Proximal upstream flanking sequences direct calcium regulation of the rat prolactin gene

Previous investigations have shown that Ca2+ strongly and specifically stimulates endogenous PRL gene expression by GH3 cells. In this study, addition of Ca2+ to Ca2+-deprived GH3 cells yielded a large (ca. 8-fold) stimulation of transient expression of a transfected PRL-chloramphenical acetyltransferase (CAT) construct containing ca. 1 kilo-base-pair of the PRL promoter region, but only a slight (less than or equal to 2-fold) nonspecific stimulation of CAT activity directed by any of three control promoters: dihydrofolate reductase, Rous sarcoma virus, or thymidine kinase. In GH3 cells never deprived of Ca2+, expression of a PRL-CAT construct was specifically stimulated and inhibited, respectively, by the dihydropyridine voltage-dependent Ca2+ channel modulators Bay K8644 and nimodipine; Ca2+ can thus regulate expression of an exogenous PRL promoter in cells incubated under physiological Ca2+ conditions. By employing a combined protocol, in which Ca2+-deprived cells are exposed to Ca2+ in the presence of Bay K8644, a very large (greater than 35-fold) but still promoter-specific induction of expression of a PRL-CAT construct was obtained. Analysis of 5'-deleted PRL-CAT constructs implied that the PRL gene Ca2+ response element is contained entirely within the first 174 base pairs of upstream flanking DNA sequence.     

hKv4.3 channel characterization and regulation by calcium channel antagonists

Relative expression pattern of short and long isoforms of hKv4.3 channels was evaluated by RT-PCR and RPA. Electrophysiological studies were performed in HEK293 cells transfected with short or long hKv4.3 cDNA. The long variant L-hKv4.3 was the only form present in lung, pancreas, and small intestine. The short variant S-hKv4.3 was predominant in brain whereas expression levels of the two isoforms were similar in cardiac and skeletal muscles. Properties of the ionic channels encoded by L-hKv4.3 and S-hKv4.3 cDNAs were essentially similar. Cadmium chloride and verapamil inhibited hKv4.3 current (with EC50s of 0.110 +/- 0.004 mM and 492.9 +/- 15.1 microM, respectively). Verapamil also accelerated current inactivation. Another calcium channel antagonist nicardipine was found inactive. In conclusion, this study confirms that both isoforms underlie the transient outward potassium current. Moreover, calcium channel inhibitors markedly affect hKv4.3 current, an effect which must be considered when evaluating transient outward potassium channel properties in native tissues.     

Evidence for a role of calmodulin in the regulation of prolactin gene expression

Epidermal growth factor (EGF) stimulates prolactin (PRL) gene expression in GH3 cells in a Ca2+-dependent manner (White, B. A., and Bancroft, F. C. (1983) J. Biol. Chem. 258, 4618-4622). The present report shows that the phenothiazine, calmidazolium (compound R 24571), blocks the ability of EGF plus Ca2+ to increase levels of PRL mRNA. Calmidazolium inhibition of this response is dose dependent in the range of 0.05-1.00 microM. Total inhibition of the response was consistently obtained at a level of calmidazolium (0.5 microM) that had no effect on total cytoplasmic RNA synthesis, total cytoplasmic protein synthesis, cell viability, or extent of EGF plus Ca2+-induced cell aggregation. The drug inhibited the increase in PRL mRNA when given immediately before or 48 h after treatment with EGF plus Ca2+. Another calmodulin inhibitor, W13, similarly blocked the ability of EGF plus Ca2+ to stimulate PRL mRNA, whereas the less active analog, W12, had little effect. These results implicate Ca2+-binding proteins such as calmodulin in the mechanism of action of EGF in GH3 cells, and, therefore, provide further evidence for a role of intracellular Ca2+ in the regulation of the expression of a specific eukaryotic gene, the PRL gene.     

Thyrotropin releasing hormone receptors in regulation of prolactin gene expression in GH cells

Calf thymus DNA polymerase β and mammalian type C retroviral DNA polymerases are strongly inhibited by low concentrations (1–2mM) of inorganic phosphate (Pi). A detailed analysis of this phenomenon revealed that Pi-mediated inhibition: a) requires the presence of Mn²⁺ (Mg²⁺ neither supports nor relieves this inhibition; b) is strongly affected by the stoichiometric relationship between Mn²⁺ and Pi concentrations; c) is competitive with respect to deoxynucleoside triphosphate (dNTP) concentration, and d) increasing the concentration of substrate or non-substrate dNTPs in reaction mixtures raised the concentration of Mn²⁺ at which significant inhibition by a fixed concentration of Pi was first seen. These findings suggested that Mn²⁺, dNTPs, and Pi may interact in Pi-mediated inhibition. Thin-layer chromatographic analysis revealed the formation of an Mn-dNTP-Pi complex, while Mg²⁺ did not participate in such complex formation. We propose that it is this tripartite complex which is responsible for the Pi-mediated inhibition of sensitive DNA polymerases.     

Calcium signaling and synaptic modulation: regulation of endocannabinoid-mediated synaptic modulation by calcium

Postsynaptic Ca2+ signal influences synaptic transmission through multiple mechanisms. Some of them involve retrograde messengers that are released from postsynaptic neurons in a Ca2+-dependent manner and modulate transmitter release through activation of presynaptic receptors. Recent studies have revealed essential roles of endocannabinoids in retrograde modulation of synaptic transmission. Endocannabinoid release is induced by either postsynaptic Ca2+ elevation alone or activation of postsynaptic Gq/11-coupled receptors with or without Ca2+ elevation. The former pathway is independent of phospholipase Cbeta (PLCbeta) and requires a large Ca2+ elevation to a micromolar range. The latter pathway requires PLCbeta and is facilitated by a moderate Ca2+ elevation to a submicromolar range. This facilitation is caused by Ca2+-dependency of receptor-driven PLCbeta activation. The released endocannabinoids then activate presynaptic cannabinoid receptor type 1 (CB1), and suppress transmitter release from presynaptic terminals. Both CB1 receptors and Gq/11-coupled receptors are widely distributed in the brain. Thus, the endocannabinoid-mediated retrograde modulation may be an important and widespread mechanism in the brain, by which postsynaptic events including Gq/11-coupled receptor activation and Ca2+ elevation can retrogradely influence presynaptic function.     

Differential modulation of L-type calcium channel subunits by oleate

Nonesterified fatty acids such as oleate and palmitate acutely potentiate insulin secretion from pancreatic islets in a glucose-dependent manner. In addition, recent studies show that fatty acids elevate intracellular free Ca(2+) and increase voltage-gated Ca(2+) current in mouse beta-cells, although the mechanisms involved are poorly understood. Here we utilized a heterologous system to express subunit-defined voltage-dependent L-type Ca(2+) channels (LTCC) and demonstrate that beta-cell calcium may increase in part from an interaction between fatty acid and specific calcium channel subunits. Distinct functional LTCC were assembled in both COS-7 and HEK-293 cells by expressing either one of the EYFP-tagged L-type alpha(1)-subunits (beta-cell Cav1.3 or lung Cav1.2) and ERFP-tagged islet beta-subunits (ibeta(2a) or ibeta(3)). In COS-7 cells, elevations in intracellular Ca(2+) mediated by LTCC were enhanced by an oleate-BSA complex. To extend these findings, Ca(2+) current was measured in LTCC-expressing HEK-293 cells that revealed an increase in peak Ca(2+) current within 2 min after addition of the oleate complex, with maximal potentiation occurring at voltages <0 mV. Both Cav1.3 and Cav1.2 were modulated by oleate, and the presence of different auxiliary beta-subunits resulted in differential augmentation. The potentiating effect of oleate on Cav1.2 was abolished by the pretreatment of cells with triacsin C, suggesting that long-chain CoA synthesis is necessary for Ca(2+) channel modulation. These results show for the first time that two L-type Ca(2+) channels expressed in beta-cells (Cav1.3 and Cav1.2) appear to be targeted by nonesterified fatty acids. This effect may account in part for the acute potentiation of glucose-dependent insulin secretion by fatty acids.     

Cloning and tissue-specific expression of the brain calcium channel beta-subunit.

A cDNA clone encoding a protein with high homology to the beta-subunit of the rabbit skeletal muscle dihydropyridine-sensitive calcium channel was isolated from a rat brain cDNA library. This rat brain beta-subunit cDNA hybridizes to a 3.4 kb message that is expressed in high levels in the cerebral hemispheres and hippocampus but is significantly reduced in cerebellum. The open reading frame encodes 597 amino acids with a predicted mass of 65 679 Da which is 82% homologous with the skeletal muscle beta-subunit. The brain cDNA encodes a unique 153 amino acid C-terminus and predicts the absence of a muscle-specific 50 amino acid internal segment. It also encodes numerous consensus phosphorylation sites suggesting a role in calcium channel regulation. The corresponding human beta-subunit gene was localized to chromosome 17. Hence the encoded brain beta-subunit, which has a primary structure highly similar to its isoform in skeletal muscle, may have a comparable role as an integral regulatory component of a neuronal calcium channel.