Cloning,Expression, Pharmacology and Tissue Distribution of the Mouse Somatostatin Receptor Subtype 5

The gene encoding the mouse somatostatin receptor subtype 5 has been isolated from a genomic library and the mRNA start point mapped to position ?95 relative to the translational start codon. The promoter region is devoid of TATA and CAAT boxes but contains putative binding sites for AP-1, AP-2 and SP1 and response elements for glucocorticoids (GRE) and phorbol esters (TRE). The encoded receptor protein with a predicted molecular weight of 42.5 kDa is comprised of 385 amino acids and thus contains 22 and 21 amino acids more than rat and human counterparts. The extra amino acids are caused by another translational initiation codon located further upstream. In the region of overlap the mouse somatostatin receptor subtype 5 displays 96.7% sequence identity to the rat and 81.7% to the human homologue. Application of somatostatin-14 and ?28 to human embryonic kidney cells expressing the recombinant receptor resulted in the inhibition of forskolin-stimulated adenylyl cyclase with comparable EC₅₀ values. Consistent with the observed sequence relationship, the mouse somatostatin receptor subtype 5 displays a pharmacological profile that resembles the rat homologue more closely than the human counterpart. mRNA for the mouse somatostatin type 5 receptor has been detected in pituitary, kidney, spleen and ovary and, to a lesser extent, in brain, stomach, intestine and thymus but was not observed in heart, pancreas and liver.     

Sequences in the proximal 5′ flanking region of the rat neuron-specific enolase (NSE) gene are sufficient for cell type-specific reporter gene expression

We investigated the regulation of the rat neuron-specific enolase gene using a transient transfection approach. Recent transgenic mouse studies have shown that a 1.8-kb segment of the ratNSE gene 5′ flanking region, including the first (noncoding) exon but not the first intron, is able to drive expression of a reporter gene in parallel with endogenousNSE. These data suggest thatcis-acting elements responsible for the spatial and temporal pattern ofNSE gene expression are located within the proximal 1.8 kb of the 5′ flanking sequence. To further investigate this region, we joined the 1.8-kb regulatory cassette to thecat reporter gene and generated a number of constructs in which the flanking sequence was progressively deleted from the 5′ end. These constructs were tested by transient transfection into neuronal and nonneuronal cells, followed by an assay for CAT activity. We found that as little as 255 bp of 5′ flanking sequence was able to confer cell type-specificity on the reporter gene. Further truncation to 120 bp of 5′ sequence resulted in a sharp downregulation of reporter activity in PC12 cells but a significant rise in both Neuro-2A neuroblastoma cells and nonneuronal Ltk- cells, indicating thatcis-acting elements controlling the regulation ofNSE in Ltk-, Neuro-2A, and PC12 cells may lie within the 135 bp region covered by this deletion. This region contains an AP-2 site and an element similar in sequence and position to a motif identified in the proximal promoter region of the neuron-specific peripherin gene. Reduction to 95 bp of 5′ sequence resulted in a slight downregulation of CAT activity in all cell lines tested, and further truncation to 65 bp of 5′ sequence caused a universal reduction to background levels of CAT activity, concomitant with the disruption of the basalNSE promoter. Our results show that the 5′ flanking region of theNSE gene is capable of conferring cell type-specificity on a heterologous gene in transfected cells and that elements responsible for this are located within the proximal 255 bp.     

Regulation of Pituitary Vasopressin V1b Receptor mRNA during Stress in the Rat

Previous studies have shown a parallel relationship between pituitary vasopressin (VP) receptor content and responsiveness of the corticotroph during chronic stress. The regulation of pituitary VP receptors was further studied by analysis of V1b VP receptor mRNA levels in pituitaries of rats subjected to chronic immobilization, i.p. hypertonic saline injection (physical stress paradigms associated with increased pituitary responsiveness), and water deprivation, or to 2% saline in the drinking water (osmotic stress paradigms associated with decreased pituitary responsiveness). Northern blot hybridization with a 363 bp ³²P-labelled fragment of the rV1b receptor cDNA coding sequence revealed two bands of about 3.7 and 3.2 Kb, whereas a probe directed to the 5′ untranslated region recognized only the 3.7 Kb band. Repeated i.p. hypertonic saline injection, 3 times in 24 h at 8 h intervals, or daily for 8 days, increased the intensity of the 3.7 Kb band by 155 ± 17.5% (P<0.01) and 118 ± 14.6% (P<0.01), respectively, while the 3.2Kb band increased by 122 ± 39.3% (P<0.01) only after 3 times injection. Smaller increases of 39 ± 11 and 33 ± 9% (P<0.05) in the 3.7 Kb band were found after repeated immobilization 3 times in 24 h and 2 h for for 8 days respectively. In situ hybridization studies confirmed significant increases (P<0.05) in V1b receptor mRNA levels after 8 and 14 days repeated immobilization (63 ± 19% and 83 ± 10%) or i.p. hypertonic saline injection (110 ± 13% and 73 ± 20%). In response to acute stress, V1b receptor mRNA increased by 77 ± 5% (3.7 Kb band) after 4 h immobilization for 1 h, whereas both bands were reduced by 49 ± 5% and 45 ± 5%, 4 h after a single i.p. hypertonic saline injection. The decrease in V1b receptor mRNA following a single i.p. hypertonic saline injection was prevented by pretreatment with a V1 receptor antagonist, suggesting that increased VP secretion may account for this effect. In spite of the decrease in V1 b receptor mRNA following i.p. hypertonic saline injection, VP binding in pituitary membrane rich fractions, and VP-stimulated inositol phosphate formation in quartered hemipituitaries were increased by 24 and 39%, respectively. V1b receptor mRNA levels were unchanged or decreased following prolonged osmotic stimulation. These studies suggest that increased V1b receptor mRNA levels contribute to the VP receptor upregulation observed during repeated immobilization and i.p. hypertonic saline injection, whereas the lack of parallelism between V1b receptor mRNA and VP binding indicates that regulation of steady-state levels of V1b receptor mRNA is not a primary determinant in the control of pituitary VP receptor concentration during stress.