Vector spherical wave function truncation in the invariant imbedding T-matrix method.

Both the computational costs and the accuracy of the invariant-imbedding T-matrix method escalate with increasing the truncation number N at which the expansions of the electromagnetic fields in terms of vector spherical harmonics are truncated. Thus, it becomes important in calculation of the single-scattering optical properties to choose N just large enough to satisfy an appropriate convergence criterion; this N we call the optimal truncation number. We present a new convergence criterion that is based on the scattering phase function rather than on the scattering cross section. For a selection of homogeneous particles that have been used in previous single-scattering studies, we consider how the optimal N may be related to the size parameter, the index of refraction, and particle shape. We investigate a functional form for this relation that generalizes previous formulae involving only size parameter, a form that shows some success in summarizing our computational results. Our results indicate clearly the sensitivity of optimal truncation number to the index of refraction, as well as the difficulty of cleanly separating this dependence from the dependence on particle shape.

Improvements in the computational efficiency and convergence of the Invariant Imbedding T-matrix method for spheroids and hexagonal prisms.

The invariant-imbedding T-matrix (II-TM) method is a numerical method for accurately computing the single-scattering properties of dielectric particles. Because of the linearity of Maxwell's equations, the incident electric field and the scattered electric field can be related through a transition matrix (T-Matrix). The II-TM method computes the T-matrix through a matrix recurrence formula which stems from an electromagnetic volume integral equation. The recurrence starts with an inscribed sphere within the particle and ends with the circumscribed sphere of the particle. For each iteration, a matrix known as the U-matrix is computed with the Gauss Legendre (GL) quadrature, and matrix inversion is subsequently performed to obtain the T-matrix corresponding to the portion of the particle enclosed by the spherical shell. We modify a commonly used scheme to avoid applying the quadrature scheme to discontinuities. Moreover, we apply a new scheme to generate nodes and weights in conjunction with the GL quadrature. This leads to a considerable improvement on convergence and computational efficiency in the cases of hexagonal prisms and spheroids. The basic shapes of ice crystals in the atmosphere are hexagonal columns and plates. The single-scattering properties of hexagonal ice prisms are important to atmospheric optics, radiative transfer, and remote sensing. We demonstrate that the present approach can significantly accelerate the convergence in simulating light scattering by hexagonal ice crystals.

A novel tool for stable nitrogen isotope analysis in aqueous samples.

RATIONALE: Bulk stable isotope analysis (BSIA) of dissolved matter (e.g. dissolved organic carbon, total nitrogen bound (TNb ), etc.) is of particular importance since this pool is a prime conduit in the cycling of N and C. Studying the two elemental pools is of importance, as transformation and transport processes of N and C are inextricably linked in all biologically mediated systems. No system able to analyze natural abundance stable carbon and nitrogen isotope composition in aqueous samples (without offline sample preparation) and simultaneously has been reported so far. Extension of the high-temperature combustion (HTC) system, to be capable of measuring TNb stable nitrogen isotope composition, is described in this study. METHODS: To extend the TOC analyzer to be capable of measuring TNb , modifications from the HTC high-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS) interface were implemented and expanded. A reduction reactor for conversion of NOx into N2 was implemented into the new developed system. The extension addresses mainly the development of the focusing unit for nitrogen and a degassing device for online separation of TNb from molecular nitrogen (N2 ) prior to injection. RESULTS: The proof of principle of the system was demonstrated with different compound solutions. In this initial testing, the δ15 NAIR-N2 values of the tested compounds were determined with precision and trueness of typically ≤0.5‰. Good results (u ≤ 0.5‰) could be achieved down to a TNb concentration of 40 mgN/L and acceptable results (u ≤ 1.0‰) down to 5 mgN/L. In addition, the development resulted in the first system reported to be suitable for simultaneous and direct δ13 C and δ15 N BSIA of aqueous samples. CONCLUSIONS: The development resulted in the first system shown to be suitable for both δ13 C and δ15 N direct BSIA in aqueous samples. This system could open up new possibilities in SIA-based research fields. Copyright © 2016 John Wiley & Sons, Ltd.

First-principles modeling of electromagnetic scattering by discrete and discretely heterogeneous random media.

A discrete random medium is an object in the form of a finite volume of a vacuum or a homogeneous material medium filled with quasi-randomly and quasi-uniformly distributed discrete macroscopic impurities called small particles. Such objects are ubiquitous in natural and artificial environments. They are often characterized by analyzing theoretically the results of laboratory, in situ, or remote-sensing measurements of the scattering of light and other electromagnetic radiation. Electromagnetic scattering and absorption by particles can also affect the energy budget of a discrete random medium and hence various ambient physical and chemical processes. In either case electromagnetic scattering must be modeled in terms of appropriate optical observables, i.e., quadratic or bilinear forms in the field that quantify the reading of a relevant optical instrument or the electromagnetic energy budget. It is generally believed that time-harmonic Maxwell's equations can accurately describe elastic electromagnetic scattering by macroscopic particulate media that change in time much more slowly than the incident electromagnetic field. However, direct solutions of these equations for discrete random media had been impracticable until quite recently. This has led to a widespread use of various phenomenological approaches in situations when their very applicability can be questioned. Recently, however, a new branch of physical optics has emerged wherein electromagnetic scattering by discrete and discretely heterogeneous random media is modeled directly by using analytical or numerically exact computer solutions of the Maxwell equations. Therefore, the main objective of this Report is to formulate the general theoretical framework of electromagnetic scattering by discrete random media rooted in the Maxwell-Lorentz electromagnetics and discuss its immediate analytical and numerical consequences. Starting from the microscopic Maxwell-Lorentz equations, we trace the development of the first-principles formalism enabling accurate calculations of monochromatic and quasi-monochromatic scattering by static and randomly varying multiparticle groups. We illustrate how this general framework can be coupled with state-of-the-art computer solvers of the Maxwell equations and applied to direct modeling of electromagnetic scattering by representative random multi-particle groups with arbitrary packing densities. This first-principles modeling yields general physical insights unavailable with phenomenological approaches. We discuss how the first-order-scattering approximation, the radiative transfer theory, and the theory of weak localization of electromagnetic waves can be derived as immediate corollaries of the Maxwell equations for very specific and well-defined kinds of particulate medium. These recent developments confirm the mesoscopic origin of the radiative transfer, weak localization, and effective-medium regimes and help evaluate the numerical accuracy of widely used approximate modeling methodologies.

The effective equivalence of geometric irregularity and surface roughness in determining particle single-scattering properties.

This study investigates the effects of geometric irregularity and surface roughness on the single-scattering properties of randomly oriented dielectric particles. Starting from a regular crystal with smooth faces, effects of roughening are compared with effects of perturbing the regular configuration of the smooth faces. Using the same slope distribution for small roughness facets and tilted faces provides a natural way to compare the effects on the single-scattering properties. It is found that the geometric irregularity and surface roughness have similar effects on the single-scattering properties of an ensemble of randomly oriented particles. In other words, particles with irregular geometries and those with surface roughness are optically equivalent if the slope distributions are the same. Furthermore, an ensemble of particles with irregular geometries can be used as an effective approximation for simulation of the scattering properties of roughened particles, and vice versa. This approach also provides new interpretation of the observed, relatively featureless and smooth, scattering phase functions of naturally occurring particles.

Modeling the scattering properties of mineral aerosols using concave fractal polyhedra.

The single-scattering properties of concave fractal polyhedra are investigated, with particle size parameters ranging from the Rayleigh to geometric-optics regimes. Two fractal shape parameters, irregularity and aspect ratio, are used to iteratively construct "generations" of irregular fractal particles. The pseudospectral time-domain (PSTD) method and the improved geometric-optics method (IGOM) are combined to compute the single-scattering properties of fractal particles over the range of size parameters. The effects of fractal generation, irregularity, and aspect ratio on the single-scattering properties of fractals are investigated. The extinction efficiency, absorption efficiency, and asymmetry factor, calculated by the PSTD method for fractal particles, with small-to-moderate size parameters, smoothly bridges the gap between those size parameters and size parameters for which solutions given by the IGOM may be used. Somewhat surprisingly, excellent agreement between values of the phase function of randomly oriented fractal particles calculated by the two numerical methods is found, not only for large particles, but in fact extends as far down in equivalent-projected-area size parameters as 25. The agreement in the case of other nonzero phase matrix elements is not as good at so small a size. Furthermore, the numerical results of ensemble-averaged phase matrix elements of a single fractal realization are compared with dust particle measurements, and good agreement is found by using the fractal particle model to represent data from a study of feldspar aerosols.

Application of a discontinuous Galerkin time domain method to simulation of optical properties of dielectric particles.

We applied a discontinuous Galerkin time domain (DGTD) method, using a fourth-order Runga-Kutta time stepping of the Maxwell equations, to the simulation of the optical properties of dielectric particles in two-dimensional (2D) geometry. As examples of the numerical implementation of this method, the single-scattering properties of 2D circular and hexagonal particles are presented. In the case of circular particles, the scattering phase matrix was computed using the DGTD method and compared with the exact solution. For hexagonal particles, the DGTD method was used to compute single-scattering properties of randomly oriented 2D hexagonal ice crystals, and results were compared with those calculated using a geometric optics method. We consider both shortwave (visible) and longwave (infrared) cases, with particle size parameters 50 and 100. In the hexagonal case, scattering results are also presented as a function of both incident and scattering angles, revealing a structure apparently not reported before. Using the geometric optics method, we are able to interpret this structure in terms of contributions from varying numbers of internal reflections within the crystal.

Mammographic pattern of microcalcifications in the preoperative diagnosis of comedo ductal carcinoma in situ: histopathologic correlation.

OBJECTIVE: The comedo subtype of ductal carcinoma in situ (DCIS) is more aggressive than noncomedo DCIS. Differentiating noncomedo DCIS from the more aggressive comedo subtypes on mammography would allow the surgeon to excise comedo DCIS with a wider margin. The mammographic features of microcalcifications associated with nonpalpable comedo DCIS, noncomedo DCIS and benign disease were compared to determine the usefulness of this finding in diagnosis of comedo DCIS. METHODS: The authors retrospectively and blindly reviewed the mammograms of 91 consecutive patients in whom DCIS was diagnosed by needle localization and surgical excision. An equal number of cases of benign microcalcifications were also reviewed. Microcalcifications were evaluated with respect to pattern, density, configuration and size. These results were correlated with the pathologic findings. RESULTS: All 16 cases (100%) of linear branching calcifications and 34 (80%) of the 43 cases of linear calcifications were associated with comedo DCIS (p < 0.001). The number of calcifications, the density and the size of clustering were not diagnostic of comedo DCIS. Granular calcifications occurred in noncomedo DCIS and in benign disease associated with noncalcifying DCIS. CONCLUSION: Comedo DCIS is suggested by the presence of linear and linear branching microcalcifications on mammography.

Regulators of G-protein signaling (RGS) 1 and 16 are induced in response to bacterial lipopolysaccharide and stimulate c-fos promoter expression.

Regulators of G-protein signaling (RGS) are negative regulators of G-protein-coupled receptor (GPCR) signaling. Sepsis is a pathophysiological condition that is induced primarily in response to bacterial infection and is associated with decreased responsiveness to a number of vasoactive GPCR agonists. Using a degenerate RT-PCR screen, we report that RGS1 and RGS16 were amplified from the heart and aorta of septic animals. By Northern blot analysis, RGS1 and RGS16 were upregulated, with their respective levels increasing 6- and 50-fold in septic hearts. Using a yeast-based bioassay, both RGS1 and RGS16 were found to be equipotent in their ability to attenuate GPCR signaling. These results suggest that both RGS1 and RGS16 contribute to the sepsis-mediated decrease in GPCR signaling. Elevated levels of some RGSs may also lead to an increase in Gbetagamma-activated signaling pathways in the absence of GPCR agonists. Using a c-fos luciferase reporter gene that is responsive to Gbetagamma-activated signaling pathways, we observed a respective 8- and 7-fold increase in the basal luciferase in serum-deprived transfected mammalian cells overexpressing RGS1 or RGS16. This suggests that RGSs play a role in promoting the sepsis-mediated increases in the activation of intracellular signal transduction pathways.

The cytoplasmic tail of the human somatostatin receptor type 5 is crucial for interaction with adenylyl cyclase and in mediating desensitization and internalization.

We have investigated the role of the cytoplasmic tail (C-tail) of the human somatostatin receptor type 5 (hSSTR5) in regulating receptor coupling to adenylyl cyclase (AC) and in mediating agonist-dependent desensitization and internalization responses. Mutant receptors with progressive C-tail truncation (Delta347, Delta338, Delta328, Delta318), Cys320 --> Ala substitution (to block palmitoylation), or Tyr304 --> Ala substitution of a putative NPXXY internalization motif were stably expressed in Chinese hamster ovary K1 cells. Except for the Tyr304 --> Ala mutant, which showed no binding, all other mutant receptors exhibited binding characteristics (Kd and Bmax) and G protein coupling comparable with wild type (wt) hSSTR5. The C-tail truncation mutants displayed progressive reduction in coupling to AC, with the Delta318 mutant showing complete loss of effector coupling. Agonist pretreatment of wt hSSTR5 led to uncoupling of AC inhibition, whereas the desensitization response of the C-tail deletion mutants was variably impaired. Compared with internalization (66% at 60 min) of wt hSSTR5, truncation of the C-tail to 318, 328, and 338 residues reduced receptor internalization to 46, 46, and 23%, respectively, whereas truncation to 347 residues slightly improved internalization (72%). Mutation of Cys320 --> Ala induced a reduction in AC coupling, desensitization, and internalization. These studies show that the C-tail of hSSTR5 serves a multifunctional role in mediating effector coupling, desensitization, and internalization. Whereas coupling to AC is dependent on the length of the C-tail, desensitization and internalization require specific structural domains. Furthermore, internalization is regulated through both positive and negative molecular signals in the C-tail and can be dissociated from the signaling and acute desensitization responses of the receptor.

Expression of somatostatin receptor subtypes in human brain tumors.

Expression of mRNA for the 5 somatostatin receptors (sst1-5) was characterized by Northern blot and RT-PCR analysis in 20 meningioma and 9 glioma samples. sst1 mRNA was detectable by Northern blots of poly-A+ RNA in meningiomas but not gliomas. In contrast, sst2 mRNA was readily detected by Northern blots of total RNA as a major 2.3 kb transcript and 2 minor 4.3 kb and 8 kb transcripts in all meningiomas and 6 out of 9 gliomas. Quantitation of the 2.3 kb sst2 mRNA showed that 15 out of 20 tumors expressed 1.3- to 33-fold higher levels than control normal human brain. Mean sst2 mRNA for the 20 meningioma samples was 978% that of normal brain. Three gliomas showed 7- to 14-fold higher sst2 mRNA than normal brain whereas the remaining samples displayed very low or undetectable levels. Immunocytochemistry of meningioma and glioma samples, with a sst2-specific antibody revealed immunoreactivity in tumor cells and peritumoral tissue, with prominent expression in blood vessels. mRNA for sst3,4,5 could not be detected by Northern blots in any of the tumors. RT-PCR analysis of meningiomas and gliomas revealed the following percent of tumors positive for a given sst mRNA: sst1 (86%), sst2 (100%), sst3 (60%), sst4 (58%), and sst5 (67%); 85% of tumors expressed 3 of the 5 subtypes. No correlation was found between the pattern of expression of sst mRNA and tumor type, location, and histology for either the meningiomas or gliomas. Our results show that meningiomas and gliomas are all positive for at least one sst subtype, the majority expressing multiple subtypes. sst2 is the most abundant isoform with a rich expression in both tumor and peritumoral tissue especially blood vessels.

Ligand binding pocket of the human somatostatin receptor 5: mutational analysis of the extracellular domains.

The ligand binding domain of G protein-coupled receptors for peptide ligands consists of a pocket formed by extracellular and transmembrane domain (TM) residues. In the case of somatostatin (SRIF), however, previous studies have suggested that the binding cavity of the octapeptide analog SMS201-995 (SMS) is lined by residues in TMs III-VII. The additional involvement of the extracellular domains for binding SMS or the natural SRIF ligands (SRIF-14, SRIF-28) has not been clarified. Using a cassette construct cDNA for the human somatostatin 5 receptor (sst5R), we systematically examined the role of exofacial structures in ligand binding by creating a series of mutants in which the extracellular portions have been altered by conservative segment exchange (CSE) mutagenesis for the extracellular loops (ECLs) and by deletion (for the NH2-terminal segment) or truncation analysis (ECL3). CHO-K1 cells were stably transfected with wild type or mutant human sst5R constructs, and agonist binding was assessed using membrane binding assays with 125I-LTT SRIF-28 ligand. Deletion of the NH2 terminus or CSE mutagenesis of ECL1 and ECL3 produced minor 2-8-fold decreases in affinity for SRIF-14, SRIF-28, and SMS ligands. Truncation of ECL3 to mimic the size of this loop in sst1R and sst4R (the two subtypes that do not bind SMS) did not interfere with the binding of SMS, SRIF-14, or SRIF-28. In contrast, both ECL2 mutants failed to bind 125I-LTT SRIF-28. Immunocytochemical analysis of nonpermeabilized cells with a human sst5R antibody revealed that the mutant receptors were targeted to the plasma membrane. Labeled SMS (125I-Tyr3 SMS) also failed to bind to the mutant ECL2 receptors. These results suggest a potential contribution of ECL2 (in addition to the previously identified residues in TMs III-VII) to the SRIF ligand binding pocket.

Agonist-dependent regulation of cloned human somatostatin receptor types 1-5 (hSSTR1-5): subtype selective internalization or upregulation.

Agonist regulation of somatostatin receptors (SSTRs) was investigated in stable CHO-K1 cells individually expressing the 5 human (h) SSTR subtypes. hSSTR 2,3,4, and 5 displayed rapid agonist-dependent internalization of [125I] LTT SST-28 ligand in a time- and temperature-dependent manner over 60 min. Maximum internalization of radioligand occurred with hSSTR3 (78%) followed by hSSTR5 (66%), hSSTR4 (29%) and hSSTR2 (20%). In contrast, hSSTR1 displayed virtually no internalization. Prolonged agonist treatment led to differential upregulation of some of the SSTRs. After 22 h, hSSTR1 was upregulated at the membrane by 110%, hSSTR2 and hSSTR4 by 26% and 22% respectively, whereas hSSTR3 and hSSTR5 showed little change. Agonist-induced recruitment of hSSTR1 to the membrane was confirmed by immunocytochemistry with hSSTR1 antibodies. These results show that SST regulates all 5 hSSTRs by differential subtype selective internalization or upregulation. Subtype selectivity for internalization and upregulation is inversely related.

Molecular biology of somatostatin receptor subtypes.

Somatostatin (SRIF) receptors (ssts) comprise a family of heptahelical membrane proteins encoded by five related genes that map to separate chromosomes and which, with the exception of sst1, are intronless. The ssts1-4 display weak selectivity for SRIF-14 binding, whereas sst5 is SRIF-28-selective. Based on structural similarity and reactivity for octapeptide and hexapeptide sst analogs, ssts2,3 and sst5 belong to a similar sst subclass; ssts1-4 react poorly with these analogs and belong to a separate subclass. All five ssts are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin-sensitive guanosine triphosphate (GTP)-binding proteins. mRNA for ssts1-5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtype-selective and tissue- and species-specific. All pituitary cell subsets express sst2 and sst5, with sst5 being more abundant. Individual pituitary cells coexpress multiple sst subtypes. The binding pocket for SRIF-14 ligand lies deep within the membrane in transmembrane domains (TMDs) 3 to 7. Except for extracellular loop 2, it does not involve the other exofacial structures. Human (h)sst2A and hsst5 undergo agonist-mediated desensitization, associated with receptor internalization. The C-tail segment of hsst5 displays positive molecular internalization signals. The ssts inhibit the growth of tumor cells directly, through blockade of mitogenic signaling leading to growth arrest and through induction of apoptosis. This process is associated with translocation of phosphotyrosine phosphatase (PTP) 1C from the cytosol to the membrane.

Expression of mRNA for somatostatin receptor (sstr) types 2 and 5 in individual rat pituitary cells. A double labeling in situ hybridization analysis.

To characterize cell specific expression of sstr subtypes in the pituitary we have analyzed mRNA for sstr1-5 in rat pituitary somatotrophs by reverse transcriptase polymerase chain reaction and determined the pattern and level of expression of mRNA for sstr subtypes 2 and 5 in individual pituitary cell subpopulations by double label in situ hybridization. Purified somatotrophs expressed mRNA for all 5 sstrs. In situ hybridization analysis revealed sstr5 mRNA in 70% of somatotrophs, 57% of thyrotrophs, 38% of corticotrophs, 33% of lactotrophs, and 21% of gonadotrophs. mRNA for sstr2 occurred in 40% of somatotrophs, 36% of thyrotrophs, 26% of lactotrophs, 3% of corticotrophs, and 8% of gonadotrophs. Not only were more cells positive for sstr5 mRNA but the average number of autoradiographic grains/cell was also higher for sstr5 than sstr2. These results show expression of multiple sstr genes in individual pituitary cells. mRNA for sstr2 and 5 occur in each of the 5 major pituitary cell subsets, sstr5 mRNA being more widely and more abundantly expressed than sstr2.

The somatostatin receptor family.

The diverse biological effects of somatostatin (SST) are mediated through a family of G protein coupled receptors of which 5 members have been recently identified by molecular cloning. This review focuses on the molecular biology, pharmacology, expression, and function of these receptors with particular emphasis on the human (h) homologs. hSSTRs are encoded by a family of 5 genes which map to separate chromosomes and which, with one exception, are intronless. SSTR2 gives rise to spliced variants, SSTR2A and 2B. hSSTR1-4 display weak selectivity for SST-14 binding whereas hSSTR5 is SST-28 selective. Based on structural similarity and reactivity for octapeptide and hexapeptide SST analogs, hSSTR2,3, and 5 belong to a similar SSTR subclass. hSSTR1 and 4 react poorly with these analogs and belong to a separate subclass. All 5 hSSTRs are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin sensitive GTP binding proteins. Some of the subtypes are also coupled to tyrosine phosphatase (SSTR1,2), Ca2+ channels (SSTR2), Na+/H+ exchanger (SSTR1), PLA-2 (SSTR4), and MAP kinase (SSTR4). mRNA for SSTR1-5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtype-selective, and tissue- and species-specific. Pituitary and islet tumors express several SSTR genes suggesting that multiple SSTR subtypes are coexpressed in the same cell. Structure-function studies indicate that the core residues in SST-14 ligand Phe6-Phe11 dock within a ligand binding pocket located in TMDs 3-7 which is lined by hydrophobic and charged amino acid residues.

Expression of mRNA for all five human somatostatin receptors (hSSTR1-5) in pituitary tumors.

Expression of mRNA for hSSTR1-5 was determined in secretory (GH, PRL, TSH, ACTH) and nonsecretory pituitary tumors, as well as normal human fetal and adult pituitary by reverse transcriptase (RT) PCR followed by Southern blots. All 5 hSSTR subtype mRNAs were expressed in fetal pituitary, while adult pituitary was positive for 4 subtypes, lacking hSSTR4 mRNA. All 15 tumors analyzed were positive for SSTR mRNA, 14 expressing more than one subtype. SSTR2 mRNA in all tissues was expressed as the 2A variant, there being no detectable transcript for SSTR2B. Amongst the 5 SSTRs, mRNA for SSTR2A was the most frequently expressed (87% of tumors) followed by SSTR1 (73%), SSTR3 (53%), SSTR5 (47%), and SSTR4 (40%). The frequency and pattern of expression of the SSTR mRNAs was virtually identical in the different tumor subclasses and did not correlate with tumor size. Since pituitary tumors are monoclonal in origin, multiple SSTR genes are expressed in individual cells. Most tumors are rich in SSTR1 and SSTR2A mRNA compared to the other subtypes. This implies that SST analogs like SMS 201-995, known to interact with SSTR2A, but not with SSTR1, act on pituitary tumors mainly via the SSTR2 subtype.

Sequence analysis of the 5'-flanking promoter region of the human somatostatin receptor 5.

We have determined the sequence of 2.2 kb of 5' flanking promoter region of the human somatostatin receptor 5 (hsstr5) gene. A number of widely distributed promoter elements were identified including AP1, AP2, AP3, E2A, GCF, and SP1 consensus sequences. hsstr5/CAT gene fusions showed that the 0.9 kb of DNA immediately upstream of the ATG functions as a promoter in rat pituitary GH3 but not in CHO ovary cells. Insertion of this hsstr5 fragment in the anti-sense orientation led to a four fold reduction in CAT activity. Dibutyryl cAMP produced a three fold induction of CAT activity whereas estradiol and retinoic acid had no significant effect. These results indicate that we have identified a DNA fragment at the 5' end of the hsstr5 gene which contains both tissue-specific and regulated elements. The absence of CRE consensus sequence suggests that the cAMP effect is mediated by the multiple AP1 and AP2 sites.

Molecular cloning, functional characterization, and chromosomal localization of a human somatostatin receptor (somatostatin receptor type 5) with preferential affinity for somatostatin-28.

Using a combination of polymerase chain reaction and genomic library screening we have cloned a human gene for a subtype of the somatostatin (SST) receptor (SSTR) termed human SSTR5 (hSSTR5), which is located on chromosome 16. The predicted amino acid sequence of hSSTR5 displays 75% sequence identity with a recently identified rat SSTR [Mol. Pharmacol. 42:939-946 (1992)], suggesting that it is the human homologue of this receptor. hSSTR5 consists of a 363-residue polypeptide exhibiting a putative seven-transmembrane domain topology typical of G protein-coupled receptors. The receptor displays considerable sequence identity to hSSTR1 (42%), hSSTR2 (48%), hSSTR3 (47%), and hSSTR4 (46%). Membranes prepared from COS-7 cells transiently expressing the hSSTR5 gene bound 125I-Leu8,D-Trp22,Tyr25-SST-28 (125I-LTT-SST-28) with high affinity and in a saturable manner. SST-14, SST-28, and various synthetic SST peptide agonists produced dose-dependent inhibition of radioligand binding with the following rank order of potency: LTT-SST-28 > SST-28 > D-Trp8-SST-14 > SST-14 approximately RC-160 approximately BIM 23014 > MK-678 > SMS 201-995. hSSTR5 bound SST-28 with a 12.6-fold greater affinity (Ki = 0.19 nM), compared with SST-14 (Ki = 2.24 nM), indicating that the receptor is SST-28 selective. Addition of GTP, guanosine-5'-O-(3-thio)triphosphate, Na+ ions, or pertusis toxin greatly reduced 125I-LTT-SST-28 binding, thereby indicating that hSSTR5 is coupled to pertussis toxin-sensitive G proteins. Both SST-14 and SST-28 displayed dose-dependent inhibition of forskolin-stimulated cAMP accumulation, consistent with functional coupling of the receptor to adenylyl cyclase inhibition. Northern blot analysis of SSTR5 mRNA revealed a 2.4-kilobase transcript in normal rat pituitary and GH3 rat pituitary tumor cells and a 4.0-kilobase transcript in normal human pituitary. Reverse transcriptase polymerase chain reaction revealed expression of the hSSTR gene in fetal human pituitary and hypothalamus but not in human cerebral cortex. In situ hybridization of the rat pituitary showed that SSTR5 mRNA is selectively localized in the anterior lobe. SSTR5 mRNA was not expressed in four human pituitary tumors (somatotroph adenoma, prolactinoma, and chromophobe adenomas) or in a human insulinoma. Although hSSTR5 displays approximately 75% sequence identity with rat SSTR5, the two receptors display significantly different pharmacological profiles, especially with respect to their binding affinities for the SST analogue SMS 201-995.

Expression of multiple somatostatin receptor genes in AtT-20 cells. Evidence for a novel somatostatin-28 selective receptor subtype.

The pattern of expression of somatostatin receptor (SSTR) genes and gene products in AtT-20 cells was characterized in an attempt to explain the SST-28 binding selectivity that typifies these cells. AtT-20 cells expressed multiple SSTR mRNAs. Paradoxically, this included mRNA for three of the four SST-14 selective receptors: SSTR2 ( +), SSTR1 (+), SSTR4 (+). The SST-28 selective SSTR5 was expressed as a 3.8-kilobase (kb) transcript of relatively low abundance (+) in contrast to normal mouse pituitary which displayed high levels ( ) of a 2.4-kb SSTR5 mRNA. Immunoblot analysis of solubilized membranes with an antipeptide SSTR2 antibody revealed a single SSTR2 protein of 72 +/- 2 kDa. Preincubation of AtT-20 cell membranes with SSTR2 antibody reduced 125I-[Leu8,D-Trp22,Tyr25]SST-28 binding sites by 38%. Residual binding sites exhibited a 4.9-fold increase in affinity for SST-28, a 2.6-fold decrease in affinity for SST-14, and an SST-28:SST-14 potency ratio of 40:1 compared with a potency ratio of 3.5:1 in control membranes. These results demonstrate the expression of four SSTR genes in AtT-20 cells of which SSTR2 predominates. Blockade of SSTR2 with antibody exposes high affinity SST-28 selective sites with comparable binding characteristics to those reported for cloned SSTR5. These SST-28 binding sites may arise from a SSTR5 variant encoded by a high molecular weight 3.8-kb transcript or more likely from another as yet undiscovered member of the SST-28 selective SSTR subfamily.