Biomedical subjects

G M Preston

Publications and source records attributed to G M Preston.

At least 19 recordsLinked to original sources

PubMed1995

Molecular cloning and characterization of AqpZ, a water channel from Escherichia coli.

The aquaporin family of molecular water channels is widely expressed throughout the plant and animal kingdoms. No bacterial aquaporins are known; however, sequence-related bacterial genes have been identified that encode glycerol facilitators (glpF). By homology cloning, a novel aquaporin-related DNA (aqpZ) was identified that contained no surface N-glycosylation consensus. The aqpZ RNA was not identified in mammalian mRNA by Northern analysis and exhibited bacterial codon usage preferences. Southern analysis failed to demonstrate aqpZ in mammalian genomic DNA, whereas a strongly reactive DNA was present in chromosomal DNA from Escherichia coli and other bacterial species and did not correspond to glpF. The aqpZ DNA isolated from E. coli contained a 693-base pair open reading frame encoding a polypeptide 28-38% identical to known aquaporins. When compared with other aquaporins, aqpZ encodes a 10-residue insert preceding exofacial loop C, truncated NH2 and COOH termini, and no cysteines at known mercury-sensitive sites. Expression of aqpZ cRNA conferred Xenopus oocytes with a 15-fold increase in osmotic water permeability, which was maximal after 5 days of expression, was not inhibited with HgCl2, exhibited a low activation energy (Ea = 3.8 kcal/mol), and failed to transport nonionic solutes such as urea and glycerol. In contrast, oocytes expressing glpF transported glycerol but exhibited limited osmotic water permeability. Phylogenetic comparison of aquaporins and homologs revealed a large separation between aqpZ and glpF, consistent with an ancient gene divergence.

Amino Acid Sequence↗

PubMed1995

Water channel properties of major intrinsic protein of lens.

The functions of major intrinsic protein (MIP) of lens are still unresolved; however the sequence homology with channel-forming integral membrane protein (CHIP) and other Aquaporins suggests that MIP is a water channel. Immunolocalizations confirmed that Xenopus oocytes injected with bovine MIP cRNA express the protein and target it to the plasma membrane. Control oocytes or oocytes expressing MIP or CHIP exhibited small, equivalent membrane currents that could be reversibly increased by osmotic swelling. When compared with water-injected control oocytes, the coefficient of osmotic water permeability (Pf) of MIP oocytes was increased 4-5-fold with a low Arrhenius activation energy, while the Pf of CHIP oocytes increased > 30-fold. To identify structures responsible for these differences in Pf, recombinant MIP proteins were expressed. Analysis of MIP-CHIP chimeric proteins revealed that the 4-kDa cytoplasmic domain of MIP did not behave as a negative regulator. Individual residues in MIP were replaced by residues conserved among the Aquaporins, and introduction of a proline in the 5th transmembrane domain of MIP raised the Pf by 50%. Thus oocytes expressing MIP failed to exhibit ion channel activity and consistently exhibited water transport by a facilitated pathway that was qualitatively similar to the Aquaporins but of lesser magnitude. We conclude that MIP functions as an Aquaporin in lens, but the protein may also have other essential functions.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues.

The Aquaporin family of water channels plays a fundamental role in transmembrane water movements in numerous plant and animal tissues. Since the molecular pathway by which water is secreted by salivary glands is unknown, a cDNA was isolated from rat submandibular gland by homology cloning. Similar to other Aquaporins, the salivary cDNA encodes a 265-residue polypeptide with six putative transmembrane domains separated by five connecting loops (A-E); the NH2- and COOH-terminal halves of the polypeptide are sequence-related, and each contains the motif Asn-Pro-Ala. A mercurial-inhibition site is present in extracellular loop E, and cytoplasmic loop D contains a cAMP-protein kinase phosphorylation consensus. In vitro translation yielded a 27-kDa polypeptide, and expression of the cRNA in Xenopus oocytes conferred a 20-fold increase in osmotic water permeability (Pf) which was reversibly inhibited by 1 mM HgCl2. Northern analysis demonstrated a 1.6-kilobase mRNA in submandibular, parotid, and sublingual salivary glands, lacrimal gland, eye, trachea, and lung. In situ hybridization revealed a strong hybridization over the corneal epithelium in eye and over the secretory lobules in salivary glands. These studies have identified a new mammalian member of the Aquaporin water channel family (gene symbol AQP5) which is implicated in the generation of saliva, tears, and pulmonary secretions.

Amino Acid Sequence↗

PubMed1995

High water permeability of human spermatozoa is mercury-resistant and not mediated by CHIP28.

A novel integral membrane protein with an apparent molecular mass of 28 kDa (CHIP28) was first isolated from human erythrocytes and is now recognized as a water channel protein. The expression of this protein has been found in several other cell types that all require high water permeability for their functions. Recent studies have shown that the water permeability (Lp) of human spermatozoa is among the highest reported for mammalian cells. Together with the low activation energy of human spermatozoa for Lp, this suggests that CHIP28 water channel may be present in the plasma membrane of human spermatozoa. However, our current studies do not support this hypothesis. Results from Western blot analysis on human sperm plasma membrane proteins, performed through use of an antibody against human erythrocyte CHIP28 protein, indicated that human spermatozoa do not express CHIP28 protein on their cell surface (n = 10). Consistent with the Western blot finding, mercuric chloride (HgCl2), a known water channel blocker, failed to reduce the osmotic water permeability of human spermatozoa. The calculated Lp values were 1.30 +/- 0.29 micron/min/atm (n = 16; mean +/- SEM) for the control group and 1.31 +/- 0.29 (n = 9; mean +/- SEM), 1.04 +/- 0.27 (n = 11; mean +/- SEM), and 1.34 +/- 0.19 (n = 6; mean +/- SEM), respectively, for the 10 microM, 30 microM, and 50 microM HgCl2-treated groups. These Lp values are not different (p > 0.05). In contrast, the same concentration of HgCl2 significantly blocked the osmotic water transport across the membrane of human erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aquaporin 1↗

PubMed1994

Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance.

The aquaporins transport water through membranes of numerous tissues, but the molecular mechanisms for sensing changes in extracellular osmolality and regulating water balance in brain are unknown. We have isolated a brain aquaporin by homology cloning. Like aquaporin 1 (AQP1, also known as CHIP, channel-forming integral membrane protein of 28 kDa), the deduced polypeptide has six putative transmembrane domains but lacks cysteines at the known mercury-sensitive sites. Two initiation sites were identified encoding polypeptides of 301 and 323 amino acids; expression of each in Xenopus oocytes conferred a 20-fold increase in osmotic water permeability not blocked by 1 mM HgCl2, even after substitution of cysteine at the predicted mercury-sensitive site. Northern analysis and RNase protection demonstrated the mRNA to be abundant in mature rat brain but only weakly detectable in eye, kidney, intestine, and lung. In situ hybridization of brain localized the mRNA to ependymal cells lining the aqueduct, glial cells forming the edge of the cerebral cortex and brainstem, vasopressin-secretory neurons in supraoptic and paraventricular nuclei of hypothalamus, and Purkinje cells of cerebellum. Its distinctive expression pattern implicates this fourth mammalian member of the aquaporin water channel family (designated gene symbol, AQP4) as the osmoreceptor which regulates body water balance and mediates water flow within the central nervous system.

Amino Acid Sequence↗

PubMed1994

Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels.

The gene aquaporin-1 encodes channel-forming integral protein (CHIP), a member of a large family of water transporters found throughout nature. Three rare individuals were identified who do not express CHIP-associated Colton blood group antigens and whose red cells exhibit low osmotic water permeabilities. Genomic DNA analyses demonstrated that two individuals were homozygous for different nonsense mutations (exon deletion or frameshift), and the third had a missense mutation encoding a nonfunctioning CHIP molecule. Surprisingly, none of the three suffers any apparent clinical consequence, which raises questions about the physiological importance of CHIP and implies that other mechanisms may compensate for its absence.

Animals↗

PubMed1994

Molecular structure of the water channel through aquaporin CHIP. The hourglass model.

Aquaporin channel-forming integral protein (CHIP) is the first characterized water channel protein (genome symbol AQP1), but the molecular structure of the aqueous pathway through CHIP remains undefined. The two halves of CHIP are sequence-related, and each has three bilayer-spanning domains with the motif asparagine-proline-alanine (NPA) at residues 76-78 (in cytoplasmic loop B) and 192-194 (in extracellular loop E). The NPA motifs are oriented 180 degrees to each other, and the second NPA is near cysteine 189, the known site where mercurials inhibit osmotic water permeability (Pf). When expressed in Xenopus oocytes, the double mutant A73C/C189S exhibited high, mercurial-sensitive Pf similar to wild-type CHIP. Conservative substitutions of slightly greater mass in or near NPA motifs in loop B or loop E in CHIP caused reduced Pf and failure of the protein to localize at the plasma membrane. Certain nonfunctional loop E mutants complemented the truncation mutant D237Z. Formation of mixed oligomers was demonstrated by velocity sedimentation, immunoprecipitation, and analysis of dimeric-CHIP polypeptides. Cellular distributions of individual mutants or complementing pairs of mutants were verified by plasma membrane isolation and confocal microscopy. An hourglass structural model is proposed in which a cytoplasmic chamber (loop B) connects within the membrane to an extracellular chamber (loop E) forming a single, narrow aqueous pathway through each of the CHIP subunits; subunit oligomerization may provide the vertical symmetry necessary for residence within the lipid bilayer.

Amino Acid Sequence↗

PubMed1994

Membrane topology of aquaporin CHIP. Analysis of functional epitope-scanning mutants by vectorial proteolysis.

CHIP is the archetypal member of the aquaporins, a widely expressed family of membrane water channels. The NH2- and COOH-terminal halves of CHIP are sequence-related, and hydropathy analysis predicted six membrane-spanning domains with five connecting loops (A-E). Here, we determined the membrane topology of CHIP expressed in Xenopus oocytes using biologically active recombinant channels. CHIP is glycosylated at Asn-42, indicating loop A is exofacial. An epitope from the coronavirus E1 glycoprotein was inserted into CHIP and localized to the outer or inner leaflet of the membrane by alpha-chymotrypsin digestion of intact oocytes or inside-out membrane vesicles. The E1 epitope at Thr-120 was protease-sensitive in intact oocytes, indicating that loop C is exofacial. The E1 epitope at Lys-6, Arg-162, or Lys-267 was protease-sensitive in inside-out membrane vesicles, confirming the cytoplasmic location of the NH2 and COOH termini and loop D. Insertions into loops B and E did not produce active water channels, but their cleavage patterns were consistent with inner (loop B) and outer (loop E) leaflet locations. This study indicates that the functional CHIP molecule is a unique structure with two internal repeats oriented 180 degrees to each other within the membrane.

Amino Acid Sequence↗

PubMed1994

Human red cell aquaporin CHIP. I. Molecular characterization of ABH and Colton blood group antigens.

Blood group antigens are structural variants in surface carbohydrate or amino acid polymorphisms on extracellular domains of membrane proteins. The red cell water channel-forming integral protein (Aquaporin CHIP) is a homotetramer with only one N-glycosylated subunit, however no CHIP-associated blood group antigens have yet been identified. Immunoblotting, monosaccharide composition analysis, and selective glycosidase digestions revealed that the CHIP-associated oligosaccharide contains ABH determinants and resembles a band 3-type glycan that cannot be cleaved from intact membranes by Peptide:N-glycosidase F. The molecular structure of the Colton antigens was previously unknown, but CHIP was selectively immunoprecipitated with anti-Coa or anti-Co(b). The DNA sequence from Colton-typed individuals predicted that residue 45 is alanine in the Co(a+b-) phenotype and valine in the Co(a-b+) phenotype. The nucleotide polymorphism corresponds to a PflMI endonuclease digestion site in the DNA from Co(a-b+) individuals. These studies have defined antigens within two blood group systems on CHIP: (a) an ABH-bearing polylactosaminoglycan attached to a poorly accessible site in the native membrane; and (b) the Colton antigen polymorphism which may permit the identification of rare individuals with defective water channel expression.

ABO Blood-Group System↗

PubMed1994

Human red cell Aquaporin CHIP. II. Expression during normal fetal development and in a novel form of congenital dyserythropoietic anemia.

Channel-forming integral protein (CHIP) is the archetypal member of the Aquaporin family of water channels. Delayed CHIP expression was shown recently in perinatal rat (Smith, B. L., R. Baumgarten, S. Nielsen, D. Raben, M. L. Zeidel, and P. Agre. 1993. J. Clin. Invest. 92:2035-2041); here we delineate the human patterns. Compared with adult, second and third trimester human fetal red cells had lower CHIP/spectrin ratios (0.72 +/- 0.12, 0.94 +/- 0.22 vs 1.18 +/- 0.11) and reduced osmotic water permeability (0.029, 0.026 vs 0.037 cm/s); CHIP was already present in human renal tubules by the second trimester. A patient with a novel form of congenital dyserythropoietic anemia (CDA) with persistent embryonic and fetal globins and absent red cell CD44 protein was studied because of reduced CHIP-associated Colton antigens. Novel CDA red cells contained < 10% of the normal level of CHIP and had remarkably low osmotic water permeability (< 0.01 cm/s), but no mutation was identified in Aquaporin-1, the gene encoding CHIP. These studies demonstrate: (a) unlike rat, human CHIP expression occurs early in fetal development; (b) red cell water channels are greatly reduced in a rare phenotype; and (c) disrupted expression of red cell CHIP and CD44 suggests an approach to the molecular defect in a novel form of CDA.

Adult↗

PubMed1993

The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel.

Water channels provide the plasma membranes of red cells and renal proximal tubules with high permeability to water, thereby permitting water to move in the direction of an osmotic gradient. Molecular identification of CHIP28 protein as the membrane water channel was first accomplished by measurement of osmotic swelling of Xenopus oocytes injected with CHIP28 RNA (Preston, G.M., Carroll, T.P., Guggino, W.B., and Agre, P. (1992) Science 256, 385-387). Since water channels are pharmacologically inhibited by submillimolar concentrations of Hg2+, site-directed mutagenesis was undertaken to demonstrate which of the 4 cysteines (87, 102, 152, or 189) is the Hg(2+)-sensitive residue in the CHIP28 molecule. Each cysteine was individually replaced by serine, and oocytes expressing each of the four mutants exhibited osmotic water permeability (Pf) equivalent to wild-type CHIP28. After incubation in HgCl2, all were significantly inhibited, except C189S exists as a multisubunit complex in the native membrane; however, although oocytes injected with mixed CHIP28 and C189S RNAs exhibited Pf corresponding to the sum of their individual activities, exposure to Hg2+ only reduced the Pf to the level of the C189S mutant. Of the six substitutions at residue 189, only the serine and alanine mutants exhibited increased Pf and had glycosylation patterns resembling wild-type CHIP28 on immunoblots. These studies demonstrated: (i) CHIP28 water channel activity is retained despite substitution of individual cysteines with serine; (ii) cysteine 189 is the Hg(2+)-sensitive residue; (iii) the subunits of the CHIP28 complex are individually active water pores; (iv) residue 189 is critical to proper processing of the CHIP28 protein.

Amino Acid Sequence↗

PubMed1993

Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney.

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.

4-Chloromercuribenzenesulfonate↗

PubMed1993

Cultured bovine corneal endothelial cells express CHIP28 water channels.

Cultured bovine corneal endothelial cells (CBCEC) transport fluid from the basal to the apical surface. In this study, we examined whether the plasma membranes of these cells have water channels. We cultured BCEC on glass plates and monitored the intensity of the light scattered (IS) by the cells. We determined the kinetic constant (k) of the change in IS on exposure to a 10% hypoosmotic challenge to calculate the osmotic permeability (Pf) of the plasma membrane. At 37 degrees C, we found values of k = 0.68 +/- 0.07 s-1 and Pf = 93.3 +/- 33 microns/S (n = 13). The sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS; 1 mM) reduced Pf by 75%; 5 mM dithiothreitol reversed such inhibition. The activation energy (Ea) of Pf in the range 10-37 degrees C was 4.7 +/- 0.7 kcal/mol (n = 5). The high Pf, values, the inhibition by pCMBS, and the low Ea strongly suggest the presence of water channels. Therefore, we tested whether the injection of poly(A)+ RNA prepared from CBCEC into Xenopus laevis oocytes results in the expression of water channels. Four days after injection, we measured oocyte Pf values from the rate of volume increase on exposure to hypoosmotic medium. In control oocytes injected with 50 nl of water, Pf was 13.4 +/- 0.3 microns/S (n = 63). In oocytes injected with poly(A)+ RNA (50 ng/oocyte in 50 nl water), Pf was 40.9 +/- 1.6 microns/S (n = 72).(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloromercuribenzenesulfonate↗

PubMed1993

Aquaporin CHIP: the archetypal molecular water channel.

Despite longstanding interest by nephrologists and physiologists, the molecular identities of membrane water channels remained elusive until recognition of CHIP, a 28-kDa channel-forming integral membrane protein from human red blood cells originally referred to as "CHIP28." CHIP functions as an osmotically driven, water-selective pore; 1) expression of CHIP conferred Xenopus oocytes with markedly increased osmotic water permeability but did not allow transmembrane passage of ions or other small molecules; 2) reconstitution of highly purified CHIP into proteoliposomes permitted determination of the unit water permeability, i.e., 3.9 x 10(9) water molecules.channel subunit-1 x s-1. Although CHIP exists as a homotetramer in the native red blood cell membrane, site-directed mutagenesis studies suggested that each subunit contains an individually functional pore that may be reversibly occluded by mercurial inhibitors reacting with cysteine-189. CHIP is a major component of both apical and basolateral membranes of water-permeable segments of the nephron, where it facilitates transcellular water flow during reabsorption of glomerular filtrate. CHIP is also abundant in certain other absorptive or secretory epithelia, including choroid plexus, ciliary body of the eye, hepatobiliary ductules, gall bladder, and capillary endothelia. Distinct patterns of CHIP expression occur at these sites during fetal development and maturity. Similar proteins from other mammalian tissues and plants were later shown to transport water, and the group is now referred to as the "aquaporins." Recognition of CHIP has provided molecular insight into the biological phenomenon of osmotic water movement, and it is hoped that pharmacological modulation of CHIP function may provide novel treatments of renal failure and other clinical problems.

Amino Acid Sequence↗

PubMed1992

Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein.

Water rapidly crosses the plasma membrane of red blood cells (RBCs) and renal tubules through specialized channels. Although selective for water, the molecular structure of these channels is unknown. The CHIP28 protein is an abundant integral membrane protein in mammalian RBCs and renal proximal tubules and belongs to a family of membrane proteins with unknown functions. Oocytes from Xenopus laevis microinjected with in vitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability; this was reversibly inhibited by mercuric chloride, a known inhibitor of water channels. Therefore it is likely that CHIP28 is a functional unit of membrane water channels.

Animals↗

PubMed1991

Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family.

CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5' untranslated nucleotide sequence, an 807-bp open reading frame, and approximately 2 kilobases of 3' untranslated sequence containing a polyadenylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DNA sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.

Amino Acid Sequence↗

PubMed1990

Effects of the DNA topoisomerase II inhibitor, VM26, on transcriptional initiation in vitro.

Inhibitors of DNA topoisomerase II have been shown to influence the expression of specific genes in vivo, leading investigators to suggest that the topological state of certain genes may affect their expression. In the present study, we have examined the effects of the topoisomerase II inhibitor, VM26, on transcriptional initiation in vitro. VM26 inhibited transcriptional initiation from the rat prolactin and adenovirus major late promoters in an in vitro transcription assay using rat pituitary tumor GH3 cell nuclear extracts. Transcriptional initiation from both supercoiled and linear DNA templates was equally inhibited by VM26 in a dose dependent manner. In order to identify sequences around the prolactin promoter recognized by topoisomerase II, we have mapped topoisomerase II cleavage sites using GH3 cell nuclear extracts as a source for the endogenous enzyme. Using a 910 bp genomic DNA fragment containing 423 bp of 5'-flanking sequences from the prolactin gene, we observed five VM26-enhanced topoisomerase II cleavage sites. Some of these sites map to positions near or within previously described regulatory DNA elements, and thus, may provide the structural basis for the sensitivity of prolactin gene expression to topoisomerase II inhibitors in vivo. These data suggest that inhibition of transcriptional initiation was a consequence of VM26 induced cleavage in the promoter region.

Animals↗

PubMed1990

Transcriptional and posttranscriptional regulation of the rat prolactin gene by calcium.

The rat prolactin gene is expressed at a high basal level in the pituitary tumor GH3 cell line. Culturing GH3 cells in a low-Ca2+, serum-free medium (SFM) depresses prolactin mRNA levels, and subsequent addition of Ca2+ to the SFM results in a specific, gradual, and sustained increase in prolactin mRNA levels. We have now examined whether the observed increase in prolactin mRNA levels can be attributed solely to an increase in the transcriptional rate of the prolactin gene. Treatment of GH3 cells in SFM with 0.4 mM CaCl2 for 24 to 48 h increased cytoplasmic prolactin mRNA levels by 5- to 10-fold, whereas the transcriptional rate of the prolactin gene was increased by less than twofold over values for SFM controls. Prolactin mRNA levels increased progressively during the 24-h period after Ca2+ addition, whereas prolactin gene transcription never exceeded a twofold increase over values for SFM controls. The activities of nuclear extracts from control and Ca2(+)-induced cells were examined in an in vitro transcription assay. The two extracts directed transcription from the prolactin promoter and the adenovirus major late promoter equally well. Cycloheximide had no effect on the ability of Ca2+ to increase or maintain prolactin mRNA levels. In dactinomycin mRNA clearance experiments, prolactin mRNA was cleared at the same rate in the absence and presence of Ca2+. These results demonstrate that although Ca2+ has a small effect on the transcriptional rate of the prolactin gene, Ca2+ produces a significant increase in prolactin mRNA levels by acting at a posttranscriptional site(s). Furthermore, Ca(2+) appears to increase prolactin mRNA levels by posttranslational modification of a stable protein, probably at a nuclear site.

Animals↗