Constitutive expression of functional GABA(B) receptors in mIL-tsA58 cells requires both GABA(B(1)) and GABA(B(2)) genes.

Studies of gamma-aminobutyric acid (GABA)(B) receptor function in heterologous cell systems have suggested that expression of two distinct seven transmembrane G-protein coupled receptor subunits is necessary for receptor activation and signal transduction. Some results suggest that both receptor proteins must be inserted into the plasma membrane to create heterodimers; however, it is possible that subunit monomers or homodimers are functional in cells which constitutively express GABA(B) receptors. A new pituitary intermediate lobe melanotrope cell clone (mIL tsA58) has been isolated which constitutively expresses GABA(B), D(2) and corticotrophin releasing factor receptors. Here, we report on characterization of the GABA(B) receptors. Solution hybridization-nuclease protection assays reveal the presence of GABA(B(1)) and GABA(B(2)) transcripts. Western blots show GABA(B(1a)) and one of two GABA(B(2)) proteins. Addition of the GABA(B) agonist baclofen to cultured mIL-tsA58 (mIL) cells inhibits high voltage activated Ca(2+) channels, as measured by agonist-induced inhibition of the K(+)-depolarization-stimulated increase in Ca(2+) influx. CGP55845, a GABA(B) antagonist, blocks the response to baclofen. Knockdown of either GABA(B(1)) or GABA(B(2)) subunits with selective antisense oligodeoxynucleotides reduced GABA(B) protein levels and completely abolished the GABA(B) receptor response in the mIL cells. Taken together, these results indicate that functionally active GABA(B) receptors in mIL cells require the constitutive expression of both GABA(B) genes. This is a physiologic validation of results from recombinant overexpression in naive cells and shows that the mIL cell line is a useful model for studying GABA(B) receptor expression, regulation and function.

Dopamine D2-receptor isoforms expressed in AtT20 cells inhibit Q-type high-voltage-activated Ca2+ channels via a membrane-delimited pathway.

Dopamine D2 receptors both acutely and chronically inhibit high-voltage-activated Ca2+ channels (HVA-CCs). Two alternatively spliced isoforms, D2L (long) and D2S (short), are expressed at high levels in rat pituitary intermediate lobe melanotropes but are lacking in anterior lobe corticotropes. We stably transfected D2L and D2S into corticotrope-derived AtT20 cells. Both isoforms coupled to inhibition of Q-type calcium channels through pertussis toxin-sensitive G proteins. Thus, we have created a model system in which to study the kinetics of D2-receptor regulation of Ca2+ channels. Rapid inhibition of HVA-CCs was characterized using a novel fluorescence video imaging technique for the measurement of millisecond kinetic events. We measured the time elapsed (lag time) between the arrival of depolarizing isotonic 66 mM K+, sensed by fluorescence from included carboxy-X-rhodamine (CXR), and the beginning of increased intracellular Ca2+ levels (sensed by changes in indo 1 fluorescence ratio). The lag time averaged 350-550 ms, with no significant differences among cell types. Addition of the D2-agonist quinpirole (250 microM) to the K+/CXR solution significantly increased the lag times for D2-expressing cells but did not alter the lag time for AtT20 controls. The increased lag times for D2L- and D2S-transfected cells suggest that at least a fraction of the Ca2+ channels was inhibited within the initial 350-550 ms. As this inhibition time is too fast for a multistep second messenger pathway, we conclude that inhibition occurs via a membrane-delimited diffusion mechanism.

GABA(B)R1a/R1b-type receptor antisense deoxynucleotide treatment of melanotropes blocks chronic GABA(B) receptor inhibition of high voltage-activated Ca2+ channels.

GABA(B) and dopamine D2 receptors, both of which acutely inhibit adenylyl cyclase and high voltage-activated Ca2+ channels (HVA-CCs), are found in high levels in the melanotrope cells of the pituitary intermediate lobe. Chronic D2 receptor agonist application in vitro has been reported to result in inhibition of HVA-CC activity by down-regulation. Here we report that chronic GABA(B), but not GABA(A), agonist treatment also resulted in HVA-CC inhibition. Two GABA(B) receptor variants have been cloned and shown to inhibit adenylyl cyclase in HEK-293 cells. We have constructed an antisense deoxynucleotide knockdown-type probe that is complementary to 18 bp from the point at which the two sequences first become homologous. Chronic coincubation with baclofen and GABA(B) antisense nucleotide completely eliminated the inhibition of the channels by baclofen alone but had no reversing effect on HVA-CC inhibition by the D2 agonist quinpirole. A scrambled, missense nucleotide also had no reversing effect. Incubation with a D2 antisense knockdown probe eliminated the ability of a D2 agonist to inhibit the channels but had no effect on baclofen blockade. These results show the existence an R1a/R1b type of GABA(B) receptor, which, like the D2 receptor, is coupled to chronic HVA-CC inhibition in melanotropes.

Amyloid beta-peptide(25-35) changes [Ca2+] in hippocampal neurons.

Insoluble aggregates of the amyloid beta-peptide (A beta) is a major constituent of senile plaques found in brains of Alzheimer disease (AD) patients. The detrimental effects of aggregated A beta is associated with an increased intracellular Ca2+ concentration ([Ca2+]i). We examined the effects of A beta(25-35) on [Ca2+]i and intracellular H+ concentration ([H+]i) in single hippocampal neurons by real time fluorescence imaging using the Ca(2+)- and H(+)-specific ratio dyes, indo-1 and SNARF-1. Incubation of these cultures with A beta(25-35) for 3-12 days in vitro increased [Ca2+]i and [H+]i in large, NMDA-responsive neurons.

Heterogeneity in POMC expression among explanted melanotropes decreases with time in culture and bromocriptine treatment.

The biosynthetic activity of rat intermediate lobe melanotropes in vivo is inhibited by stimulation of dopamine D2 receptors. Individual melanotropes are innervated differentially by dopaminergic axons and vary in their levels of pro-opiomelanocortin (POMC) mRNA. We tested the hypothesis that placement of the lobe in primary culture, which removes the inhibitory innervation, would increase POMC mRNA levels and abolish the heterogeneity in POMC expression. POMC mRNA levels increased successively in untreated melanotropes when tested on culture Days 10, 16, and 20; however, some heterogeneity in POMC expression persisted. If treated with a D2 receptor agonist (1 microM bromocriptine) from culture Day 1, POMC mRNA levels were decreased significantly throughout the testing period when compared to untreated cells with the same time in culture. Although some melanotropes still expressed high POMC levels, preparations appeared more homogeneous by Day 20. Melanotrope responses were reversible, since POMC mRNA levels were down-regulated by application and up-regulated by withdrawal of a D2 receptor agonist. A short agonist treatment resulted in subpopulations that responded differently to the agonist, possibly representing a mechanism for fine-tuning peptide hormone release.

Types and activities of voltage-operated calcium channels change during development of rat pituitary neurointermediate lobe.

Cultures of pituitary neurointermediate lobe cells were established from rats aged 1, 12, and 42 days to identify the types and assess the activities of Ca2+ channels present in melanotropes, glial-like cells, and fibroblasts during development. Day 12 represents the time at which dopaminergic axons have become distributed throughout the lobe, glial cells begin to lose their radial orientation, and melanotropes robustly express the short isoform of the dopamine D2 receptor. Thus, we studied Ca2+ channels in relation to the event of innervation of melanotropes. Real-time fluorescence video microscopy, in the presence of pharmacological agents, which block L-, N-, P-, and T-type channels, was used as an indirect measurement of channel activity. Assessment of cell type was verified by triple-label fluorescence immunohistochemistry. In melanotropes, extracellular Ca2+ addition caused Ca2+ influx through omega-conotoxin GVIA-sensitive, N-type channels on days 1 and 12 but not on day 42. The K+ depolarization induced an increase in intracellular Ca2+ concentration in all age-groups. This effect was decreased by nifedipine, an L-type channel blocker, at all ages, and by omega-agatoxin IVa, a P-type blocker, only on day 42. These results demonstrate that the predominance of N- or P-type channels on melanotropes is age-dependent and can be correlated with other developmental changes. The T-type blocker, NiSO4, had no effect. In glial-like cells of all ages, extracellular Ca2+ addition resulted in an increase in intracellular Ca2+ concentration, which was inhibited only by NiSO4. The percentage of responsive glial-like cells was equally high in days 1 and 12 cultures, then declined by day 42. The K+ depolarization had no effect on glial-like cells. Fibroblasts did not respond significantly to extracellular Ca2+ or K+ depolarization, indicating little detectable activity by this methodology from functional voltage-operated Ca2+ channels.

Dopamine D2 receptors regulate in vitro melanotrope L-type Ca2+ channel activity via c-fos.

Dopamine D2 receptor stimulation of cultured primary melanotropes was found to depress L-type calcium channel activity, whereas D2 receptor antagonist application increased it. When tested on culture days 10, 16, and 20, control cells displayed increasing rises of intracellular Ca2+ in response to K+ depolarization, indicating an increase in channel activity in the absence of dopaminergic regulation. When treated with 1 microM bromocriptine from culture day 1, cells showed minimal increase in channel activity. When bromocriptine was added on day 16, intracellular Ca2+ response to high K+ declined by day 20; removal of the agonist on day 16 resulted in the reappearance of increased responsiveness. Thus, in vitro inhibitions could be initiated or reversed with application or withdrawal of dopamine D2 receptor agonist. Cultured melanotropes were treated with antisense oligodeoxynucleotides directed against the start sequences of the D2 receptor and c-fos messenger RNA. D2 receptor antisense nucleotide prevented the depressive effect on channel activity induced by D2 agonist treatment. c-fos antisense oligodeoxynucleotide blocked the rise in channel activity. The dopamine D2 receptor antagonist haloperidol, which increased channel activity, could not reverse the c-fos antisense deoxynucleotide block. These results strongly support the idea that the chronic suppression of secretion-related activities by dopaminergic stimulation seen in the intermediate lobe in vivo is effected by chronic suppression of c-fos by D2 receptors.

Simultaneous imaging of intracellular [Ca2+] and pH in single MDCK and glomerular epithelial cells.

The interrelationships between changes in intracellular calcium concentration ([Ca2+]i) and intracellular pH in Madin-Darby canine kidney cells and kidney glomerular epithelial cells exposed to various stimuli were analyzed simultaneously using a new design of a fluorescence video microscope. Cells were double labeled with indo 1 and SNARF 1 dyes and were excited simultaneously at 350 and 540 nm. Images at four emission wavelengths were captured simultaneously at 405, 475, 575, and 640 nm at 30 frames/s for the two ratio dyes. SNARF sensitivity to pH between 6.5 and 8.0 was unchanged by [Ca2+]i. The SNARF ratio maps were used to correct the pH-dependent changes in the calculation of local cell calcium. NH4Cl loading produced the expected alkalinization and a concurrent rise in [Ca2+]i. When the NH4Cl was removed and the cells became acidic, a second rise in [Ca2+]i was recorded. Both changes in [Ca2+]i were from intracellular stores since they persisted in the absence of extracellular calcium. The findings demonstrate the need for pH correction of indo 1 recordings.

Calcium regulation of intracellular pH in pituitary intermediate lobe melanotropes.

The regulatory activities of both intracellular calcium ([Ca2+]i) and intracellular pH (pHi) have greatly increased interest in the study of their interdependence. We have designed an epifluorescence video microscope that will image the fluorescence from two ratio dyes, indo-1 (for [Ca2+]i) and SNARF-1 (for pHi) at video rates. We examined primary cultures of pituitary intermediate lobe melanotropes loaded with both dyes. After experimentation, cells were positively identified by fluorescence immunohistochemistry. K(+)-induced depolarization of melanotropes produced increases in [Ca2+]i due to activation of L-type Ca channels. A secondary Ca2+ peak or oscillations were often seen. After treatment with carbonyl cyanide m-chlorophenylhydrozone, depolarization produced a rise in intracellular [Ca2+]i as well as oscillations. After thapsigargin or cyclopiazonic acid treatment, depolarization produced a primary Ca2+ elevation, but the secondary Ca2+ changes disappeared. This suggests that the oscillations were due to Ca2+ release from an endoplasmic reticulum type of intracellular store. All of these increases in [Ca2+]i were also directly coupled to a rise in intracellular H+. The close association between intracellular Ca2+ and H+ suggests that the observed pHi changes were due to the release of H+ upon binding of Ca2+ to intracellular buffers. This direct obligate coupling of intracellular Ca2+ and H+ suggests the possibility that pH-dependent cellular processes are directly activated by sudden increases in intracellular Ca2+ levels. This second messenger type of signaling system would be activated whether the Ca2+ was released from intracellular stores or entered the cell via plasma membrane Ca2+ channels.

Calcium influx mediated by the Escherichia coli heat-stable enterotoxin B (STB).

The heat-stable enterotoxin B (STB) of Escherichia coli is a 48-amino acid extracellular peptide that induces rapid fluid accumulation in animal intestinal models. Unlike other E. coli enterotoxins that elicit cAMP or cGMP responses in the gut [heat-labile toxin (LT) and heat-stable toxin A (STA), respectively], STB induces fluid loss by an undefined mechanism that is independent of cyclic nucleotide elevation. Here we studied the effects of STB on intracellular calcium concentration ([Ca2+]i), another known mediator of intestinal ion and fluid movement. Ca2+ and pH measurements were performed on different cell types including Madin-Darby canine kidney (MDCK), HT-29/C1 intestinal epithelial cells, and primary rat pituitary cells. Ca2+ and pH determinations were performed by simultaneous real-time fluorescence imaging at four emission wavelengths. This allowed dual imaging of the Ca(2+)- and pH-specific ratio dyes (indo-1 and SNARF-1, respectively). STB treatment induced a dose-dependent increase in [Ca2+]i with virtually no effect on internal pH in all of the cell types tested. STB-mediated [Ca2+]i elevation was not inhibited by drugs that block voltage-gated Ca2+ channels including nitrendipine, verapamil (L-type), omega-conotoxin (N-type), and Ni2+ (T-type). The increase in [Ca2+]i was dependent on a source of extracellular Ca2+ and was not affected by prior treatment of MDCK cells with thapsigargin or cyclopiazonic acid, agents that deplete and block internal Ca2+ stores. In contrast to these results, somatostatin and pertussis toxin pretreatment of MDCK cells completely blocked the STB-induced increase in [Ca2+]i. Taken together, these data suggest that STB opens a GTP-binding regulatory protein-linked receptor-operated Ca2+ channel in the plasma membrane. The nature of the STB-sensitive Ca2+ channel is presently under investigation.