HERG K(+) currents in human prolactin-secreting adenoma cells.

To investigate the presence and possible function of ether-à-go-go-related gene (erg) K(+) channels in human lactotroph cells (HERG channels), primary cultures were prepared from human prolactinoma tissue. In almost all primary cultures, HERG currents could be recorded in identified prolactin cells using an external high-K(+) solution. The antiarrhythmic agent E-4031, a specific blocker of erg channels, served to isolate HERG currents as the drug-sensitive currents. In cells of two tumours tested, thyrotropin-releasing hormone significantly reduced the amplitude of the HERG currents. The potential dependence of HERG current availability and the deactivation kinetics differed significantly even between prolactin cells derived from one adenoma. For comparison, corresponding values were obtained for heterologously expressed rat erg1, erg2 and erg3 channels. The expression of the three HERG channel subunits was investigated in nine human adenomas using RT-PCR. Transcripts for HERG1 were present in all adenomas and although transcripts for HERG2 and HERG3 were also detected, their expression level was more variable. The results demonstrate the functional expression of HERG channels in human prolactin-secreting tumours and are compatible with a physiological role for these channels in the control of prolactin secretion, as has been shown in normal rat lactotroph cells.

Ca2+ channels in clonal rat anterior pituitary cells (GH3/B6).

In clonal rat somatomammotroph cells (GH3/ B6) Ca2+ influx through voltage-dependent Ca2+ channels is important for regulating the Ca2+ concentration that mediates hormone secretion. To study the Ca2+ channel subtypes in GH3/B6 cells, Ca2+ channel currents were recorded with the whole-cell configuration of the patch-clamp technique using Ba2+ as the charge carrier. Forty-nine percent of the total Ba2+ current amplitude was mediated by a nifedipine-sensitive current (L-type). In addition, three other high-voltage-activated Ca2+ channel current components could be distinguished pharmacologically: 10 nM omega-agatoxin-IVA-sensitive current (22%; P-type), omega-conotoxin-MVIIC-sensitive current (18%; Q-type), and toxin-resistant current (24%). Since omega-conotoxin GVIA (2 microM) had no blocking effect, N-type Ca2+ channels are assumed not to be present in GH3/B6 cells. The T-type Ca2+ channel current was either absent or very small. Different pore-forming alpha1 subunits of Ca2+ channels were found to be expressed in GH3/B6 cells, which could be the molecular correlates of the different Ba2+ current subtypes: alpha1G of T-type, alpha1C, alpha1D and alpha1S of L-type, and alpha1A of P/Q-type current. In addition, transcripts for beta1, beta2 and beta3 subunits were detected. Blockage of L-type channels with 10 microM nifedipine or P/Q-type channels with 10 nM omega-agatoxin MVIIC + 200 nM omega-conotoxin blocked action potential firing in GH3/B6 cells and decreased basal prolactin secretion.

Modulation of rat erg1, erg2, erg3 and HERG K+ currents by thyrotropin-releasing hormone in anterior pituitary cells via the native signal cascade.

The mechanism of thyrotropin-releasing hormone (TRH)-induced ether-a-go-go-related gene (erg) K+ current modulation was investigated with the perforated-patch whole-cell technique in clonal somatomammotroph GH3/B6 cells. These cells express a small endogenous erg current known to be reduced by TRH. GH3/B6 cells were injected with cDNA coding for rat erg1, erg2, erg3 and HERG K+ channels. The corresponding erg currents were isolated with the help of the specific erg channel blockers E-4031 and dofetilide and their biophysical properties were determined. TRH (1 M) was able to significantly reduce the different erg currents. The voltage dependence of activation was shifted by 15 mV (erg1), 10 mV (erg2) and 6 mV (erg3) to more positive potentials without strongly affecting erg inactivation. TRH reduced the maximal available erg current amplitude by 12% (erg1), 13% (erg2) and 39% (erg3) and accelerated the time course of erg1 and erg2 channel deactivation, whereas erg3 deactivation kinetics were not significantly altered. The effects of TRH on HERG currents did not differ from those on its rat homologue erg1. In addition, coinjection of rat MiRP1 with HERG cDNA did not influence the TRH-induced modulation of HERG channels. Rat erg1 currents recorded in the cell-attached configuration were reduced by application of TRH to the extra-patch membrane in the majority of the experiments, confirming the involvement of a diffusible second messenger. Application of the phorbol ester phorbol 12-myristate 13-acetate (PMA; 1 M) shifted the voltage dependence of erg1 activation in the depolarizing direction, but it did not reduce the maximal current amplitude. The voltage shift could not be explained by a selective effect on protein kinase C (PKC) since the PKC inhibitor bisindolylmaleimide I did not block the effects of TRH and PMA on erg1. In addition, cholecystokinin, known to activate the phosphoinositol pathway similarly to TRH, did not significantly affect the erg1 current. Various agents interfering with different known TRH-elicited cellular responses were not able to completely mimic or inhibit the TRH effects on erg1. Tested substances included modulators of the cAMP-protein kinase A pathway, arachidonic acid, inhibitors of tyrosine kinase and mitogen-activated protein kinase, sodium nitroprusside and cytochalasin D. The results demonstrate that all three members of the erg channel subfamily are modulated by TRH in GH3/B6 cells. In agreement with previous studies on the TRH-induced modulation of the endogenous erg current in prolactin-secreting anterior pituitary cells, the TRH effects on overexpressed erg1 channels are not mediated by any of the tested signalling pathways.

Erg1, erg2 and erg3 K channel subunits are able to form heteromultimers.

Clonal somato-mammotroph GH3/B6 cells and lactotroph MMQ cells express two (ergl, erg2) of the three cloned rat ether-à-go-go-related gene (erg) K channel subunits. To study whether the erg subunits form heteromultimers, dominant-negative mutants of erg and erg2 were constructed by point mutation (erg1G630S, erg2G480S). After co-expression of these mutants with the wild-type erg1, erg2, or erg3 in Chinese hamster ovary (CHO) cells no erg currents could be detected. In contrast, in co-expression experiments with members of the other ether-à-go-go (EAG) subfamilies (eagl, elkl) the mutant erg1G630S had no effect. These results strongly suggest that erg channel subunits are able to form heteromultimers within the erg channel subfamily. Suppression of the endogenous E-4031-sensitive currents in GH3/B6 and MMQ cells by erg1G630S confirms that they are mediated by erg channels despite the differences in gating kinetics in these cells. Reduction of the erg current in GH3/B6 cells by erg2G480S indicates that erg heteromultimers can also be formed in these cells.

Expression of mRNA for voltage-dependent and inward-rectifying K channels in GH3/B6 cells and rat pituitary.

The expression of mRNA for voltage-dependent (Kv) and inward-rectifying K channels (Kir) was studied in clonal rat somato-mammotroph cells (GH3/B6 cells) and rat pituitary using reverse transcription-polymerase chain reaction (RT-PCR). In GH3/B6 cells transcripts for 16 different Kv channel alpha-subunits (seven Shaker-related: Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv3.2, Kv4.1, Kv5.1; six EAG: eag1, erg1, erg2, elk1-elk3; three KCNQ: KCNQ1-KCNQ3) and for five different Kir channel alpha-subunits (Kir1.1, Kir2.3, Kir3.2, Kir3.3, Kir6.2) were found. In addition, transcripts for a short isoform of Kvbeta2 and transcripts for Kvbeta3 subunits were present. In rat pituitary transcripts for 21 different Kv channel alpha-subunits (11 Shaker-related: Kv1.3, Kv1.4, Kv1.6, Kv2.1, Kv2.2, Kv3.2, Kv3.4, Kv4.1, Kv4.2, Kv4.3, Kv6.1; seven EAG: eag1, erg1-erg3, elk1-elk3; three KCNQ: KCNQ1-KCNQ3) and nine Kir channel alpha-subunits (Kir1.1, Kir2.2, Kir3.1-Kir3.4, Kir4.1, Kir6.1, Kir6. 2) were found. In addition, all tested auxiliary subunits (Kvbeta1-Kvbeta3, minK, SUR1, SUR2) are expressed in the pituitary. The results indicate that the macroscopic K currents in GH3/B6 and pituitary cells are presumably mediated by K channels constructed by a larger number of K channel alpha-subunits and auxiliary beta-subunits than previously distinguished electrophysiologically and pharmacologically.

Separation of M-like current and ERG current in NG108-15 cells.

Differentiated NG108-15 neuroblastoma x glioma hybrid cells were whole-cell voltage-clamped. Hyperpolarizing pulses, superimposed on a depolarized holding potential (-30 or -20 mV), elicited deactivation currents which consisted of two components, distinguishable by fitting with two exponential functions. Linopirdine [DuP 996, 3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one), a neurotransmitter-release enhancer known as potent and selective blocker of the M-current of rat sympathetic neurons, in concentrations of 5 or 10 microM selectively inhibited the fast component (IC50 = 14.7 microM). The slow component was less sensitive to linopirdine (IC50>20 microM). The class III antiarrhythmics [(4-methylsulphonyl)amido]benzenesulphonamide (WAY-123.398) and 1-[2-(6-methyl-2-pyrydinil)ethyl]-4-(4-methylsulphonylaminobenz oyl) piperidine (E-4031), selective inhibitors of the inwardly rectifying ERG (ether-à-go-go-related gene) potassium channel, inhibited predominantly the slow component (IC50 = 38 nM for E-4031). The time constant of the WAY-123.398-sensitive current resembled the time constant of the slow component in size and voltage dependence. Inwardly rectifying ERG currents, recorded in K+ -rich bath at strongly negative pulse potentials, resembled the slow component of the deactivation current in their low sensitivity to linopirdine (28% inhibition at 50 microM). The size of the slow component varied greatly between cells. Accordingly, varied the effect of WAY-123.398 on deactivation current and holding current. RNA transcripts for the following members of the ether-à-go-go gene (EAG) K+ channel family were found in differentiated NG108-15 cells: ERG1, ERG2, EAGI, EAG-like (ELK)1, ELK2; ERG3 was only present in non-differentiated cells. In addition, RNA transcripts for KCNQ2 and KCNQ3 were found in differentiated and non-differentiated cells. We conclude that the fast component of the deactivation current is M-like current and the slow component is deactivating ERG current. The molecular correlates are probably KCNQ2/KCNQ3 and ERG1/ERG2, respectively.

The erg-like potassium current in rat lactotrophs.

1. The ether-à-go-go-related gene (erg)-like K+ current in rat lactotrophs from primary culture was characterized and compared with that in clonal rat pituitary cells (GH3/B6). The class III antiarrhythmic E-4031 known to block specifically erg K+ channels was used to isolate the erg-like current as the E-4031-sensitive current. The experiments were performed in 150 mM K+ external solution using the patch-clamp technique. 2. The erg-like K+ current elicited with hyperpolarizing pulses negative to -100 mV consisted of a fast and a pronounced slowly deactivating current component. The contribution of the slow component to the total current amplitude was potential dependent and varied from cell to cell. At -100 mV it ranged from 50 to 85% and at -140 mV from 21 to 45%. 3. The potential-dependent channel availability curves determined with 2 s prepulses were fitted with the sum of two Boltzmann functions. The function related to the slowly deactivating component of the erg-like current was shifted by more than 40 mV to more negative membrane potentials compared with that of the fast component. 4. In contrast to that of native lactotrophs studied under identical conditions, the erg-like K+ current of GH3/B6 cells was characterized by a predominant fast deactivating current component, with similar kinetic and steady-state properties to the fast deactivating current component of native lactotrophs. 5. Thyrotrophin-releasing hormone reduced the erg-like current in native lactotrophs via an intracellular signal cascade which seemed to involve a pathway independent from protein kinase A and protein kinase C. 6. RT-PCR studies on cytoplasm from single lactotrophs revealed the presence of mRNA of the rat homologue of the human ether-à-go-go-related gene HERG (r-erg1) as well as mRNA of the two other cloned r-erg cDNAs (r-erg2 and r-erg3) in different combinations. In GH3/B6 cells, only the transcripts of r-erg1 and r-erg2 were found.

RERG is a molecular correlate of the inward-rectifying K current in clonal rat pituitary cells.

The rat homologue of the human ether-ä-go-go-related gene (r-erg) was cloned from rat brain using homology screening. RERG has a 96% amino acid identify to HERG. Membrane currents recorded in CHO cells after previous injection of r-erg showed that the voltage- and time-dependent properties are indistinguishable from h-erg-induced currents expressed in the same system. RT-PCR revealed the presence of r-erg mRNA in clonal rat pituitary cells (GH3/B6 cells). These cells exhibit a voltage-dependent inward-rectifying K current (IK, IR) which is highly sensitive to the class III antiarrhythmic E-4031. IK, IR recorded in GH3/B6 cells and ERG currents in CHO cells were compared using similar experimental conditions (same pulse protocols and isotonic KCl as extracellular solution). The voltage- and time-dependent properties of both currents were found to be almost identical. These results strongly suggest that RERG channels mediate IK, IR in GH3/B6 cells.

Molecular and pharmacological characterization of somatostatin receptor subtypes in adrenal, extraadrenal, and malignant pheochromocytomas.

SRIH receptors were quantified by radioautography in 33 pheochromocytomas and 5 normal adrenals. Binding was evenly distributed over the tumors, whereas it was more intense in adrenal medulla than cortex. Binding levels were significantly higher in tumoral than in normal tissue, but did not differ among tumors. At 100 nmol/L, SRIH-14 and octreotide (or BIM23014 in cross-linking experiments to a 57-kilodalton component) comparably displaced SRIH binding, BIM23042 and BIM23052 were less potent, and BIM23056 was inefficient. In increasing doses, the rank order of potency was SRIH-14 > SRIH-28 > octreotide > BIM23052 >> BIM23042 >> > BIM23056. All five species of SRIH receptor (SSTR1-5) messenger ribonucleic acids (mRNAs) were measurable in pheochromocytomas and normal adrenals, SSTR2 and SSTR4 mRNA were the most expressed moieties. The proportion of SSTR5 mRNA species was higher in normal adrenals (21%) than in pheochromocytomas (6%). In the presence of guanylylimidodiphosphate, SRIH binding was reduced by 83%. However, SRIH did not alter basal or forskolin-stimulated adenylyl cyclase activity. Taken together, these pharmacological and molecular data indicate that SRIH binding on pheochromocytomas depends on a mixed population of receptors, mainly of the SSTR2 and SSTR4 subtypes, efficiently coupled to G proteins, but not to adenylyl cyclase inhibition.

Sequence analysis of the promoter region of the rat somatostatin receptor subtype 1 gene.

Somatostatin receptor (SSTR) subtype genes are differentially expressed in brain and various peripheral tissues. RNA blotting and semiquantitative PCR analyses have revealed low levels of SSTR1 mRNA in the gastrointestinal tract and relatively high levels in GH3 anterior pituitary cells. As a first step in the investigation of the regulation of SSTR1 gene expression, we isolated a genomic fragment that contains the promoter region and determined the transcriptional initiation site. The SSTR1 gene lacks introns and TATA and CAAT motifs, but possesses several consensus recognition sequences for the transcription factors GCF and AP-2. The presence, also, of two Pit-1 binding sites could explain the high SSTR1 mRNA levels in GH3 cells.

Expression patterns of rat somatostatin receptor genes in pre- and postnatal brain and pituitary.

The relative abundances of mRNAs encoding four different somatostatin receptors were examined using PCR techniques during postnatal development of the rat brain and hypophysis. In most tissues, somatostatin receptor 1 and 4 mRNAs are more abundant than those encoding somatostatin receptor 2 and 3. Transcript levels of somatostatin receptor subtype 4 are relatively high in the cortex, hippocampus, and striatum, those of subtype 1 in the cortex and brainstem, and those of subtype 3 in the cerebellum. In situ hybridization revealed the presence of significant amounts of somatostatin receptor 1 mRNA, as early as prenatal day 14, in the trigeminal ganglion and in the neuroepithelial layers surrounding the lateral, third, and fourth ventricles. In the developing cortex a morphological change in the sites of somatostatin receptor 1 gene expression occurs; mRNA is present superficially in the cortex at prenatal stages, appears in all layers shortly after birth, and in adult rats is restricted to the deep cortical layers. In the cerebellum, somatostatin receptor 1 mRNA levels are highest around birth, declining thereafter. In contrast, cerebellar somatostatin receptor 3 transcripts are absent at birth, become detectable around postnatal day 7, and reach a maximal level during maturation.

Molecular cloning of a somatostatin-28 receptor and comparison of its expression pattern with that of a somatostatin-14 receptor in rat brain.

The tetradecapeptide somatotropin-release inhibiting factor somatostatin-14 regulates the release of peptide hormones and also functions as neurotransmitter. The octacosapeptide somatostatin-28, the N-terminally extended form of somatostatin-14, shows similar biological activities yet with different potencies. Both peptides most likely function through distinct receptors. Here we report on the molecular and functional characterization of a somatostatin-28 receptor (SSR-28) cloned from a rat brain cDNA library. The nucleotide sequence contains an open reading frame for a protein of 428 amino acid residues with a predicted molecular mass of 47 kDa. Binding assays using radiolabeled somatostatin-14 and membranes from COS cells transfected with the cloned cDNA show that this receptor, SSR-28, has a higher binding affinity for somatostatin-28 (IC50 = 0.24 nM) than for somatostatin-14 (IC50 = 0.89 nM). RNA blot analysis reveals a 4.4-kilobase mRNA in rat cerebellum and at significantly lower abundance in other brain regions. In situ hybridization indicates that SSR-28 mRNA is present in the granular and Purkinje cell layers of the cerebellum and in the large cells of the hypoglossal nucleus of the brain stem. Signals for SSR-28 mRNA do not overlap with those of a previously cloned rat receptor that preferentially binds somatostatin-14 (SSR-14). SSR-14 mRNA is found in the medial cerebellar nucleus, horizontal limb of the diagonal band, various hypothalamic nuclei, and in layers IV and V of the cortex. In the rat cerebellum, SSR-14 and SSR-28 mRNAs are developmentally regulated; the levels of the former are highest around birth and levels of the latter are highest at the adult stage.