Autosomal dominant GH deficiency type II (IGHDII) is often associated with mutations in the human GH gene (GH1) that give rise to products lacking exon-3 ((Deltaexon3)hGH). In the heterozygous state, these act as dominant negative mutations that prevent the release of human pituitary GH (hGH). To determine the mechanisms of these dominant negative effects, we used a combination of transgenic and morphological approaches in both in vitro and in vivo models. Rat GC cell lines were generated expressing either wild-type GH1 (WT-hGH-GC) or a genomic GH1 sequence containing a G->A transition at the donor splice site of IVS3 ((Deltaexon3)hGH-GC). WT-hGH-GC cells grew normally and produced equivalent amounts of human and rGH packaged in dense-cored secretory vesicles (SVs). In contrast, (Deltaexon3)hGH-GC cells showed few SVs but accumulated secretory product in amorphous cytoplasmic aggregates. They produced much less rGH and grew more slowly than WT-hGH-GC cells. When cotransfected with an enhanced green fluorescent protein construct (GH-eGFP), which copackages with GH in SVs, WT-hGH-GC cells showed normal electron microscopy morphology and SV movements, tracked with total internal reflectance fluorescence microscopy. In contrast, coexpression of (Deltaexon3)hGH with GH-eGFP abolished the vesicular targeting of GH-eGFP, which instead accumulated in static aggregates. Transgenic mice expressing (Deltaexon3)hGH in somatotrophs showed an IGHD-II phenotype with mild to severe pituitary hypoplasia and dwarfism, evident at weaning in the most severely affected lines. Hypothalamic GHRH expression was up-regulated and somatostatin expression reduced in (Deltaexon3)hGH transgenic mice, consistent with their profound GHD. Few SVs were detectable in the residual pituitary somatotrophs in (Deltaexon3)hGH transgenic mice, and these cells showed grossly abnormal morphology. A low copy number transgenic line showed a mild effect relatively specific for GH, whereas two severely affected lines with higher transgene copy numbers showed early onset, widespread pituitary damage, macrophage invasion, and multiple hormone deficiencies. These new in vitro and in vivo models shed new light on the cellular mechanisms involved in IGHDII, as well as its phenotypic consequences in vivo.

Analysis of small dorsal root ganglion (DRG) neurons revealed novel functions for vanilloid receptor 1 (VR1) in the regulation of cytosolic Ca2؉ . The VR1 agonist capsaicin induced Ca 2؉ mobilization from intracellular stores in the absence of extracellular Ca 2؉ , and this release was inhibited by the VR1 antagonist capsazepine but was unaffected by the phospholipase C inhibitor xestospongins, indicating that Ca 2؉ mobilization was dependent on capsaicin receptor binding and was not due to intracellular inositol-1,4,5-trisphosphate generation. Confocal microscopy revealed extensive expression of VR1 on endoplasmic reticulum, consistent with VR1 operating as a Ca 2؉ release receptor. The main part of the capsaicin-releasable Ca 2؉ store was insensitive to thapsigargin, a selective endoplasmic reticulum Ca 2؉ -ATPase inhibitor, suggesting that VR1 might be predominantly localized to a thapsigargin-insensitive endoplasmic reticulum Ca 2؉ store. In addition, VR1 was observed to behave as a store-operated Ca 2؉ influx channel. In DRG neurons, capsazepine attenuated Ca 2؉ influx following thapsigargin-induced Ca 2؉ store depletion and inhibited thapsigargin-induced inward currents. Conversely, transfected HEK-293 cells expressing VR1 showed enhanced Ca 2؉ influx and inward currents following Ca 2؉ store depletion. Combined data support topographical and functional diversity for VR1 in the regulation of cytosolic Ca 2؉ with the plasma membraneassociated form behaving as a store-operated Ca 2؉ influx channel and endoplasmic reticulum-associated VR1 possibly functioning as a Ca 2؉ release receptor in sensory neurons.Vanilloid receptor 1 (VR1 1 or TRPV1) belongs to the transient receptor potential (TRP) family of nonspecific cation channels and has been proposed to be analogous to the capsaicin receptor of sensory neurons (1). VR1 confers several sensory functions in these cells, including the transduction of chemical (vanilloids and pH) and physical (heat) stimuli resulting in the generation of action potentials in nociceptive nerve endings, which are ultimately responsible for the sensations of heat and thermal/inflammatory pain (2, 3). Although VR1 exhibits Ca 2ϩ -dependent desensitization and has been proposed to be modulated by protein kinase C (4, 5), it is possible that this receptor is subject to many other regulatory mechanisms that may be of fundamental importance to its functioning in sensory neurons. Although the activation of certain members of the TRP family of cation channels is reliant on the state of filling of intracellular Ca 2ϩ stores (6), with these channels being activated by intracellular Ca 2ϩ store depletion, it remains controversial whether VR1 has an innate capacity to function as a store-operated Ca 2ϩ channel (SOCC) in sensory neurons. To date, there is no published evidence to support this, a previous study failing to detect activation of expressed VR1 following Ca 2ϩ store depletion by the endoplasmic reticulum Ca 2ϩ -ATPase inhibitor thapsigargin in Xenopus oocytes (1).In addition to funct...

In stable transfection experiments in the GH-producing GC cell line, a construct containing the entire signal peptide and the first 22 residues of human GH linked in frame with enhanced green fluorescent protein (eGFP), produced brightly fluorescent cells with a granular distribution of eGFP. This eGFP reporter was then inserted into a 40-kb cosmid transgene containing the locus control region for the hGH gene and used to generate transgenic mice. Anterior pituitaries from these GH-eGFP transgenic mice showed numerous clusters of strongly fluorescent cells, which were also immunopositive for GH, and which could be isolated and enriched by fluorescence-activated cell sorting. Confocal scanning microscopy of pituitary GH cells from GH-eGFP transgenic mice showed a markedly granular appearance of fluorescence. Immunogold electron microscopy and RIA confirmed that the eGFP product was packaged in the dense cored secretory vesicles of somatotrophs and was secreted in parallel with GH in response to stimulation by GRF. Using eGFP fluorescence, it was possible to identify clusters of GH cells in acute pituitary slices and to observe spontaneous transient rises in their intracellular Ca2+ concentrations after loading with Ca2+ sensitive dyes. This transgenic approach opens the way to direct visualization of spontaneous and secretagogue-induced secretory mechanisms in identified GH cells.

The hypothalamic GHRH neurons secrete pulses of GHRH to generate episodic GH secretion, but little is known about the mechanisms involved. We have made transgenic mice expressing enhanced green fluorescent protein (eGFP) specifically targeted to the secretory vesicles in GHRH neurons. GHRH cells transported eGFP from cell bodies in the arcuate nucleus to extensively arborized varicose fiber terminals in the median eminence. Patch clamp recordings from visually identified GHRH cells in mature animals showed spontaneous action potentials, often firing in short bursts up to 10 Hz. GHRH neurons received frequent synaptic inputs, as demonstrated by the recording of abundant inward postsynaptic currents, but spikes were followed by large after-hyperpolarizations, which limited their firing rate. Because many GHRH neurons lie close to the ventral hypothalamic surface, this was examined by wide-field binocular epifluorescence stereomicroscopy. This approach revealed an extensive horizontal network of GHRH cells at low power and individual fiber projections at higher power in the intact brain. It also showed the dense terminal projections of the GHRH cell population in the intact median eminence. This model will enable us to characterize the properties of individual GHRH neurons and their structural and functional connections with other neurons and to study directly the role of the GHRH neuronal network in generating episodic secretion of GH.

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