivo or in vitro.     

Gi3 mediates somatostatin-induced activation of an inwardly rectifying K+ current in human growth hormone-secreting adenoma cells

SRIF activates an inwardly rectifying K+ current in human GH-secreting adenoma cells. Activation of this K+ current induces hyperpolarization of the membrane and abolishment of action potential firing. This mechanism is an essential mechanism for SRIF-induced decrease in intracellular Ca2+ concentration and inhibition of GH secretion. The activation of the inwardly rectifying K+ current is mediated by a pertussis toxin-sensitive G protein. In this article, the expression of the pertussis toxin-sensitive G protein alpha-subunits in the human GH-secreting adenoma cells were analyzed by RT-PCR, and the G protein transducing the SRIF-induced activation of this inwardly rectifying K+ current was investigated. RT-PCR of the messenger RNA from two human GH-secreting adenomas revealed that all G alpha(i1), G alpha(i2), G alpha(i3), and G alpha(o) were expressed in these adenomas. Primary cultured cells from these two adenoma cells were investigated under the voltage clamp of the whole-cell mode. Specific antibodies against the carboxyl terminus of G protein alpha-subunits were microinjected into the cells. Microinjection of antibody against the carboxyl terminal sequence of G alpha(i3) attenuated the SRIF-induced activation of the inwardly rectifying K+ current, whereas antibody against the common carboxyl terminal sequence of G alpha(i1) and G alpha(i2) did not. These data indicate that the G protein transducing the SRIF-induced activation of the inwardly rectifying K+ current is Gi3.     

Corticotropin-releasing hormone receptor expression and functional coupling in neonatal cardiac myocytes and AT-1 cells

CRH is the principal mediator of the stress response in mammals. In addition to pituitary and central nervous system effects, peripheral effects of CRH have been observed involving the immune and cardiovascular systems. Two CRH receptor subtypes, CRH-R1 and CRH-R2, have been cloned and show significant amino acid homology (69%), but differ in their tissue distribution. CRH-R1 is expressed predominantly in the brain and pituitary, whereas the CRH-R2 subtype is highly expressed in heart and skeletal muscle. To investigate the role of CRH in cardiac signaling, we analyzed the effect of CRH on freshly isolated neonatal rat cardiomyocytes and murine atrial cardiomyocyte tumor cells, AT-1, which express CRH-R2 messenger RNA. We show that stimulation of these cells with CRH and the CRH-related peptides, sauvagine from frog and urotensin I from fish, elicits large increases in the intracellular level of cAMP. This stimulation is transient, reaching a maximum in 5-15 min in neonatal cardiomyocytes and in 2-4 min in AT-1 cells, followed by a rapid decline. We show that stimulation of AT-1 cells by these peptides is specific for CRH receptors, as the CRH antagonist, alpha-helical CRH-(9-41) inhibits cAMP increases. Furthermore, we show that CRH, sauvagine, and urotensin I stimulations are dose dependent in both neonatal cardiomyocytes and AT-1 cells. Sauvagine and urotensin I are more potent than CRH at stimulating an increase in intracellular cAMP in neonatal cardiomyocytes (EC50 = 1.74, 2.61, 6.42 nM, respectively) and AT-1 cells (EC50 = 16.2, 15.8, and 149 nM, respectively). This rank order is consistent with that previously demonstrated in CRH-R2-transfected HEK293 cells and parallels the in vivo vasodilatory activity of these peptides. In summary, this is the first evidence that CRH, sauvagine, and urotensin I act directly on cardiac myocytes to stimulate increases in intracellular cAMP, presumably through CRH-R2. In addition, these results indicate that cardiac myocytes may be an informative in vitro model to investigate the effects of CRH and its role in the cardiovascular response to stress.     

The 5-HT1-like receptors mediating inhibition of sympathetic vasopressor outflow in the pithed rat: operational correlation with the 5-HT1A, 5-HT1B and 5-HT1D subtypes

1. It has been suggested that the inhibition of sympathetically-induced vasopressor responses produced by 5-hydroxytryptamine (5-HT) in pithed rats is mediated by 5-HT1-like receptors. The present study has re-analysed this suggestion with regard to the classification schemes recently proposed by the NC-IUPHAR subcommittee on 5-HT receptors. 2. Intravenous (i.v.) continuous infusions of 5-HT and the 5-HT1 receptor agonists, 8-OH-DPAT (5-HT1A), indorenate (5-HT1A), CP 93,129 (5-HT1B) and sumatriptan (5-HT(1B/1D)), resulted in a dose-dependent inhibition of sympathetically-induced vasopressor responses. 3. The sympatho-inhibitory responses induced by 5-HT, 8-OH-DPAT, indorenate, CP 93,129 or sumatriptan were analysed before and after i.v. treatment with blocking doses of the putative 5-HT receptor antagonists, WAY 100635 (5-HT1A), cyanopindolol (5-HT(1A/1B)) or GR 127935 (5-HT(1B/1D)). Thus, after WAY 100635, the responses to 5-HT and indorenate, but not to 8-OH-DPAT, CP 93,129 and sumatriptan, were blocked. After cyanopindolol, the responses to 5-HT, indorenate and CP 93,129 were abolished, whilst those to 8-OH-DPAT and sumatriptan (except at the lowest frequency of stimulation) remained unaltered. In contrast, after GR 127935, the responses to 5-HT, CP 93,129 and sumatriptan, but not to 8-OH-DPAT and indorenate, were abolished. 4. In additional experiments, the inhibition induced by 5-HT was not modified after 5-HT7 receptor blocking doses of mesulergine. 5. The above results suggest that the 5-HT1-like receptors, which inhibit the sympathetic vasopressor outflow in pithed rats, display the pharmacological profile of the 5-HT1A, 5-HT1B and 5-HT1D, but not that of 5-HT7, receptors.     

Stimulation of 5-HT2A receptors on astrocytes in primary culture opens voltage-independent Ca2+ channels

A two-step exocytosis/endocytosis protocol was used in rat pancreatic acini to study membrane trafficking events at the apical plasma membrane (APM) as a function of extracellular pH. Exocytosis, as measured by cholecystokinin (CCK)-8-induced release of amylase into the incubation medium, was relatively insensitive to changes in extracellular pH from 5.5 to 9.0. In contrast, endocytosis, as measured by temperature-dependent uptake of horseradish peroxidase (HRP), was robust at pH values between 6.5 and 8.3 but abolished at acidic pH values of 5.5 to 6.0. Energy metabolism and cell viability were maintained during pH 6-induced cessation of HRP uptake, and the vesicular block could be reversed upon raising the luminal pH to 7.4. Histochemical and morphometric studies of HRP uptake examined by electron microscopy indicated that extracellular pH regulates endocytosis at the apical plasma membrane. At pH 6.0 in prestimulated cells, HRP uptake at the APM was abolished, and acinar lumen membranes remained markedly dilated with decreased density of microvilli and "arrested" exocytic images. At pH 7.4, HRP was taken up into endolysosomal structures within the Golgi complex, and acinar lumen membranes were contracted. Cleavage of GP2, a glycosyl phosphatidylinositol-anchored protein, was associated with the pH-dependent activation of HRP uptake. These studies demonstrate that acinar lumen pH regulates endocytic but not exocytic activity at the APM and suggest that alkalinization of the acinar lumen by duct cells is required for retrieval of exocytic membranes into the acinar cell via vesicular uptake mechanisms. The role of acid-base interactions within the acinar lumen provides a novel basis for understanding the cellular and luminal defects observed within the exocrine pancreas in cystic fibrosis.     

Spatial memory deficits following stimulation of hippocampal 5-HT1B receptors in the rat

The authors review the principles of reconstructive surgery for lower limb salvage after severe lower limb trauma to determine factors that have been used as decision-making criteria for limb salvage or amputation in severe lower extremity injuries and the methods of reconstruction and their outcome. The use of scoring systems and their value in acute decision making (primary amputation or limb salvage) are described. Soft-tissue reconstructive techniques, with emphasis on the use of flaps and the importance of selecting the best technique and time for the reconstruction are reviewed. Skeletal reconstructive techniques are described, including available options and currently held views on indications and use of the best contemporary methods. It is essential for the physician to make a good initial decision on the need for primary amputation or limb salvage. A multidisciplinary approach is fundamental to successful salvage.     

Effects of volatile anesthetic isoflurane on ATP-sensitive K+ channels in rabbit ventricular myocytes

It has been suggested that volatile anesthetic, isoflurane mediates cardioprotective effects via activation of the ATP-sensitive K+ (KATP) channels. However, no direct evidence has been provided to define whether isoflurane activates cardiac KATP channels using patch-clamp technique. We examined the effects of isoflurane on the KATP channels in rabbit ventricular myocytes by use of patch-clamp technique. Contrary to the results of the in vivo experiments, isoflurane inhibited the channel activity without a change in the single-channel conductance. Isoflurane decreased the channel activity by a decrease in burst duration and an increase in the inter-burst duration. On the other hand, isoflurane diminished the ATP sensitivity of KATP channels, indicating an increased probability of KATP channel opening for a given concentration of ATP after isoflurane anesthesia. The result supports, at least in part, the hypothesis that isoflurane mediates cardioprotective effects via KATP channel activation.     

Soluble or bound laminin elicit in human neuroblastoma cells short- or long-term potentiation of a K+ inwardly rectifying current: relevance to neuritogenesis

Changes in the resting potential (VREST) and in the underlying ionic conductances were measured by the patch-clamp technique in SH-SY5Y human neuroblastoma cells exposed to substrate-bound or soluble Laminin (bLN; sLN), as compared to integrin-independent substrates (polylysine (PL); bovine serum albumin (BSA)). While PL and BSA were ineffective, both forms of LN caused an early (5-15 min) activation of a peculiar type of Inwardly Rectifying K+ current (IIR) characterised by a voltage-dependent inactivation in the range of membrane potentials around -50/0 mV. IIR was blocked by Cs+ ions and by the antiarrhythmic drug E-4031, a specific inhibitor of the HERG-codified channels. In cells adherent to bLN, IIR potentiation (85%) persisted for 90-120 min and was accompanied by a similar, but transient, increase in the leakage conductance (GL). Successively, the persistence of a high IIR conductance and the decrease of GL progressively bring VREST from -12 to approximately -30 mV in about 120 min. On the other hand, in cells adherent to PL, exposure to sLN produced a similar but not persistent activation of IIR, without any increase in GL: this caused a rapid, transient hyperpolarisation of VREST. The effects of bLN and sLN were mimicked by antibodies raised against the integrin beta 1 subunit, suggesting a specific integrin-mediated mechanism. In fact, when bound to the culture dishes, these antibodies simultaneously activated the IIR and GL, whereas in soluble form they only activated IIR. Cells adherent to bLN emitted neurites, a process impaired by the block of IIR by E-4031 and Cs+. On the whole data suggest that the integrin-mediated activation of IIR plays a crucial role in the commitment to neuritogenesis of neuroblastoma cells, independently on the effects of this activation on VREST.     

Differential membrane targeting and pharmacological characterization of chimeras of rat serotonin 5-HT1A and 5-HT1B receptors expressed in epithelial LLC-PK1 cells

The serotonin 5-HT1A and 5-HT1B receptors are two structurally related but pharmacologically distinguishable 5-HT receptor types. In brain, the 5-HT1A receptor is localized on the soma and dendrites of neurons, whereas the 5-HT1B receptor is targeted to the axon terminals. We previously showed that these two receptors are targeted in different membrane compartments when stably expressed in the epithelial LLC-PK1 cell line. Further investigations on the mechanisms responsible for their differential targeting were done by constructing chimeras of 5-HT1A and 5-HT1B receptors still able to bind specifically [3H]lysergic acid diethylamide and selective agonists and antagonists. Their cellular localization examined by confocal microscopy suggests that the third intracellular domain of the 5-HT1B receptor was responsible for its Golgi-like localization in transfected LLC-PK1 cells. In contrast, the third intracellular domain of the 5-HT1A receptor apparently allowed the sorting of the chimeras to the plasma membrane. Further inclusion of the C-terminal domain of the 5-HT1A receptor in their sequence led to a basolateral localization, whereas that of the 5-HT1B receptor allowed an apical targeting, suggesting the existence of a targeting signal in this portion of the receptor(s).     

Functional coupling of endogenous serotonin (5-HT1B) and calcitonin (C1a) receptors in CHO cells to a cyclic AMP-responsive luciferase reporter gene

A cyclic AMP-responsive reporter cell line has been established through the stable expression of a luciferase reporter plasmid in Chinese hamster ovary (CHO) cells. Reporter cells showed a dose-dependent expression of luciferase in response to incubation with forskolin. These CHO cells were screened for endogenous G protein-coupled receptors capable of stimulating or inhibiting adenylyl cyclase, by monitoring changes in luciferase expression. Serotonin (5-HT) receptor agonist ligands caused an inhibition of forskolin-stimulated luciferase expression in the rank order 5-carboxamidotryptamine > 5-HT > sumatriptan > 8-hydroxy-2-(di-n-propylamino)tetralin. The response to 5-HT was reversed by the 5-HT1 receptor antagonists cyanopindolol and pindolol, but not the 5-HT2 receptor antagonist ketanserin. Calcitonin was more potent than calcitonin gene-related peptide (CGRP) at stimulating luciferase expression in this cell line, and these responses were insensitive to the CGRP receptor antagonist, CGRP (8-37). These results were consistent with the presence of 5-HT(1B-like) and calcitonin (C1a-like) receptors in CHO cells, with the responses to 5-HT and CGRP being pertussis and cholera toxin-sensitive, respectively. This reporter gene assay gave the expected pharmacological profile for these receptors when compared with cyclic AMP accumulation assays, confirming its value as a functional assay for G protein-coupled receptors linked to adenylyl cyclase.