Pituitary cell line GH3 expresses two somatostatin receptor subtypes that inhibit adenylyl cyclase: functional expression of rat somatostatin receptor subtypes 1 and 2 in human embryonic kidney 293 cells

Using a polymerase chain reaction approach, we have studied the expression of somatostatin receptor (SSTR) subtypes in the GH3 rat pituitary cell line, a well established in vitro model for the cellular effects of somatostatin. We found that the previously identified SSTR1 and SSTR2 are the major subtypes expressed in this cell line. No other SSTR subtype was detected by our analysis. Northern blots confirmed that both subtypes, but not SSTR3, are expressed in GH3 cells. We studied the functional expression of both SSTR subtypes by transfection of their cDNAs into human embryonic kidney 293 cells. We found that somatostatin inhibited cAMP accumulation in human embryonic kidney 293 cells only when cells were transfected with either SSTR1 or SSTR2. This inhibition was blocked by treatment of the transfected cells with pertussis toxin, demonstrating that it is mediated by G proteins sensitive to this toxin. In addition, we provide pharmacological evidence that the endogenous SSTR2 subtype mediates inhibition of cAMP accumulation in intact GH3 cells. Our results contradict previous reports that concluded thsat neither SSTR1 nor SSTR2 is involved in inhibition of adenylyl cyclase. The reasons for this apparent contradiction are discussed. We conclude that both SSTR1 and SSTR2 are capable of coupling to pertussis toxin-sensitive G proteins to inhibit adenylyl cyclase.     

Biological response to ErbB ligands in nontransformed cell lines correlates with a specific pattern of receptor expression

The human epidermal growth factor receptor (HER or ErbB) family consists of four distinct members, including the epidermal growth factor (EGF) receptor (EGFR, HER1, or ErbB1), ErbB2 (HER2 or neu), ErbB3 (HER3), and ErbB4 (HER4). Activation of these receptors plays an important role in the regulation of cell proliferation, differentiation, and survival in several different tissues. Binding of a specific ligand to one of the ErbB receptors triggers the formation of specific receptor homo- and heterodimers, with ErbB2 being the preferred signaling partner. We analyzed the levels of various ErbB receptor messenger RNAs in a series of nontransformed cell lines by real time quantitative RT-PCR. The cell lines chosen were derived from a variety of tissues, including pancreas, lung, heart, and nervous system. Further, we measured biological responses in these cell lines upon treatment with EGF, betacellulin, and two types of neuregulins, heregulin and sensory and motor neuron-derived factor. All cell lines examined expressed detectable levels of ErbB2. High levels of expression of ErbB3 were correlated with responsiveness to heregulin and sensory and motor neuron-derived factor, whereas high levels of EGFR expression were correlated with responsiveness to EGF and betacellulin. Moreover, the sensitivity of a cell line to ErbB ligands was also correlated with the levels of expression of the appropriate ErbB receptors in that cell line. These results are consistent with our hypothesis that appropriate biological responsiveness to ErbB ligands is determined by the levels of expression of specific ErbB receptor combinations within a given tissue.     

Cardiac senescence is associated with enhanced expression of angiotensin II receptor subtypes

Recent studies have pointed out the differential role of angiotensin II (Ang II) receptor subtypes, AT1 and AT2, in cardiac hypertrophy and fibrosis during pathological cardiac growth. Because senescence is characterized by an important cardiovascular remodeling, we examined the age-related expression of cardiac Ang II receptors in rats. AT1 and AT2 receptor subtype messenger RNA (mRNA) levels were quantitated by RT-PCR. In parallel, specific Ang II densities were determined in competition binding experiments using specific antagonists. AT1a and AT1b mRNA levels were markedly up-regulated (5.6-fold) in the left ventricle of 24-month-old rats compared with 3-month-old rats, but not in the right ventricle. In contrast, AT2 gene expression was increased in both ventricles of senescent rats (4.2- and 2.8-fold in the left and right ventricles, respectively). Similarly, AT1 and AT2 gene expression was increased 2.3- and 2-fold, respectively, in freshly isolated cardiomyocytes from aged rats. Furthermore, AT1 and AT2 specific binding was increased in the aged left ventricular myocardium. Even though the mechanistic pathway of this up-regulation of Ang II receptor subtype gene expression might be intrinsic to developmental gene reprogramming, the up-regulation of AT1 mRNA accumulation in the left ventricle during aging could also be secondary to age-related hemodynamic changes, whereas increased AT2 gene expression in both ventricles may depend upon hormonal and humoral factors.     

Definition of the role of somatostatin receptor scintigraphy in gastrointestinal neuroendocrine tumor localization

There are six major steps in the management of patients with neuroendocrine tumors (NETs) (carcinoids and pancreatic endocrine tumors). One of the steps that is increasing in its importance is the need to assess primary tumor location and tumor extent in these patients. Without such information, it is not possible to adequately manage these patients. Conventional imaging studies (CT scan, MRI, ultrasound, angiography), functional localization studies measuring hormonal gradients, endoscopic ultrasound, and most recently, somatostatin receptor scintigraphy (SRS) with [125I-DTPA-DPhe1]-octreotide have all been advocated to localize NETs in different studies. Whereas it is now established that for all NETs, except insulinomas, SRS has the greatest sensitivity, it remains unclear whether this increased sensitivity translates into increased clinical usefulness. It, therefore, remains unclear based on fiscal and clinical considerations what should be the recommended algorithm for the use of the different localization methods. To address this issue, we have recently performed two prospective studies on patients with gastrinomas. In this paper, the methods and results of each are summarized and based on these results, an algorithm for localization studies in NETs is proposed. One study assessed the role of SRS in management in 122 patients and shows that the use of SRS changed management in 47 percent of patients according to six different criteria when the patients were stratified according to their principal management problem. Determining whether liver metastases were present is one of the major goals of tumor localization studies and is frequently a source of confusion because of the difficulty in distinguishing small NETs liver metastases from hemangiomas. In the second study, the ability of SRS and other tumor localization methods to distinguish these two possibilities was assessed in 15 patients with small hemangiomas and 15 patients with small hepatic metastases (mean size 1.3 cm). SRS correctly identified 93 percent of the patients with liver metastases and was not positive in any patient with a hemangioma, suggesting it was not a liver metastases. SRS had greater negative and positive predictive value than conventional studies. Based on these two studies, and SRS's greater sensitivity and fiscal considerations, it is proposed that SRS should be the initial tumor imaging study in all NETs except insulinomas, and algorithms for the use of other localization studies in both NETs and insulinomas are proposed.     

Comorbidity of panic disorder with agoraphobia and specific phobia: relationship with the subtypes of specific phobia

The study objectives were to determine comorbidity rates for various subtypes of specific phobia (SP) in a sample of patients with the principal diagnosis of panic disorder with agoraphobia (PDA) and to examine the possible etiologic relatedness of these SP subtypes to PDA. Ninety consecutive day clinic patients with PDA were administered the Structured Clinical Interview for DSM-III-R (SCID) modified for DSM-IV. The overall comorbidity rate for SP was 65.6%. The most frequent subtypes of SP were situational phobia and dental phobia, followed by natural environment phobia, phobia of funerals, cemeteries, dead bodies, and other death-related phenomena and objects (referred to as death-related phobia), and blood-injection-injury phobia. Except for death-related phobia, other subtypes of SP clearly tended to precede the onset of PDA, often by many years. The smallest difference between the age of onset for PDA and particular subtypes of SP (temporal distance) was found for death-related phobia, whereas the temporal difference was longer for situational phobia, hospital phobia, and blood-injection-injury phobia. The frequency and temporal distance data suggest that death-related phobia may constitute a risk factor for developing PDA or that it is a prodrome of PDA, whereas situational phobia, hospital phobia, and blood-injection-injury phobia appear to predispose to PDA to a lesser degree. Of the three broadly conceived groups of SP, mutilation phobias (which include death-related phobia, hospital phobia, blood-injection-injury phobia, and dental phobia) appear most etiologically relevant for PDA, with the group of situational phobias (which also includes the natural environment subtype of SP) being less relevant, and animal phobias showing a negligible etiologic relatedness to PDA.     

Localization of the somatostatin receptor SST2A in rat brain using a specific anti-peptide antibody

Biological actions of somatostatin are exerted via a family of receptors, for which five genes recently have been cloned. However, none of these receptor proteins has been visualized yet in the brain. In the present-study, the regional and cellular distribution of the somatostatin sst2A receptor was investigated via immunocytochemistry in the rat central nervous system by using an antibody generated against a unique sequence of the receptor protein. Specificity of the antiserum was demonstrated by immunoblot and immunocytochemistry on rat brain membranes and/or on cells transfected with cDNA encoding the different sst receptor subtypes. In rat brain sections, sst2A receptor immunoreactivity was concentrated either in perikarya and dendrites or in axon terminals distributed throughout the neuropil. Somatodendritic labeling was most prominent in the olfactory tubercle, layers II-III of the cerebral cortex, nucleus accumbens, pyramidal cells of CA1-CA2 subfields of the hippocampus, central and cortical amygdaloid nuclei, and locus coeruleus. Labeled terminals were detected mainly in the endopiriform nucleus, deep layers of the cortex, claustrum, substantia innominata, subiculum, basolateral amygdala, medial habenula, and periaqueductal gray. Electron microscopy confirmed the association of sst2A receptors with perikarya and dendrites in the former regions and with axon terminals in the latter. These results provide the first characterization of the cellular distribution of a somatostatin receptor in mammalian brain. The widespread distribution of the sst2A receptor in cerebral cortex and limbic structures suggests that it is involved in the transduction of both pre- and postsynaptic effects of somatostatin on cognition, learning, and memory.     

sst2 somatostatin receptor expression reverses tumorigenicity of human pancreatic cancer cells

Among the five cloned somatostatin receptor subtypes (sst1 to sst5), sst2 mediates the antiproliferative effect of somatostatin analogues in vitro. Somatostatin analogues have been shown to inhibit cell growth in vitro and in vivo in pancreatic cancer models that expressed sst2. We recently demonstrated the loss of sst2 gene expression in human pancreatic adenocarcinomas and most of the derived pancreatic cancer cell lines. In the present study, we corrected the sst2 defect in human pancreatic cancer BxPC-3 and Capan-1 cells by stable transfection with human sst2 cDNA. In the absence of exogenous ligand, both BxPC-3 and Capan-1 cells expressing sst2 showed a significant reduction in cell growth. This inhibitory effect was blocked by treatment with antiserum to somatostatin. sst2-expressing cells produced somatostatin-like immunoreactivity that mainly corresponded to somatostatin 14, indicating the induction of a negative autocrine loop. In other respects, sst2 expression in Capan-1 cells induced a significant reduction of clonogenicity in soft agar. Moreover, a significantly reduced (Capan-1 cells) or suppressed (BxPC-3 cells) tumor growth in athymic nude mice was observed. The reversal of tumorigenicity induced by the restoration of sst2 expression suggests that the loss of sst2 contributes to the malignancy of human pancreatic cancers.     

Determination of somatostatin receptor subtype 2 in carcinoid tumors by immunohistochemical investigation with somatostatin receptor subtype 2 antibodies

We have shown previously that expression of mRNA for somatostatin receptor subtype 2 (sst2) detected by in situ hybridization correlates to therapeutic outcome in patients with carcinoid tumors treated with somatostatin analogues. However, in situ hybridization is laborious and not practical in clinical routine work. We have, therefore, developed polyclonal antibodies directed against sst2 that may be used for immunohistochemistry on tissue specimens. The staining is specific and is highly correlated to expression of mRNA for sst2 (P < 0.01) as well as to tracer uptake at somatostatin receptor scintigraphy (P < 0.01). There is also a good correlation to the therapeutic response in carcinoid patients treated with somatostatin analogues (P < 0.05). Of 35 patients with carcinoid tumors included in this investigation, 25 stained positive with the antibodies. Twenty-two of these were investigated by somatostatin receptor scintigraphy and showed tracer uptake in metastases. An additional two patients that did not stain with the antibodies showed pathological uptake of the tracer in metastases, which might indicate binding to somatostatin receptor subtype 5. None of the 10 patients without positive immunostaining responded to somatostatin analogue treatment, whereas patients with a positive stain had a biochemical response or remained stable during treatment. Thus, these antibodies may be used to determine the presence of sst2 in carcinoid tumors and to select patients suitable for somatostatin analogue treatment. The method is easily applicable in clinical practice.     

Molecular cloning, functional characterization and mRNA expression analysis of the murine chemokine receptor CCR6 and its specific ligand MIP-3alpha

We have cloned the murine CCR6 receptor and its ligand, the beta-chemokine mMIP-3alpha. Calcium mobilization assays performed with mCCR6 transfectants showed significant responses upon addition of mMIP-3alpha. Murine MIP-3alpha RNA is expressed in thymus, small intestine and colon, whereas mCCR6 RNA is expressed in spleen and lymph nodes. RT-PCR analysis of FACS-sorted lymphoid and antigen presenting cell subsets showed mCCR6 expression mainly in B cells, CD8- splenic dendritic cells and CD4+ T cells. The cloning and functional characterization of the mCCR6 and mMIP-3alpha will allow the study of the role of these proteins in mouse models of inflammation and immunity.     

Alpha1-adrenergic receptors in human spinal cord: specific localized expression of mRNA encoding alpha1-adrenergic receptor subtypes at four distinct levels

alpha1-Adrenergic receptors (alpha1ARs) are important in lower urinary tract syndromes such as benign prostatic hypertrophy and bladder irritability. Spinal cord alpha1ARs have been postulated to play a role in modulating these diseases, yet alpha1AR subtype (alpha1a, alpha1b, alpha1d) neuronal localization in human spinal cord has not been described. We therefore tested the hypothesis that alpha1AR subtype distribution varies according to specific spinal cord tract and level. In situ hybridization was performed to identify cell bodies containing alpha1AR subtype mRNA at four levels of human spinal cord (cervical enlargement, thoracic, lumbar, sacral). alpha1AR mRNA is present in ventral gray matter only (ventral>dorsal; sacral>lumbar=thoracic>cervical). Signaling cell bodies were detected in anterior horn motor neurons at all levels; dorsal nucleus of Clarke and intermediolateral columns in cervical enlargement, thoracic and lumbar spinal cord regions; and parasympathetic nucleus in sacral spinal cord. Although all three alpha1AR subtypes are present throughout human spinal cord, alpha1d mRNA predominates overall. If confirmed at a protein level, these findings may contribute to the development of new therapeutic strategies in the treatment of several human diseases.     

Differential gene expression of angiotensin II receptor subtypes in the epididymides of mature and immature rats

Electrospray Ionization Mass Spectrometry can be applied to detect aberrant proteins using intact molecules. Direct examination of hemolysate might well facilitate rapid ascertainment of a variant hemoglobin (Hb) provided that the mass difference between normal and abnormal chains is larger than the resolution power of standard instruments (i.e. = 10 Da). We propose immunoprecipitation as a preparation method of plasma and cell proteins other than Hb prior to MS. Amino acid sequences of various variants detected by MS were determined by MS/MS. Some of these variants were new. These new variants were; 1: Hb Sagami[beta 139(H17)Asn-->Thr]. 2: Hb Hokusetsu[beta 52(D3)Asp-->Gly]. 3: a variant transthyretin, amyloidogenic, [38Asp-->Ala]. 4: a variant transthyretin, non-amyloidogenic, [101Gly-->Ser]. The abundance of ion peaks showed the approximate ratio of each component, which was in agreement with the ratio obtained by chromatography and by ESIMS in the analyses of glycated hemoglobin. Samples with low kidney function (BUN > 50 mg/dl, creatinine > 2.5 mg/dl) showed higher values of glycated Hb on routine HPLC than the MS method. Samples containing high carbamylated Hb might cause this discrepancy.     

Suppression of tumor growth with recombinant murine angiostatin

PURPOSE: To assess visualization of the extrahepatic segment of the ligamentum teres on plain radiographs, the ligamentum teres sign, which is an indicator of pneumoperitoneum. MATERIALS AND METHODS: The ligamentum teres was recognized in 12 patients with pneumoperitoneum. Findings from supine radiographs were correlated with those from oblique radiographs and computed tomograms. RESULTS: The extrahepatic segment of the ligamentum teres, which extended between the umbilicus and the ligamentum teres notch at the inferior border of the liver, was visualized on supine radiographs. The ligamentum teres appeared in the right upper quadrant as an obliquely oriented, straight, or slightly arcuate interface with a sharply demarcated inferolateral margin or as a similarly positioned, well-defined band of soft tissue of varying length. The falciform ligament was also seen in six of the 12 patients as a long, thin line that ran vertically to the right of the midline and joined the ligamentum teres caudally near or at the umbilicus. Even though there were other signs of pneumoperitoneum in all cases, the ligamentum teres sign was the most prominent in three cases. CONCLUSION: Visualization of the extrahepatic segment of the ligamentum teres on supine radiographs requires the anterior accumulation of extraluminal, intraperifoneal gas and is another sign of moderate to massive amounts of free air.     

Temporal and spatial pattern of expression of the HDL receptor SR-BI during murine embryogenesis

Cerebellar long-term depression (LTD) is a model system for neuronal information storage that has an absolute requirement for activation of protein kinase C (PKC). It has been claimed to underlie several forms of cerebellar motor learning. Previous studies using various knockout mice (mGluR1, GluRdelta2, glial fibrillary acidic protein) have supported this claim; however, this work has suffered from the limitations that the knockout technique lacks anatomical specificity and that functional compensation can occur via similar gene family members. To overcome these limitations, a transgenic mouse (called L7-PKCI) has been produced in which the pseudosubstrate PKC inhibitor, PKC[19-31], was selectively expressed in Purkinje cells under the control of the pcp-2(L7) gene promoter. Cultured Purkinje cells prepared from heterozygous or homozygous L7-PKCI embryos showed a complete blockade of LTD induction. In addition, the compensatory eye movements of L7-PKCI mice were recorded during vestibular and visual stimulation. Whereas the absolute gain, phase, and latency values of the vestibulo-ocular reflex and optokinetic reflex of the L7-PKCI mice were normal, their ability to adapt their vestibulo-ocular reflex gain during visuo-vestibular training was absent. These data strongly support the hypothesis that activation of PKC in the Purkinje cell is necessary for cerebellar LTD induction, and that cerebellar LTD is required for a particular form of motor learning, adaptation of the vestibulo-ocular reflex.     

Readiness of zebrafish brain neurons to regenerate a spinal axon correlates with differential expression of specific cell recognition molecules

We analyzed changes in the expression of mRNAs for the axonal growth-promoting cell recognition molecules L1.1, L1.2, and neural cell adhesion molecule (NCAM) after a rostral (proximal) or caudal (distal) spinal cord transection in adult zebrafish. One class of cerebrospinal projection nuclei (represented by the nucleus of the medial longitudinal fascicle, the intermediate reticular formation, and the magnocellular octaval nucleus) showed a robust regenerative response after both types of lesions as determined by retrograde tracing and/or in situ hybridization for GAP-43. A second class (represented by the nucleus ruber, the nucleus of the lateral lemniscus, and the tangential nucleus) showed a regenerative response only after proximal lesion. After distal lesion, upregulation of L1.1 and L1.2 mRNAs, but not NCAM mRNA expression, was observed in the first class of nuclei. The second class of nuclei did not show any changes in their mRNA expression after distal lesion. After proximal lesion, both classes of brain nuclei upregulated L1.1 mRNA expression (L1.2 and NCAM were not tested after proximal lesion). In the glial environment distal to the spinal lesion, labeling for L1.2 mRNA but not L1.1 or NCAM mRNAs was increased. These results, combined with findings in the lesioned retinotectal system of zebrafish (Bernharnhardt et al., 1996), indicate that the neuron-intrinsic regulation of cell recognition molecules after axotomy depends on the cell type as well as on the proximity of the lesion to the neuronal soma. Glial reactions differ for different regions of the CNS.