Regulation of G protein mRNA levels by thyroliberin,vasoactive intestinal peptide and somatostatin in prolactin-producing rat pituitary adenoma cells

We have investigated the regulation of mRNA levels of alpha- and beta-subunits of guanine nucleotide-binding regulatory proteins (G proteins) by peptide hormones in prolactin producing rat pituitary adenoma cells (GH₃ cells) in culture. The cells were treated with thyroliberin (1 μm), vasoactive intestinal peptide (1 μm) or somatostatin (10 μM) for 6 to 48 hours. Thyroliberin and vasoactive intestinal peptide increased the levels of G_sα G_oα, G_i-2α, G_i-3α, G_xα, Gβ₃₆ and Gβ₃₅ mRNAs. The effect of vasoactive intestinal peptide was however earlier and more pronounced. G_i-2α mRNA levels showed the quantitatively largest alterations. Somatostatin upregulated G_sα and downregulated G_oα and G_i-2 mRNAs. G protein mRNAs for G_i-2α and G_oα were increased by exposure of the cells to a medium devoid of serum. We conclude that G protein mRNA levels are subjected to alterations by hormones that act through the corresponding G proteins in the regulation of prolactin synthesis and secretion.     

Modulation of G proteins and second messenger responsiveness by steroid hormones in GH3 rat pituitary tumour cells.

We have investigated the modulation of different G protein alpha- and beta-subunit levels in prolactin (PRL) and growth hormone producing rat pituitary adenoma cells (GH3 cells) in culture after prolonged exposure (6-48 h) to the steroid hormones 17 beta-oestradiol and dexamethasone. Gi-3 alpha- and G beta-subunits were the only G protein subunits which increased in response to 10(-6) M oestradiol (to approximately 150 and 200% of controls, respectively), while the other alpha-subunits investigated (Gs alpha, Gi-2 alpha and G(o) alpha) remained relatively unchanged. Thyroliberin (TRH)--and guanosine 5'-[beta gamma-imido]trisphosphate (Gpp(NH)p)-elicited adenylyl cyclase (AC) activities were reduced during 6-12 h of oestradiol treatment (by 60 and 20%, respectively), while the inhibitory effect of somatostatin (SRIF) increased by approximately 100%. Dexamethasone (10(-6) M) increased levels of the stimulatory G protein Gs alpha (to approximately 340%) and decreased levels of Gi-3 alpha (to 25%). After 48 h, the AC response to TRH was reduced by approximately 70%, whereas the effect of the other modulators remained close to controls. We conclude that G protein subunits in GH3 cells are subject to specific regulation by steroid hormones and that this may be important in the tuning of the responsiveness of PRL secretion to hormones in the in vivo situation.     

Effects of thyroliberin and 4-aminopyridine on action potentials and prolactin release and synthesis in rat pituitary cells in culture

Effects of thyroliberin (TRH) and 4-aminopyridine (4AP) were studied on prolactin (PRL) secreting rat pituitary tumour cells in culture (GH₃ cells). Intracellular recordings obtained from the same cell before and during TRH stimulation showed this peptide to increase the spontaneous firing frequency and prolong the Ca²⁺ dependent action potentials. These effects were mimicked by 4 AP, which acts by interfering selectively with voltage dependent ionic channels without affecting resting membrane properties. Optimal doses of TRH and 4AP approximately doubled the release of PRL. In contrast, TRH increased PRL synthesis 1.9-fold while 4AP had no effect. Increased PRL synthesis is thus not a direct consequence of the hormone release. We conclude that TRH and 4AP both stimulates PRL release via the facilitating effects on the action potentials. TRH has additional intracellular effects which lead to increased synthesis of the hormone. The effects of TRH responsible for stimulation of PRL synthesis are not causally related to the impulse activity of the surface membrane of the cell.     

Different G proteins are involved in the biphasic response of clonal rat pituitary cells to thyrotropin-releasing hormone

In rat anterior pituitary tumour cells (GH₃/B₆) thyrotropin-releasing hormone (TRH) elicits a biphasic response. First, a release of intracellularly stored Ca²⁺ induces a hyperpolarization of the cell. Second, a depolarization thought to be induced by a reduction of the inward-rectifying K⁺ current (K_IR) causes an increase in action potential frequency and a plateau-like increase in [Ca²⁺]_i. It has been proposed that the two phases are induced by the actions of inositol 1,4,5-trisphosphate (InsP ₃) and protein kinase C (PKC), respectively, but we demonstrate here that PKC is not responsible for the second phase increase in [Ca²⁺]_i and suggest that the pathways diverge at the level of receptor and G protein coupling. Both phases of the TRH response were insensitive to pertussis toxin, but cholera toxin (CTX) selectively affected the second phase. After CTX pretreatment cells had a high spontaneous spiking frequency and smaller K_IR amplitude. In these cells TRH failed to increase the action potential frequency after the first phase hyperpolarization, elicited only a transient peak increase in [Ca²⁺]_i with no plateau phase and could only slightly reduce K_IR. These effects of CTX are not mediated by its ability to increase cAMP via activation of G_S, as increased cAMP levels neither inhibit K_IR nor prevent its reduction by TRH. In addition, inhibition of protein kinase A activation did not block the second phase increase in [Ca²⁺]_i induced by TRH, suggesting that the CTX-sensitive G protein mediating the second phase of the TRH response is not G_S.On leave from Uchtomsky-Institute, St. Petersburg University, Russia     

Cell type influences the molecular mechanisms involved in hormonal regulation of ERG K<Superscript>+</Superscript> channels

While the thyrotropin-releasing hormone (TRH) effect of raising intracellular Ca²⁺ levels has been shown to rely on G_q/11 and PLC activation, the molecular mechanisms involved in the regulation of ERG K⁺ channels by TRH are still partially unknown. We have analysed the effects of βγ scavengers, Akt/PKB inactivation, and TRH receptor (TRH-R) overexpression on such regulation in native and heterologous expression cell systems. In native rat pituitary GH₃ cells β-ARK/CT, Gα_t, and phosducin significantly reduced TRH inhibition of rERG currents, whereas in HEK-H36/T1 cells permanently expressing TRH-R and hERG, neither of the βγ scavengers affected the TRH-induced shift in V _1/2. Use of specific siRNAs to knock Akt/PKB expression down abolished the TRH effect on HEK-H36/T1 cell hERG, but not on rERG from GH₃ cells. Indeed, wortmannin or long insulin pretreatment also blocked TRH regulation of ERG currents in HEK-H36/T1 but not in GH₃ cells. To determine whether these differences could be related to the amount of TRH-Rs in the cell, we studied the TRH concentration dependence of the Ca²⁺ and ERG responses in GH₃ cells overexpressing the receptors. The data indicated that independent of the receptor number additional cellular factor(s) contribute differently to couple the TRH-R to hERG channel modulation in HEK-H36/T1 cells. We conclude that regulation of ERG currents by TRH and its receptor is transduced in GH₃ and HEK-H36/T1 cell systems through common and different elements, and hence that the cell type influences the signalling pathways involved in the TRH-evoked responses.     

Combined pituitary function-test with four hypothalamic releasing hormones

Summary Anterior pituitary function was investigated in ten healthy subjects by administering a combination of 200 µg thyrotropin releasing hormone (TRH), 100 µg gonadotropin releasing hormone (GnRH), 100 µg growth hormone releasing factor (GRF^1–44), and 100 µg human corticotropin releasing factor (CRF). The same test protocol was performed in all subjects after pretreatment with 0.25 mg terguride. Five subjects were tested only with TRH and GnRH, five only with CRF, and six only with GRF. There was a prompt increase in all hormones after the administration of the four releasing hormones (RH). Pretreatment with terguride lowered the prolactin (PRL) increase (p<0.01) as well as the thyrotropin (TSH) peak (p<0.05) compared with the test without dopamine agonist pretreatment. The PRL levels after combined RH administration were significantly higher than after TRH and GnRH alone. Although four of the five subjects had higher TSH levels after combined RH administration than after TRH and GnRH alone, the difference was not significant. Other hormones were not significantly influenced by the combined RH administration or dopamine agonist pretreatment. Despite the fact that the interaction of the different releasing hormones and dopamine agonists influences the pituitary hormone response, combined RH administration seems to be a useful test for evaluating pituitary function also in patients receiving dopamine agonist therapy.Abbreviations ACTH Adrenocorticotropic hormone - CRF Human corticotropin releasing factor - DA Dopamine - FSH Follicle-stimulating hormone - GH Human growth hormone - GnRH Gonadotropin releasing hormone - GRF; GRF^1–44 Growth hormone releasing factor - LH Luteinizing hormone - PRL Prolactin - RH Releasing hormone (s) - RIA Radioimmunoassay - SE Standard error - TRH Thyrotropin releasing hormone - TSH Thyrotropin Supported by Deutsche Forschungsgemeinschaft (We 439/5-1 and Mu 585/2-2).     

TGF-B and Estrogen Regulate P27(Kip1) and Cyclin D(1) in Normal and Neoplastic Rat Pituitary Cells

Pituitary hyperplasia and tumor growth are regulated by various hormones and growth factors. Estrogen (E₂) stimulates pituitary cell proliferation and prolactin (PRL) production. Estrogen also regulates transforming growth factor-β (TGF-β) effects in the pituitary. TGF-β in turn regulates various cell cycle proteins including p15 and p27^Kip1 (p27). To better understand the regulatory role of growth factors and hormones in the cell cycle we analyzed cyclin D₁, cyclin E, and p27 expression in normal and neoplastic rat pituitary cells. An in vitro analysis using cultured normal pituitary cells and GH₃ tumor cells and an in vivo analysis of estrogen-treated normal pituitary and implanted GH₃ cells were performed. Semiquantitative RT-PCR was used to analyze mRNA expression for cyclin D₁, cyclin E, and p27 in cultured pituitary cells and E₂-treated pituitaries in vivo. Cyclin D₁ and p27 were localized in the nuclei of normal pituitary cells by immunocytochemistry (ICC). Very weak or absent immunostaining for cyclin D₁ and p27 was present in GH₃ cells. Both normal pituitary and GH₃ cells had strong nuclear staining for cyclin E. Normal pituitary had a 20-fold greater amount of cyclin D₁ mRNA and a 3-fold greater amount of p27 mRNA compared to GH₃ cells, whereas GH₃ cells had slightly (1.5-fold) more cyclin E than normal pituitary cells. E₂ treatment in vivo stimulated cell proliferation and decreased cyclin D₁ mRNA levels in normal pituitary. GH₃ tumor cells, implanted subcutaneously in the same animal, showed increased proliferation after E₂ treatment, but there was no change in cyclin D₁ mRNA in GH₃ cells. Cyclin E and p27 mRNA levels did not change significantly in normal pituitary or in GH₃ cells after E₂ treatment in vivo. Treatment of normal pituitary cells with 10⁻⁹ M TGF-β1 for 3 d in vitro led to significant decreases in cyclin D₁ and p27 mRNAs (p < 0.05), whereas cyclin E levels were unchanged. These results indicate that cyclin D₁ and p27 mRNAs are present at significantly higher levels in normal pituitary compared to GH₃ cells, and that both E₂ and TGF-β1 can downregulate cyclin D₁ mRNA levels in normal pituitary cells, suggesting that these factors regulate G1 to S phase transition in pituitary cells. The lower levels of specific cell cycle regulators in GH₃ cells may explain the decreased regulatory control by E₂ in GH₃ tumor cells.     

Demonstration of an inwardly rectifying K+ current component modulated by thyrotropin-releasing hormone and caffeine in GH3 rat anterior pituitary cells

 Reduction of an inwardly rectifying K⁺ current by thyrotropin-releasing hormone (TRH) and caffeine has been considered to be an important determinant of electrical activity increases in GH₃ rat anterior pituitary cells. However, the existence of an inwardly rectifying K⁺ current component was recently regarded as a misidentification of an M-like outward current, proposed to be the TRH target in pituitary cells, including GH₃ cells. In this report, an inwardly rectifying component of K⁺ current is indeed demonstrated in perforated-patch voltage-clamped GH₃ cells. The degree of rectification varied from cell to cell, but both TRH and caffeine specifically blocked a fraction of current with strong rectification in the hyperpolarizing direction. Use of ramp pulses to continuously modify the membrane potential demonstrated a prominent blockade even in cells with no current reduction at voltages at which M-currents are active. Depolarization steps to positive voltages at the maximum of the inward current induced a caffeine-sensitive instantaneous outward current followed by a single exponential decay. The magnitude of this current was modified in a biphasic way according to the duration of the previous hyperpolarization step. The kinetic characteristics of the current are compatible with the possibility that removal from inactivation of a fast-inactivating delayed rectifier causes the hyperpolarization-induced current. Furthermore, the inwardly rectifying current was blocked by astemizole, a potent and selective inhibitor of human ether-á-go-go -related gene (HERG) K⁺ channels. Along with other pharmacological and kinetic evidence, this indicates that the secretagogue-regulated current is probably mediated by a HERG-like K⁺ channel. Addition of astemizole to current-clamped cells induced clear increases in the frequency of action potential production. Thus, an inwardly-rectifying K⁺ current and not an M-like outward current seems to be involved in TRH and caffeine modulation of electrical activity in GH₃ cells. Received: 15 May 1997 / Received after revision and accepted: 24 July 1997     

Characteristics and modulation by thyrotropin-releasing hormone of an inwardly rectifying K+ current in patch-perforated GH3 anterior pituitary cells

Hyperpolarization of patch-perforated GH₃ rat anterior pituitary cells in high-K⁺ Ca²⁺-free medium reveals an inwardly rectifying K⁺ current. This current showed potential-dependent activation and inactivation kinetics, complete inactivation during strong hyperpolarization and rectification at depolarized potentials. The current was blocked by millimolar concentrations of external Cs⁺, Ba²⁺, Cd²⁺ and Co²⁺, but it was almost insensitive to tetraethylammonium, 4-aminopyridine and two dihydropyridines, nisoldipine and nitrendipine. Verapamil and methoxyverapamil produced a strong and reversible inhibition of the current. In the presence of 100 nM thyrotropin-releasing hormone (TRH), the current was reduced. This reduction was increased by holding the cell at more negative potentials and was accompanied by a shift in steady-state voltage dependence of inactivation towards more positive voltages. Furthermore, the current slowly returned to the initial levels upon washout. Treatment of the cell with the protein phosphatase inhibitor okadaic acid increased the magnitude of the inhibition caused by TRH. Moreover, the current did not return towards the control level during a 30-min washout period. It is concluded that protein phosphatases participate in modulation of the GH₃ cell inwardly rectifying K⁺ channels by TRH. Furthermore, these data indicate that either a protein phosphatase or a factor necessary for its activation is lost under whole-cell mode, which could account for the permanent reduction of the current in response to TRH.     

Contribution of different Ca2+‐activated K+ channels to the first phase of the response to TRH in clonal rat anterior pituitary cells

Aims: Thyrotropin‐releasing hormone (TRH) induces biphasic changes in electrical activity, cytosolic free Ca²⁺ level ([Ca²⁺]_i), and prolactin secretion from both clonal GH cells and native lactotrophs. The first phase of the TRH response is characterized by hyperpolarization because of activation of Ca²⁺‐activated K⁺ channels (K_Ca). In the present study, the relative contribution of BK, SK, and IK channels to the first phase of the TRH response in GH₄ cells was assessed. Methods: The expression of IK channels was confirmed by PCR with specific primers for SK4 (IK). The response to TRH was studied using the perforated patch technique and Ca²⁺ microfluoromety (fura‐2). The involvement of different K_Ca channels was estimated by employing the specific channel blockers iberiotoxin (BK), apamin (SK) and clotrimazole (IK). Results: Application of 100 nm iberiotoxin, 1 μm apamin, and 10 μm clotrimazole reduced the peak value of the outward K⁺ current during the first phase of the TRH response by 33, 26, and 33%, respectively. Clotrimazole also shortened the duration of the outward current response by 60%, causing a reduction of total charge movement by 73%. All these toxin‐induced reductions were significant (P < 0.05). A combination of all three toxins abolished the current response almost completely. Conclusion: All the three main types of K_Ca channels are involved in the first phase of the TRH response, with IK as the major contributor. This is the first demonstration of a dominant role of IK compared with BK and SK channels in excitable cells.     

Reduction of the allergenic protein in soybean meal by enzymatic hydrolysis

The effects of Alcalase and Trypsin hydrolysis on the allergenic protein and its solubility of soybean meal (SBM) were investigated by SDS-PAGE and ELISA. After 10 min of hydrolysis with Alcalase, the solubility of the protein was 87.19%, the α′, α and β-subunits of β-conglycinin disappeared and the acidic and basic subunits of glycinin decreased obviously by SDS-PAGE pattern. However, with Trypsin for 10 min, the solubility of the protein was 72.74%. The contents of α′, α subunits of β-conglycinin decreased slightly in SDS-PAGE, and the acidic and basic subunits of glycinin were almost not affected. For the insoluble hydrolysates prepared by Alcalase, the α′, α subunits of β-conglycinin and acidic subunit of glycinin decreased significantly, that by trypsin did not. The content of immune-reactive β-conglycinin obtained by ELISA was consistent with them. These results confirmed that the allergenic proteins of SBM were more sensitive to Alcalase than to Trypsin.     

Single-compartment model analysis of thyrotropin-releasing hormone kinetics in hyper- and hypothyroid patients

Summary The pharmacokinetics of thyrotropin-releasing hormone (TRH) were assessed following an i.v. injection in blood of ten hyperthyroid, ten hypothyroid, and six normal subjects. A single-compartment model was employed. After methanol extraction, TRH concentrations were analyzed using a specific radioimmunoassay technique combined with fast protein liquid chromatography (FPLC). As for the basal levels of TRH, no differences were observed in either study group. Peak concentrations were always present two min after the injection of TRH. In the euthyroid subjects, TRH blood levels had a half-life (t _1/2) of 6.5±0.41 min, mean±SD, whilet _1/2 was 7.2±0.62 min in the hyperthyroid andt _1/2 was 12±1.67 min (p<0.001) in the hypothyroid patients. The metabolic clearance rate (MCR) (82.2±15.3 liters/m²/day vs. 89.8±17.2) and the volume of distribution (V_d) (7.1±4.2 liters vs. 7.3±3.4) were approximately the same in the normal subjects and in the hyperthyroid group. MCR (66.2±15.3 Iiters/m²/day) and V_d (6.2±3.3 liters) were found to be lower in the hypothyroid patients. In FPLC, when TRH was added to plasma, it eluted in one peak. Blood samples taken 5 min after TRH i.v. injection had an elution profile of 9.94 ml. These data indicate that 1) TRH has a very short half-life, 2) hypothyroidism can prolong thet _1/2 of exogenous TRH, and 3) when TRH should be used in clinical studies, the function of the thyroid gland has to be taken into consideration.Abbreviations TRH Thyrotropin-releasing hormone - RIA radioimmunoassay - FPLC Fast protein liquid chromatography - TRH-IR Thyrotropin releasing hormone-immunoreactivity - t _1/2 half-life - MCR metabolic clearance rate - V_d volume of distribution - AUC area under the curve - BW body weight This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Pf-38/3-1)     

Scn3b knockout mice exhibit abnormal sino-atrial and cardiac conduction properties

Aim: In contrast to extensive reports on the roles of Na_v1.5 α-subunits, there have been few studies associating the β-subunits with cardiac arrhythmogenesis. We investigated the sino-atrial and conduction properties in the hearts of Scn3b^−/− mice. Methods: The following properties were compared in the hearts of wild-type (WT) and Scn3b^−/− mice: (1) mRNA expression levels of Scn3b, Scn1b and Scn5a in atrial tissue. (2) Expression of the β₃ protein in isolated cardiac myocytes. (3) Electrocardiographic recordings in intact anaesthetized preparations. (4) Bipolar electrogram recordings from the atria of spontaneously beating and electrically stimulated Langendorff-perfused hearts. Results: Scn3b mRNA was expressed in the atria of WT but not Scn3b^−/− hearts. This was in contrast to similar expression levels of Scn1b and Scn5a mRNA. Immunofluorescence experiments confirmed that the β₃ protein was expressed in WT and absent in Scn3b^−/− cardiac myocytes. Lead I electrocardiograms from Scn3b^−/− mice showed slower heart rates, longer P wave durations and prolonged PR intervals than WT hearts. Spontaneously beating Langendorff-perfused Scn3b^−/− hearts demonstrated both abnormal atrial electrophysiological properties and evidence of partial or complete dissociation of atrial and ventricular activity. Atrial burst pacing protocols induced atrial tachycardia and fibrillation in all Scn3b^−/− but hardly any WT hearts. Scn3b^−/− hearts also demonstrated significantly longer sinus node recovery times than WT hearts. Conclusion: These findings demonstrate, for the first time, that a deficiency in Scn3b results in significant atrial electrophysiological and intracardiac conduction abnormalities, complementing the changes in ventricular electrophysiology reported on an earlier occasion.     

G<Subscript>i2</Subscript>α protein deficiency: A model for inflammatory bowel disease

Mice deficient for the G protein subunit G_i2α were obtained by gene targeting. They displayed a growth retardation that was apparent at 6 weeks of age. They subsequently developed diffuse colitis with clinical and histopathological features closely resembling those of ulcerative colitis in humans. Seven of 20 G_i2α-deficient mice with colitis also developed adenocarcinomas of the colon. G_i2α-deficient thymocytes displayed two-to fourfold increases in mature CD4⁺8^? and CD4^?8⁺ phenotypes, an approximately threefold increase in highintensity CD3 staining and enhanced proliferative responses to T-cell receptor stimuli. Stimulation of G_i2α-deficient peripheral T cells induced a hyperresponsive profile of interleukin-2, tumor necrosis factor, and interferon-γ production, which may reflect a heightened response of primed cells or a defective negative regulation. We suggest that G_i2α-deficient mice may represent a useful animal model for dissecting the pathomechanisms of inflammatory bowel disease and also for the development of novel therapeutic strategies.     

Pap,papG and prsG DNA sequences in Escherichia coli from the fecal flora and the urinary tract

The pap gene clusters encode P fimbriae and fimbriae-associated G adhesins. DNA sequence analysis has resolved three G adhesin variants (papG_J96, papG_IA2 and prs G_J96) that differ in receptor specificity and therefore in binding to epithelial cells. In this study, DNA probes specific for the pap gene cluster or the papG_J96, pap G_IA2 and prsG_J96 adhesin sequences were used to examine 74 fecal and 204 urinary Escherichia coli isolates (67 from acute pyelonephritis, 71 from acute cystitis and 66 from asymptomatic bacteriuria). In accordance with previous studies, a higher frequency of pap⁺ strains was found in the urinary strains (71%) than in the fecal (20%) E. coli isolates. The papG_IA2, and prs G_J96 sequences were more frequent among urinary (42% pap G⁺_IA2, 23% prs G⁺_J96) than among fecal (18% pap G⁺_IA2, 5% prs G⁺_J96) isolates. None of the isolates hybridized with the papG_J96 probe. Pap⁺ strains accounted for 82% of the pyelonephritis, 69% of the cystitis and 61% of the asymptomatic bacteriuria strains. The papG_IA2 genotype dominated in acute pyelonephritis strains (72% pap G⁺_IA2, 16% prs G⁺_J96). The prsG_J96 genotype was most frequent in cystitis strains (25% pap G⁺_IA2, 37% prs G⁺_J96). The asymptomatic bacteriuria strains formed an intermediate group (30% papG⁺_IA2, 14% prs G⁺_J96). Most of the pap G⁺_IA2 strains expressed P fimbriae which agglutinated human erythrocytes, sheep erythrocytes and Galα1-4Galβ latex beads. The prsG⁺_J96 strains varied in agglutination of human and sheep erythrocytes and Galα1-4Galβ-latex beads. The results demonstrated that the papG_IA2 and prs G_J96 adhesin DNA sequences differ in disease association.     

Local changes in GTP-binding protein immunoreactivities in human epileptogenic neocortex

The relative levels of guanine nucleotide-binding protein α-subunits G_i1α, G_i2α, G_i3α, G_oα, G_sα, and G_x/zα were measured in neocortex removed at surgery from patients with intractable temporal lobe epilepsy. Immunoreactivity was quantified using specific polyclonal antisera against the Gα-subunits according to the Laurell “rocket” immunoelectrophoresis technique. We compared the G protein contents of spiking (active) and nonspiking (nonactive) cortical regions, based on intraoperative electrocorticography, within the same and different patients. There were no clear trends for lower or higher levels of G-protein subtypes to be found in the samples of protein extracts from nonspiking regions as compared to spiking regions. However, comparison of paired samples of spiking and nonspiking cortex within the same patient demonstrated that levels of certain G-protein subtypes were either increased or decreased in all patients. This indicates that cortical regions with enhanced neuronal activity may produce microzonal alterations in the levels of G proteins. Moreover, our results suggest that high levels of G_i1α and low levels of the other G-protein subtypes appear to be associated with a greater susceptibility to maintaining spiking activity. Received: 30 July 1997 / Accepted: 15 September 1997     

The role of the inwardly rectifying K+ current in resting potential and thyrotropin-releasing-hormone-induced changes in cell excitability of GH3 rat anterior pituitary cells

Exposure of GH₃ rat anterior pituitary cells to cholera toxin for 2–4 h significantly increased the thyrotropin-releasing-hormone(TRH)-induced inhibition of the inwardly rectifying K⁺ current studied in patchperforated voltage-clamped cells. On the other hand, the current reduction became almost totally irreversible after washout of the neuropeptide. Comparison of the effects elicited by the toxin with those of 8-(4-chlorophenylthio)-cAMP or forskolin plus isobutylmethylxanthine indicated that, although the irreversibility may be due, at least in part, to elevations of cAMP levels, the enhancement of the TRH-induced inhibition of the current is not mediated by the cyclic nucleotide. Only reductions on the inwardly rectifying K⁺ current, but not those elicited by TRH on voltage-dependent Ca²⁺ currents, were increased by the treatment with cholera toxin. In current-clamped cells showing similar rates of firing, the second phase of enhanced action-potential frequency induced by TRH was also significantly potentiated by cholera toxin. Measurements of [Ca²⁺]_i oscillations associated with electrical activity, using video imaging with fura-2-loaded cells, demonstrated that cholera toxin treatment causes a clear reduction of spontaneous [Ca²⁺]_i oscillations. However, this did not prevent the stimulatory effect of TRH on oscillations due to the action potentials. In cholera-toxin-treated cells, the steady-state, voltage dependence of inactivation of the inward rectifier was shifted by nearly 20 mV to more negative values. These data suggest that the inwardly rectifying K⁺ current plays an important role in maintenance of the resting K⁺ conductance in GH₃ cells. Furthermore, the TRH-induced reductions on this current may be an important factor contributing to the increased cell excitability promoted by the neuropeptide.     

Ganglioside GM2 N-acetyl-ß-D-gafactosaminidase and asialo GM2 (GA2) N-acetyl-ß-D-galactosaminidase; studies in human skin fibroblasts

Ganglioside G_M2 and its asialo-derivative, G_A2 were radiolabeled in their N-acetyl-D-galactosaminyl moieties by oxidation with galactose oxidase and reduction with tritiated sodium borohydride. Specific activities of 6 × 10⁴ dpm/nmol (G_M2) and 1.8 × 10⁶ dpm/nmol (G_A2) were achieved. About 98% of the label was in N-acetyl-D-galactosamine. Using these substrates, an assay was developed for G_M2-N-acetyl-β-D-galactosaminidase (E.C.3.2.1.30) and G_A2-N-acetyl-β-D-galactosaminidase (E.C.3.2.1.30) activities in human cultured skin fibroblasts. The products of the G_M2 cleaving reaction were identified as N-acetylgalactosamine and ganglioside G_M3- Both G_M2 and G_A2 cleaving activities were stimulated about 5-fold by purified sodium taurocholate, and this stimulation was inhibited by neutral detergents, lipids and albumin at low concentrations. Addition of various salts, reducing agents and a protein activator factor from human liver of Li et al. (1973) did not stimulate G_M2-N-acetyl-β-D-galactosaminidase activity beyond that found with sodium taurocholate. Under optimal conditions, control fibroblast supernates cleaved ganglioside G_M2 at a rate of 3.7 nmol/mg protein/h compared to 1100 for G_A2-N-acetyl-β-D-galactosaminidase and 4700 for 4-methylumbelliferyl-N-acetyl-β-D-glucosaminidase. Supernates from two patients with Tay-Sachs disease had markedly reduced activity levels for G_M2-N-acetyl-β-D-galactosaminidase but not for the other two substrates. Supernates from two patients with Sandhoff's disease had reduced activities for all three substrates. A supernate from one patient with juvenile G_M2 gangliosidosis cleaved G_M2 at a somewhat faster rate than those from Tay-Sachs or Sandhoff's patients. Two healthy adult women with markedly reduced hexosaminidase A activities using 4MU-N-acetyl-β-D-glucosaminide as substrate had approximately half-normal activities using G_M2 as substrate. A patient with the Tay-Sachs phenotype but with a partial deficiency of hexosaminidase A using the 4-MU substrate had a profound deficiency using G_M2 as substrate. In such unusual hexosaminidase mutants, assays using G_M2 as substrate are better indicators of phenotype than those using synthetic substrates.     

BK channels in intact clonal rat pituitary cells are activated by physiological elevations of the cytosolic Ca2+ concentration at the normal resting potential

Activation of large conductance Ca²⁺-activated K⁺ channels (BK channels) in intact clonal rat pituitary cells (GH₄ cells) was investigated using the cell-attached patch-clamp configuration. This method prevents loss of intracellular factors which might influence channel activity. BK channels are generally considered to be inactive at the resting membrane potential in excitable cells. However, at the resting potential (0 mV pipette potential), 40% of the cell-attached patches displayed spontaneously active BK channels, which remained active even at 20 mV hyperpolarization. The peptide thyroliberin (TRH) elevates the cytosolic Ca²⁺ concentration ([ Ca²⁺]_i) in GH cells by IP₃-induced release of Ca²⁺ from intracellular stores. This rise in [Ca²⁺]_i occurs concomitantly with membrane hyperpolarization. TRH stimulation caused activation of BK channels in nine out of 30 silent cell-attached patches, and caused enhanced channel activity in seven out of 29 cell-attached patches containing spontaneously active BK channels. The Ca²⁺ ionophore ionomycin activated silent BK channels in three out of 10 cell-attached patches, and increased the activity of spontaneously active BK channels in seven out of 16 cell-attached patches. The pipette potential was clamped to 0 mV in all these experiments. We conclude that the BK channels in GH₄ cells may be active at the resting membrane potential and more negative membrane potentials. The channels may also be activated further by physiological elevations of [Ca²⁺]_i in the same potential range. Our results point towards new possible physiological roles for the BK channels in GH₄ cells. This is in agreement with the emerging picture of BK channels highly sensitive to [Ca²⁺]_i in a wide variety of cell types.     

Glucose stimulates the entry of Ca2+ into the insulin-producing β cells but not into the glucagon-producing α2 cells

Rat pancreatic β and α₂ cells were purified by autofluorescence-activated cell sorting and used for electrophysiological patch clamp studies and measurements of the initial uptake of ⁴⁵Ca. Both β and α₂ cells were electrically active, the action potentials of the latter cells also were detected in the absence of glucose. Furthermore, α₂ cells differed from β cells in lacking a glucose-sensitive K⁺ channel with a single channel conductance of 50–60 pS (in symmetric 140 mm K⁺ solutions). The rate of Ca²⁺ entry into the α₂ cells was slower than that into the β cells, being equivalent to 0.2 mmol, kg^-1 dry wt min^-1. Whereas raising the glucose concentration to 20 mm significantly increased the amount of Ca²⁺entering the β cells, the sugar was without effect on Ca²⁺ entry into the α₂ cells.