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Somatostatin (somatotropin release inhibiting factor) is an abundant neuropeptide, which acts on five subtypes of somatostatin receptor (SST1-SST5; nomenclature as agreed by the NC-IUPHAR Subcommittee on Somatostatin Receptors [98]). Activation of these receptors produces a wide range of physiological effects throughout the body including the inhibition of secretion of many hormones. Endogenous ligands for these receptors are somatostatin-14 (SRIF-14) and somatostatin-28 (SRIF-28). cortistatin-14 has also been suggested to be an endogenous ligand for somatostatin receptors [61].

Background: Intra articular fractures of proximal tibia are difficult to treat. Age, skin conditions, osteoporosis further increase the obstacles in the healing process. Various modalities of treatment are available but no ideal treatment has yet evolved. The aim of this study is to study the result of tibia plating in intra articular tibia fracture in terms of union, knee movement, infection and functional ability of the patient. Materials and methods: This is a prospective cohort study of 30 cases of intraarticular proximal tibia fractures, aged >18 years during the period 2018-2019 in our hospital. Fractures classified according to Schatzker Classification All are treated by open reduction and internal fixation with proximal tibial plate and screws, Post-operatively Above knee slab or removable knee brace with leg elevation given to decrease the pain and edema. Static quadriceps exercises and ankle pump exercise started on second day. The patients with stable fixation were allowed intermittent knee mobilization once the wound pain subsided, depending upon type of fracture. The patient is followed up at regular intervals of 4, 8, 12 and 16 weeks. The functional outcome is assessed at the end of 16 weeks by KNEE SOCIETY SCORE. Results: We had 73.33% excellent results, 20% good results, 6.66% fair results. Conclusion: In our study type V is the most common type of fracture pattern. Lateral locking plate with rafting screws provides support for postromedial and posterolateral fragments. Locking plate provides better stability than conventional buttress plating.

Introduction: Shaft humerus fracture is one of the most common fracture in our newly developed world. Wide range of treatment modalities available from conservative to nailing and plating. Our study done to evaluate the clinical, radiological, and functional outcome of such fractures in 20 patients with anterior bridge plating with MIPO technique. Though open reduction and plating technique of humerus shaft fracture is gold standard, this technique also gives good outcome. Materials and methods: A prospective study of anterior bridge plating ith MIPO technique was carried out at our institute, at the Department of Orthopaedics, S.S.G. hospital Vadodara between November 2018 and July 2019, were 20 patients of shaft hmerus fracture selected after applying inclusion and exclusion criteria. Fracture s were classified as per AO classification. Assessment of union, movement at shoulder and elbow joint, Quick DASH score and complications were done during regular follow up of 4 month. Results: In our study, after clinical and radiological assessment after 4 monthh follow up,out of the 20 patients in the study, 16 were males and 4 were females. The mean age was 42.35 years (range 19 to 63 years). The mean fracture union (radiological) time was 13.5 weeks (range: 10–16 weeks). According to quick DASH score 18(90%) patients has excellent result and remaining 2(10%) patients had good results. Conclusion: There is high rate of union and excellent functional outcomes by this procedure. It is concluded from the study that close reduction with internal fixation with anterior bridge plating & MIPO technique is an excellent mode of treatment for shaft humerus fracture which constantly give long term good result with less complication and early postoperative mobilization.

Introduction: Proximal humerus fractures are common and debilitating injuries, especially in elderly as osteoporosis and deforming forces of muscle attached. Wide range of treatment modalities ranging from conservative management to shoulder arthroplasty. In our study we have evaluated proximal humerus interlocking osteosynthesis (PHILOS) plate in displaced and comminuted fracture due to its rigid construction, angular stability and early post- operative rehabilitation. Objective of Study: The objective of study 1) To evaluate and analyze the functional outcomes of PHILOS plate for displaced fractures of proximal humerus 2) To improve stability in osteoporotic humeral bone 3) To preserve the biological integrity of the humeral head and to secure an anatomical reduction with multiple locking screws with angular stability 4) To study the complication rates of PHILOS plating in proximal humerus fractures Methodology: A prospective study of treatment of proximal humerus fractures treated with PHILOS plate was carried out at our institute, at the Department of Orthopaedics, S.S.G. Hospital Vadodara from June 2017 to august 2018, where after applying exclusion and inclusion criterion, 20 patients of proximal humerus fractures were included in our study. Fractures were classified as per NEER’S classification. Assessment of union, movements at shoulder, constant score and complications were done during regular follow-up of 5 month. The functional outcome was measured using Constant and Murley Shoulder Scoring System. Results: In present study, after clinical and radiological assessment after 5 months follow-up, mean Constant and Murley score of 84.85 was achieved.9 patients (45%) had excellent result with score more than 90 and 7 (35%) patients had good result with score more than 80, 2 patients (10%) had fair result with score more than 70. 2(10%) patients had poor results with score less than 70 may be due to with medial cortical comminution and osteoporosis in which malreduction and shoulder stiffness are most frequent complication. Conclusions: The divergent and convergent orientation of the locking screws of PHILOS plate provides stable biological fixation with high union rate to use for treatment of fractures of the proximal humerus in Neer’s 2-part, 3-part, and 4-part with better functional and radiological outcome especially in osteoporotic bone due to low complication and early postoperative mobilization. Potential minimal complication can be prevented by advanced surgical skill and expertise and rotator cuff tying.

Objectives: A prospective study of management of fracture distal radius by external fixator using the principle of ligamentotaxis was conducted from July 2016 to August 2017 at our institute to evaluate the clinical efficacy of external fixator with ligamentotaxis in distal radius fracture reduction, fracture healing, functional recovery after surgery. Material and Methods: 30 cases of fracture distal radius were treated with ligamentotaxis. Inclusion criterias were 1) skeletally mature patients having age more than 18 years 2) closed/unstable fractures. 3) Frykman type 1 to 8 fractures. 4) Fractures with history of trauma

Introduction: Stress fractures have continued to plague competitive athletes and military recruits, especially at the beginning of military service. The underlying pathophysiology of stress fractures occurs when the rate of repetitive microtrauma exceeds that of osseous remodeling. The prevention of lower extremity stress fractures remains a long-standing problem in the military, and it represents a significant socioeconomic burden. Treatment strategies include early identification of the symptoms, early diagnosis, a sufficiently long training pause. The present study aimed at determining the incidence and type of Stress Fractures in Border Security Force cadets (BSF) recruited in Indore through clinical and radiological diagnosis. Material and Methods: In a prospective study of Stress Fractures, a group of 1000 BSF cadets training in Indore was selected and those presenting with symptoms were evaluated clinically and radiologically. The SF was graded in to 4 groups based on the suggested clinicoradiological classification by Agarwal. Results: One hundred and seventy four (174) out of 1000 consecutive recruits had symptoms of SFs. Out of 150 diagnosed with SF 60 (40%) were Grade I, 54 (36%) was Grade II, 29 (19.33%) were Grade III and 7 (4.67%) were Grade IV. The significant higher incidence of SFs has been attributed to training with maximum stress on running, jumping, parade on hard ground, and gymnastics. The maximum occurrence of Stress fracture in our study was found at 10 weeks of training. Conclusion: There is a high incidence of SF in military cadets which tends to remain unreported otherwise. Proper education of trainees, trainers and instructors, modification in training procedures, use of better equipments can reduce occurrence of these fractures. Early reporting to hospital and treatment is also necessary as it can help in early return to full activity.

Background: Clavicle fractures are common injuries in young, active individuals which were treated conservatively with a sling or figure of 8 brace. In the present study, we have treated displaced clavicle fractures with open reduction and internal fixation using anatomical locking clavicle plate. Materials and Methods: All the patients were evaluated clinically according to DASH SCORING and radiologically at 5 months post operatively Results: In majoring of the patient, fractures were united within 7 to 8 weeks. Mean union time is 7.3 weeks. Majority had FINAL DASH SCORE abound 32. All 20 patients had full range of motion at final follow up without any limitation Discussion: Result of previous studies compared with present study Conclusion: Precontoured Anatomical Locking Clavicle Plate is preferred for the treatment of displaced or non united clavicle fractures with better functional outcome and faster recovery compared to other modes of treatment.

Background and Objectives: Fractures of shaft of humerus consists of 1% - 3% of all fractures. Despite the numerous surgical modalities, plate osteosynthesis remains the gold standard for fixation of humerus shaft fractures. The aim of this study is to see the functional results of humerus shaft fractures treated with plating, reemphasizes the effectiveness of plate osteosynthesis in the management of humeral shaft fractures so as to restore the patient structurally and functionally to near normal status. Materials and Methods: This is retrospective and prospective study of 30 cases of fractures of shaft of humerus admitted at S.S.G. Hospital, Vadodara, Gujarat in between july 2015 to june 2016. Cases were sleceted as per inclusion and exclusion criteria. All patients were operated using posterior or anterolateral approach. They were assessed radiologically and clinically for fracture union at regular intervals of 6 wks, 12 wks and 18wks by using DR. R.N. Deweshwar’s Score.Results: Our study of 30 cases, there were 25 men and 5 women, with average age of 39 years.16 (53.33%) patients were admitted due to fall from height,12 (40%) due to road traffic accidents and 2 (6.66%) patients due to assult with predominance of right side 20(66.66%) patients. Out of 30 cases 21(70%) patients of middle third, 7 (23.33%) were lower third, and 2 (6.66%) were upper third. 28 patients having close fractures and 2 have open injury. simple transverse fractures were most common that is 12(40%).10 (33.33%) patients were having associate injuries. The fracture united in 29(96.66%) patients with 1 (3.33%) patient going for non union. Good and full range of shoulder and elbow movements was present in 28 (93.33%) patients with 2 patients having fair shoulder range of movement and 2 patients having fair elbow range of movement. Conclusion: Open reduction internal fixation with plating is an excellent mode of treatment in fracture of shaft of humerus, which consistently gives long term good results.

Background: Distal tibial fractures including tibial pilon present a challenge due to subcutaneous location of tibia and precarious blood supply of distal leg. MIPO (Minimal invasive plating osteosynthesis) has evolved as a newer concept to treat distal tibial fractures with minimal articular comminution and minimal soft tissue damage Method: A prospective study for the management of distal tibial fractures by MIPO technique by using LCP (locking compression plate) was done, in 20 patients. Patients were followed and scored according to TEENY and WISS scoring criteria. Result: Among 20 patients, there were 16 males and 4 females with a mean age of 41.4 yrs (range from 20-76yrs). High energy trauma (road traffic accident) predominated causing 11 fractures. Superficial wound infections were seen in 02 cases, surgical wound breakdown with implant exposure in 01 case. 15 patients had excellent, 3 patients had good outcome and 02 patients had fair outcome. Conclusion: We conclude that MIPO is an effective technique for the management of distal tibial fractures.

Somatostatin (somatotropin release inhibiting factor) is an abundant neuropeptide, which acts on five subtypes of somatostatin receptor (SST1-SST5; nomenclature as agreed by the NC-IUPHAR Subcommittee on Somatostatin Receptors [89]). Activation of these receptors produces a wide range of physiological effects throughout the body including the inhibition of secretion of many hormones. Endogenous ligands for these receptors are somatostatin-14 (SRIF-14) and somatostatin-28 (SRIF-28). cortistatin-14 has also been suggested to be an endogenous ligand for somatostatin receptors [56].

Soils contain natural reserves of plant nutrients but these reserves are largely in different forms which are used for plant growth. Also, Plant nutrients are not available to plants, because of the extra use of the chemical fertilizers. Chemicals make the nutrients, which remain in soil, in active and so are not used by plants, also make the soil polluted. The using of chemical fertilizer since long time results in the soil being full of chemicals thus damaging the production and full of harmful chemicals to the human body. As an option to all this use of biofertilizers can help us get back our soil health by natural way ultimately the health of organisms.

Background: The implants removal after fracture healing has always been an issue of controversy. After union, the implant ceases to be important and can be removed. Nevertheless, some patients require metallic implant removal due to various implant-related difficulties. Our study was aimed to identify the most common causes for removal of implant.Methods: The patients admitted for implant removal in our department were consented and included in the study. Pre-operative radiological images collected and evaluated. Post-operatively, images were taken and followed for resolution of symptoms or appearance of new problems.Results: A total of 60 patients were studied. Of these, 47 were males and 13 were females. Mean age was 33.7 years (range 4-70 years). Patient request (35% of patients) was the main indication for removal of implants. Reasons were found to be discomfort due to implant, infection, failure of implants and others.Conclusions: Belief regarding hardware removal has been set and most of them are agreed that routine removal should not be performed unless obviously indicated. In our attempt to fill this gap, we trust that routine removal should not be performed in ‘asymptomatic’ patients. The procedure should not need a big procedure than the implant fixation surgery. Procedure should be sought also as a risk like refracture, bleeding, infection, neurovascular injury and prior to surgery, proper consent to be taken and patient should be well explained. Sometimes indicated results expected after surgery can’t be fulfilled, and instead, complication can results.

Background: The intertrochanteric fractures are extra capsular fractures of proximal femur in the trochanteric region. Different fixation techniques were tried for intertrochanteric fractures, with variety of implants but the dynamic hip screw fixation is most widely accepted treatment. However, several authors have concluded that sliding compression screws may be associated with several complications such as perforation of the femoral head, loss of reduction caused by excessive sliding of the lag screw, non-union, shortening of the affected limb and pain. This study was carried out to ascertain the factors that contributed to mechanical failure at our institute.Methods: We retrospectively reviewed 92 patients with unilateral intertrochanteric fracture treated with a sliding hip screw between July 2015 and April 2017. Postoperative radiographs were studied for any loss of reduction, which was defined as a varus deformity greater than 10°, perforation of the femoral head, extrusion of the lag screw of more than 20 mm, or metal failure. The Pearson chi-square test was used to assess the relationship between failure and osteoporosis. A p value of less than 0.05 was considered to be significant.Results: Results revealed a significant relationship between failure and osteoporosis. A possible relationship between the stability of the fracture on Evans’ classification and osteoporosis on Singh’s index was investigated which revealed a high positive correlation between the failure rates of unstable fractures with osteoporosis.Conclusions: An unstable fracture combined with osteoporosis, has higher percentage of fixation failure leading to other methods of treatment like hemiarthroplasty.

The actions of somatostatin (SST) in the nervous system are mediated by specific high affinity SST receptors (SSTR1-5). However, the role of this hormone and the distribution of its receptor subtypes have not yet been defined in neural structures of the human fetus. We have analyzed four neural tissues (CNS, hypothalamus, pituitary and spinal cord) from early to midgestation for the expression of five human SSTR mRNAs, using a reverse transcription-polymerase chain reaction and Southern blot approach. These fetal neural tissues all express mRNA for multiple SSTR subtypes from as early as 16 weeks of fetal life but the developmental patterns of expression vary considerably. Transcripts for SSTR1 and SSTR2A are the most widely distributed, being expressed in all four neural tissues. SSTR2A is often the earliest transcript to be detected (7.5 weeks in CNS). SSTR3 mRNA is confined to the pituitary, hypothalamus, and spinal cord. SSTR4 is expressed in fetal brain, hypothalamus and spinal cord but not pituitary. SSTR5 mRNA is detectable in the pituitary and spinal cord by 14-16 weeks of fetal life. This mapping of SSTR mRNA expression patterns in human fetal neural tissues is an important first step toward our goal of determining the role of SST in the nervous system during early stages in human development.

OBJECTIVE To compare the expression and cellular distribution of D(2)-like dopamine receptors in the kidney of the spontaneously hypertensive rat (SHR) and normotensive Wistar-Kyoto (WKY) rat. DESIGN Renal D(2)-like receptor protein expression and distribution has not been studied in the SHR. Since changes in D(2)-like receptor expression and/or distribution may contribute to the dysregulation of renal dopamine and D(1A) receptor function, we examined the expression of the three subtypes of D(2)-like receptors (D(2), D(3) and D(4)) in SHR and WKY rat kidneys. METHODS Western blot analysis and confocal immunocytochemistry with specific polyclonal antipeptide antibodies directed against the receptor subtypes, were used to assess protein expression. RESULTS There were no differences in protein expression and cellular immunolocalization of the D(2) receptor subtypes between SHR and WKY rats. Expression of the 50 kDa D(3) receptor was reduced in the cortex of the SHR; no differences in D(3) receptor levels were seen in the inner medulla of SHR and WKY rats. The D(4) receptor polypeptides were overexpressed in the cortex of SHR, while in the inner medulla no difference in expression of the D(4) receptor proteins was observed between SHR and WKY rats. Immunocytochemistry also showed increased immunostaining of D(4) receptors in tubular structures in the cortex, but diminished staining in the SHR inner medulla. CONCLUSION The observed differences in expression and distribution of D(3) and D(4) dopamine receptors between cortex and inner medulla of the kidneys of SHR and WKY rats may contribute to the aberrant state of dopaminergic-mediated natriuresis in SHR.

Apolipoprotein D (apoD), a member of the lipocalin superfamily of ligand transporters, has been implicated in the transport of several small hydrophobic molecules including sterols and steroid hormones. We have previously established that apoD is a secreted protein from cultured mouse astrocytes and that treatment with the oxysterol 25-hydroxycholesterol markedly stimulates apoD release. Here, we have investigated expression and cellular processing of apoD in the Niemann-Pick type C (NPC) mouse, an animal model of human NPC, which is a genetic disorder affecting cellular cholesterol transport. NPC is phenotypically characterized by symptoms of chronic progressive neurodegeneration. ApoD gene expression was up-regulated in cultured NPC astrocytes and in NPC brain. ApoD protein levels were also increased in NPC brain with up to 30-fold higher apoD content in the NPC cerebellum compared with control mice. Subcellular fractionation of NPC brain homogenates revealed that most of the apoD was associated with the myelin fraction. ApoD was found to be a secreted protein from cultured normal astrocytes and treatment with the oxysterol, 25-hydroxycholesterol, markedly stimulated apoD release (by five- to 10-fold). By contrast, secretion of apoD from NPC astrocytes was markedly reduced and could not be stimulated by oxysterol treatment. Secretion of apoE, another apolipoprotein normally produced by astrocytes, was similar in NPC and control cells. Furthermore, apoE secretion was not potentiated by oxysterol treatment in either cell type. Plasma levels of apoD were sixfold higher in NPC, whereas hepatic levels were substantially reduced compared with controls, possibly reflecting reduced hepatic clearance of the circulating protein. These results reveal hitherto unrecognized defects in apoD metabolism in NPC that appear to be linked to the known defects in cholesterol homeostasis in this disorder.

The sections in this article are: 1 Anatomical Distribution of Somatostatin Cells 1.1 Localization 1.2 Pancreatic Somatostatin Cells 2 Biosynthesis, Processing, and Intracellular Targeting 2.1 Somatostatin Genes and Gene Products 3 Regulation of Islet Somatostatin 3.1 Regulation of Secretion 3.2 Regulation of Gene Expression 4 Actions and Mechanism of Action of Somatostatin 4.1 Islet Cell Actions 4.2 Extra-Islet Actions 4.3 Somatostatin Agonists 4.4 Somatostatin Receptors 5 Metabolism of Somatostatin 6 Circulating Somatostatin 7 Islet Somatostatin Function 7.1 Paracrine Regulation 7.2 Regulation via the Microcirculation 7.3 Gap Junctional Coupling 7.4 Independent Regulation by Somatostatin-14 and Somatostatin-28 8 Somatostatin and Diabetes 8.1 Experimental Insulinopenic Diabetes 8.2 Experimental Hyperinsulinemic Diabetes 8.3 Human Diabetes 9 Concluding Remarks

Pulsatile growth hormone (GH) secretion differs between males and females and regulates the sex-specific expression of cytochrome P450s in liver. Sex steroids influence the secretory dynamics of GH, but the neuroendocrine mechanisms have not been conclusively established. Because periventricular hypothalamic somatostatin (SST) expression is greater in males than in females, we generated knockout (Smst-/-) mice to investigate whether SST peptides are necessary for sexually differentiated GH secretion and action. Despite marked increases in nadir and median plasma GH levels in both sexes of Smst-/- compared with Smst+/+ mice, the mutant mice had growth curves identical to their sibling controls and retained a normal sexual dimorphism in weight and length. In contrast, the liver of male Smst-/- mice was feminized, resulting in an identical profile of GH-regulated hepatic mRNAs between male and female mutants. Male Smst-/- mice show higher expression of two SST receptors in the hypothalamus and pituitary than do females. These data indicate that SST is required to masculinize the ultradian GH rhythm by suppressing interpulse GH levels. In the absence of SST, male and female mice exhibit similarly altered plasma GH profiles that eliminate sexually dimorphic liver function but do not affect dimorphic growth. Fil: Low, Malcolm J.. Oregon Health And Science University; Estados Unidos Fil: Otero Corchon, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Oregon Health And Science University; Estados Unidos Fil: Parlow, Albert. F.. University of California at Los Angeles; Estados Unidos Fil: Ramirez, Jose L.. McGill University; Canadá Fil: Kumar, Ujenda. McGill University; Canadá Fil: Patel, Yogesh C.. McGill University; Canadá Fil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

BACKGROUND We have analysed the distribution of the five somatostatin receptors (sst1–5) by immunohistochemistry in a large retrospective series of 51 medullary carcinoma of the thyroid (MCT) specimens and correlated the pattern of sst expression with expression of somatostatin (SRIF) peptide, tumour pathology and clinical outcome. MEASUREMENTS Immunohistochemistry was performed with rabbit polyclonal antipeptide antibodies directed against the extracellular domains or cytoplasmic tail of human (h) sst1–5. SRIF immunoreactivity was investigated in parallel paraffin sections. RESULTS Eighty-five percent of the tumours were positive for one or more sst, localized to both tumour cells as well as surrounding peritumoural structures, especially blood vessels. Forty-nine percent of the tumours were positive for sst1, 43% for sst2, 47% for sst3, 4% for sst4, and 57% for sst5. Fifty-one percent of tumours expressed one or two sst subtypes; 33% were positive for three or more sst isoforms. All five sst receptors were detected in only two cases. Tumours expressing octreotide sensitive subtypes (sst2,3,5) accounted for 75% of the series. 50% of the tumours co-expressed SRIF suggesting tumour cell regulation by endogenous SRIF via paracrine/autocrine circuits. There was no correlation between sst1–5 expression and age, sex, tumour size or stage, histological type or clinical outcome. Simultaneous analysis of primary tumour and lymph node metastases revealed a similar pattern of sst immunoreactivity indicating that sst expression is not modified in the course of disease progression. CONCLUSIONS With the exception of sst4, medullary carcinoma of the thyroid display a rich but heterogeneous expression of sst subtypes. Immunohistochemical typing of sst receptor expression using specific antireceptor antibodies represents an ideal approach for characterizing sst subtype expression in medullary carcinoma of the thyroid for optimizing receptor targeted diagnosis and therapy with somatostatin analogs.

Activation of initiator and effector caspases, mitochondrial changes involving a reduction in its membrane potential and release of cytochrome c (cyt c) into the cytosol, are characteristic features of apoptosis. These changes are associated with cell acidification in some models of apoptosis. The hierarchical relationship between these events has, however, not been deciphered. We have shown that somatostatin (SST), acting via the Src homology 2 bearing tyrosine phosphatase SHP-1, exerts cytotoxic action in MCF-7 cells, and triggers cell acidification and apoptosis. We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP-1-mediated caspase-8 activation is required for SST-induced decrease in pH(i). (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pH(i) is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis. These data reveal that SHP-1-/caspase-8-mediated acidification occurs at a site other than the mitochondrion and that SST-induced apoptosis is not dependent on disruption of mitochondrial function and caspase-9 activation.

Niemann–Pick type C disease (NP-C) is an inherited neurovisceral lipid storage disorder characterized by progressive neurodegeneration. Most cases of NP-C result from inactivating mutations of NPC1 , a recently identified member of a family of genes encoding membrane-bound proteins containing putative sterol sensing domains. By using a specific antipeptide antibody to human NPC1, we have here investigated the cellular and subcellular localization and regulation of NPC1. By light and electron microscopic immunocytochemistry of monkey brain, NPC1 was expressed predominantly in perisynaptic astrocytic glial processes. At a subcellular level, NPC1 localized to vesicles with the morphological characteristics of lysosomes and to sites near the plasma membrane. Analysis of the temporal and spatial pattern of neurodegeneration in the NP-C mouse, a spontaneous mutant model of human NP-C, by amino–cupric–silver staining, showed that the terminal fields of axons and dendrites are the earliest sites of degeneration that occur well before the appearance of a neurological phenotype. Western blots of cultured human fibroblasts and monkey brain homogenates revealed NPC1 as a 165-kDa protein. NPC1 levels in cultured fibroblasts were unchanged by incubation with low density lipoproteins or oxysterols but were increased 2- to 3-fold by the drugs progesterone and U-18666A, which block cholesterol transport out of lysosomes, and by the lysosomotropic agent NH 4 Cl. These studies show that NPC1 in brain is predominantly a glial protein present in astrocytic processes closely associated with nerve terminals, the earliest site of degeneration in NP-C. Given the vesicular localization of NPC1 and its proposed role in mediating retroendocytic trafficking of cholesterol and other lysosomal cargo, these results suggest that disruption of NPC1-mediated vesicular trafficking in astrocytes may be linked to neuronal degeneration in NP-C.

We have developed a panel of rabbit polyclonal antipeptide antibodies against the five human somatostatin receptor subtypes (hSSTR1-5) and used them to analyze the pattern of expression of hSSTR1-5 in normal human islet cells by quantitative double-label confocal fluorescence immunocytochemistry. All five hSSTR subtypes were variably expressed in islets. The number of SSTR immunopositive cells showed a rank order of SSTR1 > SSTR5 > SSTR2 > SSTR3 > SSTR4. SSTR1 was strongly colocalized with insulin in all beta-cells. SSTR5 was also an abundant isotype, being colocalized in 87% of beta-cells. SSTR2 was found in 46% of beta-cells, whereas SSTR3 and SSTR4 were relatively poorly expressed. SSTR2 was strongly colocalized with glucagon in 89% of alpha-cells, whereas SSTR5 and SSTR1 colocalized with glucagon in 35 and 26% of alpha-cells, respectively. SSTR3 was detected in occasional alpha-cells, and SSTR4 was absent. SSTR5 was preferentially expressed in 75% of SST-positive cells and was the principal delta-cell SSTR subtype, whereas SSTR1-3 were colocalized in only a few delta-cells, and SSTR4 was absent. These studies reveal predominant expression of SSTR1, SSTR2, and SSTR5 in human islets. Beta-cells, alpha-cells, and delta-cells each express multiple SSTR isoforms, beta-cells being rich in SSTR1 and SSTR5, alpha-cells in SSTR2, and delta-cells in SSTR5. Although there is no absolute specificity of any SSTR for an islet cell type, SSTR1 is beta-cell selective, and SSTR2 is alpha-cell selective. SSTR5 is well expressed in beta-cells and delta-cells and moderately well expressed in alpha-cells, and thereby it lacks the islet cell selectivity displayed by SSTR1 and SSTR2. Subtype-selective SSTR expression in islet cells could be the basis for preferential insulin suppression by SSTR1-specific ligands and of glucagon inhibition by SSTR2-selective compounds.

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.

The human somatostatin receptor subtype 5 (hSSTR5) gene has previously been cloned and localized to chromosome 16 p13.3. This region is evolutionarily conserved in all vertebrate genomes from the puffer fish (Fugu rubripes) to human, and also contains loci for genes associated with two common multisystemic disorders, adult polycystic kidney disease (PKD1) and tuberous sclerosis (TSC2). Analysis of the 5′ flanking region of the hSSTR5 gene has revealed consensus sequences for a number of transcription factors as well as Alu-like repeat elements. In the present study, genomic DNA from 53 unrelated individuals was analysed by PCR and Southern blots probed with radiolabeled fragments generated from different segments of the hSSTR5 gene. We have identified two restriction fragment length polymorphisms (RFLP) with high heterozygosity values at the 5′ flanking region of the hSSTR5 gene. These RFLP markers will be useful for determining the allelic loss of genetic material from this region. The observed polymorphism in the promoter region may affect the function of the hSSTR5 gene.

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.

Using quantitative double-label fluorescence immunocytochemistry and confocal microscopy, we have analysed the pattern of expression of SSTR1-5 in normal rat pituitary somatotrophes. Antipeptide rabbit polyclonal antibodies were produced against the extracellular domains of SSTR1-5. SSTR antigens were colocalized in GH positive cells using rhodamine conjugated secondary antibody for SSTRs and FITC-conjugated secondary antibody for GH. SSTR5 was the predominant subtype which was expressed in 86 +/- 9.7% of GH cells followed by SSTR2 in 42 +/- 6.4% of GH positive cells. SSTR4 and SSTR3 were modestly expressed in 23 +/- 4.7% and 18 +/- 3.2% of somatotrophes respectively whereas SSTR1 was the least expressed subtype occurring in only 5 +/- 1.2% of somatotrophes. These results demonstrate variable expression of the 5 SSTRs in somatotrophes. The preponderance of the SST-28 preferring SSTR5 subtype correlates with the reported higher potency of SST-28 than SST-14 for inhibiting GH secretion.

We have characterized the biosynthetic origin of somatostatin-14 (SS-14), SS-28, and pro-SS [1-10] from pro-SS (PSS) in 1027B 2 rat islet tumor cells. Because these cells lack regulated secretion and show unresponsiveness of the SS gene to cAMP, we have additionally carried out morphological and functional studies to elucidate the molecular defect in cAMP signalling and to localize the sites of PSS maturation along the secretory pathway. Cell extracts and secretion media were analysed by high performance liquid chromatography and specific C- and N-terminal radioimmunoassays. Electron microscopic sampling of 1027B 2 cell cultures showed that most cells had very few dense core secretory granules for heterogeneous sizes. The cells expressed the endoproteases furin, PC1, and PC2 and contained large quantities of fully processed SS-14 and SS-28 with very little unprocessed PSS (ratio SS-14:SS-28:PSS=39:51:10%). They secreted high concentrations of SS-14, SS-28, and PSS [1-10] constitutively along with PC1 and PC2. Pulse-chase studies demonstrated that PSS is rapidly (within 15 min), and efficiently processed to SS-14, SS-28, and PSS [1-10] via separate biosynthetic pathways: PSS→SS-14+8 kDa; PSS→SS-28+7 kDa; PSS→PSS [1-10] . Monensin reduced intracellular SS-like immunoreactivity without altering processing efficiency. Transfection with the catalytic subunit of protein kinase A (PKA-C) activated SS promoter-CAT activiting indicating that the defect in cAMP-dependent signaling in 1027B 2 cells lies at the level of PKA-C. PKA-C overexpression failed to alter the ratio of processed SS-14 and SS-28. These results demonstrate that SS-14, SS-28, and PSS [1-10] are independently synthesized from PSS and that efficient precursor processing can occur within the constitutive secretory pathway in the relative absence of dense core secretory vesicles.

This paper examines the role of medical expertise in clinical reasoning, using a complete workup of a patient with an endocrine disorder. Endocrinologists, housestaff, and final year medical students were asked to develop the case. This consisted of history-taking, interpreting physical examination results, request for tests and their interpretations, and providing therapeutic and patient management plans, and an explanation of the pathophysiology underlying the problem. The subjects were also asked to provide explanations supporting their decisions during the patient workup. A variety of techniques deriving from cognitive psychology were used to analyze the data. The main concern was how expertise affected the building of relationship between the components of the workup. Experts formed integrated knowledge-rich structures that were generated during the history-taking and used consistently throughout the workup. Housestaff formed more tentative and less integrated representations which were modified during the patient encounter. Students representations superficially resembled those of experts, but were knowledge-lean. The results are interpreted in terms of clinical performance of endocrinologists, housestaff, and students during the workup, and its relationship to the explanation task as an indicator of clinical competence of the patient problem provided by the subjects. It is proposed that the explanation given after the problem solving process could be used as an indicator of clinical competence.

Somatostatin (SST) exerts direct antiproliferative effects in tumor cells, triggering either growth arrest or apoptosis. The cellular actions of SST are transduced through a family of five distinct somatostatin receptor subtypes (SSTR1-5). Whereas growth inhibition has been reported to follow stimulation of protein tyrosine phosphatase via SSTR2 or inhibition of Ca2+ channels via SSTR5 in heterologous expression systems, the subtype selectivity for signaling apoptosis has not been investigated. The tumor suppressor protein p53 and the protooncogene product c-Myc regulate cell cycle progression (growth factors present) or apoptosis (growth factors absent). The p53-induced G1 arrest requires induction of p21, an inhibitor of cyclin-dependent kinases, whereas apoptosis requires induction of Bax. c-Myc is capable of abrogating p53-induced G1 arrest by interfering with the inhibitory action of p21 on cyclin-dependent kinases. We have, therefore, investigated the regulation of p53, p21, c-Myc, and Bax and cellular apoptosis in relation to cell cycle progression in CHO-K1 cells stably expressing individual human SSTR1-5. We demonstrate that apoptosis is signaled uniquely through human SSTR3 and is associated with dephosphorylation-dependent conformational change in wild-type (wt) p53 as well as induction of Bax. The induction of wt p53 occurs rapidly and precedes the onset of apoptosis. We show that the increase in wt p53 is not associated with the induction of p21 or c-Myc when octreotide-induced apoptosis becomes evident, suggesting that such apoptosis does not require G1 arrest and is not c-Myc dependent. These findings provide the first evidence for hormonal induction of wt p53-associated apoptosis via G protein-coupled receptor in a subtype-selective manner.

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 sst 1 , are intronless. The ssts 1–4 display weak selectivity for SRIF-14 binding, whereas sst 5 is SRIF-28—selective. Based on structural similarity and reactivity for octapeptide and hexapeptide sst analogs, ssts 2,3 and sst 5 belong to a similar sst subclass; ssts 1–4 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 ssts 1–5 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 sst 2 and sst 5 , with sst 5 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)sst 2 A and hsst 5 undergo agonist-mediated desensitization, associated with receptor internalization. The C-tail segment of hsst 5 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.

The role of PC2 in prosomatostatin (PSS) processing was investigated in GH3/GH4C1 pituitary cells. These cells are sparsely granulated, express different amounts of PC2 and no PC1. We described heterologous processing of rat PSS (rPSS) co-expressed with PC2 in stably transfected cells, correlate PC2 protein levels under different conditions of transfection with efficiency of PSS processing to somatostatin-14 (SS-14), determine the effect of modulating cell granularity on enzyme expression and PSS processing, and compare the relative potency of PC2 with that of PC1, PSS and cleavage products were monitored by HPLC and radioimmunoassay of SS-like immunoreactivity (SSLI). Radioimmunoassay analysis of N-terminal PC2-like immunoreactivity (PC2 LI) in GH4C1:rPSS, GH4C1:rPSS + PC2 and GH3:rPSS transfectants showed a gradient of PC2 protein of 1:2.6:3.4 in cell extracts and 1:4.7:9 in secretion media from these cells respectively. The concentration of PC2 protein correlated with SS-14 conversion efficiency was 36 +/- 3% in GH4C1:rPSS cells, 56 +/- 7% in GH4C1:rPSS-PC2 cells and 100% in GH3:rPSS cells. Treatment of GH4C1:rPSS + PC2 cells with epidermal growth factor, insulin, and beta-estradiol to induce granules, significantly increased basal and forskolin-stimulated co-release of SS LI and PC2 LI, but had no influence on SS-14 processing efficiency. Hormone treatment led to a small increase in the ratio of mature PC2 (68 kDa) to proPC2 (75 kDa) forms. PC1 stably transfected in GH4C1 cells produced significantly greater SS-14 conversion (62% in cells, 66% in media) compared with PC2 transfectants (53% in cells, 47% in media) These results provide the first proof that PC2 can effect dibasic processing of mammalian PSS, and, along with PC1, qualifies as an authentic SS-14 convertase. The activity of PC2 requires the milieu of the secretory cell but not the secretory granule.

We have previously reported that rat prosomatostatin (rPSS) undergoes conversion at Arg decreases and Lys decreases monobasic sites to SS-28 and PSS-(1-10) respectively in COS-7 cells, and have proposed furin or a related enzyme of the constitutive secretory pathway as the endoproteinase responsible. Here we have tested directly the ability of furin to cleave rPSS at the two monobasic sites as well as at the RXRK dibasic site of SS-14 conversion (a furin motif, except for Lys substituting for Arg at P1). Recombinant vaccinia virus (VV) vectors were used to co-express rPSS with graded doses of furin in COS-7 cells and LoVo colon carcinoma cells deficient in furin. PSS and cleavage products in cell extracts and media were characterized by HPLC analysis and C-terminal [SS-14-like immunoreactivity (SS-14 LI)] and N-terminal [PSS-(1-10) LI] directed radioimmunoassays. There was a dose-dependent increase in SS-28 production from rPSS by furin in COS-7 cells from 29% (control) to 58% (high-dose furin) associated with a progressive decrease in unprocessed PSS from > 60% to approximately 20% of total SS-14 LI. Significant SS-14 production occurred only at high levels of furin infection. Control LoVo cells infected with VV:rPSS exhibited production of approximately 21% SS-28, approximately 15% PSS-(1-10) and 3.5% SS-14. Infection of LoVo cells with VV:hfurin (hfurin = human furin) enhanced SS-28 production to 30-34%. SS-14 synthesis also increased to 25-40%, probably by conversion from SS-28. Overexpression of furin in COS-7 or LoVo cells failed to increase PSS-(1-10) production. These results show that furin is a candidate SS-28 convertase. Arginine is the preferred residue at the P1 site of furin cleavage. Furin does not process rPSS to PSS-(1-10), suggesting the existence of another monobasic convertase with a preference for Lys rather than Arg at P1. Such an enzyme could also explain the presence of endogenous SS-28-, PSS-(1-10)- and SS-14-producing activities in LoVo cells.

Expression of the lipophilic ligand transporter, apolipoprotein D (apoD) by primary astrocyte cultures derived from neonatal mouse brain was investigated. Western blot analysis of cell lysates and media showed that apoD is constitutively secreted by astrocytes with little intracellular storage. The secreted apoD floated primarily at density 1.063-1.21 g ml -1 upon sequential ultracentrifugation indicating its association with lipids. Treatment of astrocytes with the carboxylic ionophore, monensin, resulted in intracellular retention and decreased secretion of apoD that was of slightly reduced M r . Progesterone, a steroid hormone that binds to apoD with high affinity (10 -6 mol l -1 ) and the oxysterol, 25-hydroxycholesterol which is a potent regulator of cellular cholesterol homeostasis in mammalian cells, differentially stimulated apoD, but not apoE secretion. These results show that astrocytes synthesize and constitutively secrete apoD and suggest a physiologic role for this lipocalin in cholesterol metabolism in the nervous system

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.

Recent reports have suggested that only some of the cloned somatostatin receptors (SSTRs) are coupled to adenylyl cyclase. These studies have used both stable and transiently transfected cells or cells lacking appropriate Gi alpha and are controversial. To investigate SSTR signalling mechanisms, we have established stably transfected CHO-K1 cells expressing human genes for SSTR1-5. The effect of 0.1-100 nM SST-14 and SST-28 on forskolin (1 microM) stimulated cAMP accumulation was determined and compared to their receptor binding affinities. The 5 expressed hSSTRs bound SST-14 and SST-28 with high affinity (IC50 1.1-2.1 nM for SST-14; IC50 0.25-5.4 nM for SST-28). hSSTR1-4 bound SST-14 > SST-28 whereas hSSTR5 bound SST-28 > SST-14. Radioligand binding to hSSTR1-5 was significantly inhibited by GTP, GTP gamma S and pertussis toxin. Both SST-14 and SST-28 inhibited forskolin-induced cAMP stimulation with ED50 values which paralleled their binding affinities for the individual hSSTR subtypes. These results demonstrate that all 5 human SSTRs are functionally coupled to inhibition of adenylyl cyclase in CHO-K1 cells via pertussis toxin sensitive G proteins.

The drug cysteamine (CHS) induces a profound loss of somatostatin-14 (SS-14) biological and immunological (SS-14 LI) activity from somatostatin cells in vivo and in vitro. The present study was designed to determine (i) whether CHS induced loss of somatostatin is accompanied by secondary increases in SS-mRNA perhaps through loss of autoinhibition of somatostatin cells; (ii) whether CHS exerts additional direct effects on SS gene regulation. CHS was administered to rats in vivo or applied in vitro to primary cultures of rat islet cells, rat islet somatostatin-producing tumor cells (1027 B 2 ), and endogenous or in vitro synthesized SS-mRNA. In vivo administration of CHS led to 80% reduction in tissue SSLI by 4 h. These changes were accompanied by significant alterations in SS-mRNA that were both tissue-specific and time-dependent. The pattern in brain and intestine was typified by a significant 60% increase in SS-mRNA at 2 h followed by a gradual reduction to ∼55% of control at 8 h. Stomach showed a significant 95% increase in SS-mRNA at 4 h followed by a 37% decrease by 8 h. Pancreatic SS-mRNA displayed a sustained 25–65% reduction for 8 h. Pretreatment of islet cell cultures with CHS reproduced the in vivo findings with pancreas viz. decreased SSLI (80–90% of control) accompanied by a parallel reduction in SS-mRNA (40–50% of control) sustained from 2–72 h. CHS also induced a reduction in immunoreactive insulin and insulin mRNA in cultured islet cells. As with normal islet cells, CHS treatment of 1027 B 2 islet tumor cells led to a profound and sustained decrease in SSLI and SS-mRNA. These changes occurred in the absence of any alteration in intracellular cAMP levels. CHS was without effect when incubated directly with SS-mRNA isolated from 1027 B 2 cells or with in vitro synthesized SS-mRNA. We conclude that in addition to its effect on SSLI, CHS also induces time- and tissue-dependent alterations in SS-mRNA. The mechanism of CHS action on SS-mRNA is complex and may involve both an indirect effect secondary to loss of somatostatin autoinhibition (to account for SS-mRNA increases) and/or a direct inhibition of SS gene expression (to explain SS-mRNA reduction). The precise site of direct CHS action on SS gene regulation remains to be defined. Since CHS inactivates cellular somatostatin and produces longterm reductions in SS-mRNA in all tissues, it represents a useful tool for inducing functional lesions of somatostatin cells at both gene and peptide levels.

Pharmacological studies have suggested that the somatostatin (SS) receptor is heterogeneous and exhibits SS-14-and SS-28-selective subtypes. Whether such subtypes arise from molecular heterogeneity of the receptor protein has not been definitively established. Previous reports characterizing the molecular properties of the SS receptor by the cross-linking approach have yielded divergent size estimates ranging from 27 kDa to 200 kDa. In order to resolve this discrepancy, as well as to determine whether SS-14 and SS-28 interact with specific receptor proteins, we have cross-linked radioiodinated derivatives of [125I-Tyr11]SS-14 (T*-SS-14) and [Leu8,D-Trp22,125I-Tyr25]SS-28 (LTT*-SS-28) to membrane SS receptors in rat brain, pituitary, exocrine pancreas and adrenal cortex using a number of chemical and photoaffinity cross-linking agents. The labelled cross-linked receptor proteins were analysed by SDS/PAGE under reducing conditions followed by autoradiography. Our findings indicate that the pattern of specifically labelled cross-linked SS receptor proteins is sensitive to the concentration of chemical cross-linking agents such as disuccinimidyl suberate and dithiobis-(succinimidyl propionate). Labelled high-molecular-mass complexes of cross-linked receptor-ligand proteins were observed only when high concentrations of these cross-linkers were employed. Using optimized low concentrations of cross-linkers, however, two major labelled bands of 58 +/- 3 kDa and 27 +/- 2 kDa were detected. These two bands were identified as specifically labelled SS receptor proteins subsequent to cross-linking with a number of photoaffinity cross-linking agents as well. We demonstrate here that the 58 kDa protein is the major SS receptor protein in the rat pituitary, adrenal and exocrine pancreas, whereas the 27 kDa moiety represents the principal form in the brain. Additionally, the presence of a minor specifically labelled band of 32 kDa was detected uniquely in the brain, and a minor labelled protein of 42 kDa was observed in the pancreas. The labelling pattern obtained with LTT*-SS-28 was identical to that observed with T*-SS-14. Labelling of the 27 kDa band by either ligand was inhibited by SS-14 and SS-28 in a dose-dependent manner. Densitometric quantification showed that SS-14 exhibited greater than 2-fold greater potency than SS-28 for inhibiting the labelling of the 27 kDa species. These findings emphasize the need for careful interpretation of cross-linking data obtained for SS receptors, and provide evidence for molecular heterogeneity and for a tissue-specific distribution of the two principal SS receptor proteins.

Striatal atrophy in Huntington's disease (HD) is characterized by selective preservation of a subclass of neurons colocalizing NADPH-diaphorase (NADPH-d), somatostatin (SS), and neuropeptide Y (NPY), which have been reported to show three- to fivefold increases in SS-like immunoreactivity (SSLI) and NPY content. Since HD brain is capable of producing excessive quantities of the excitotoxin quinolinic acid (Quin), an N-methyl-D-aspartate (NMDA) receptor agonist, and since experimental Quin lesions show neuronal loss with sparing of NADPH-d/SS/NPY neurons, it has been suggested that Quin may be important in the pathogenesis of HD. In the present study we determined whether Quin stimulates SS gene function in cultured cortical cells known to be rich in NADPH-d/SS/NPY neurons. Cultures of dispersed fetal rat cortical cells were exposed to Quin (1 and 10 mM) with or without (-)-2-amino-5-phosphon-ovaleric acid (APV; 0.5 mM), an NMDA receptor antagonist, NMDA (0.2 and 0.5 mM), and glutamate (Glu; 0.5 mM). Medium and cellular SSLI was determined by radioimmunoassay and SS mRNA by Northern analysis with a cRNA probe. Quin induced significant (p < 0.01) 1.6- and 2.5-4 fold increases in SSLI and SS mRNA accumulation, respectively, which were abolished by APV. Release of SSLI into the culture medium was stimulated two- to fivefold by Quin over a 2- to 20-h period. The increase in SS mRNA produced by Quin was time and dose dependent. A similar dose-dependent increase in SS mRNA comparable with that observed with Quin was induced by NMDA. These increases were selective for SS mRNA and were not accompanied by any change in β-actin mRNA. By contrast, glutamate at the single dose tested was without effect on both SSLI and SS mRNA. These results demonstrate that Quin induces a selective NMDA receptor-mediated stimulation of SS gene expression and SS biosynthesis in vitro and suggest a similar mechanism for the augmented striatal SSLI in HD.

Mammalian prosomatostatin (pro-S) undergoes extensive processing at the C-terminal segment where the somatostatin-like biological activities (S-14 and S-28) reside. The recent discovery of pro-S-(1-10) (antrin) as a prominent mature product in the stomach suggests that pro-S may also be processed at the N-terminus. In the present study we have developed an antibody directed against the N-terminal segment of pro-S-(1-10) capable of detecting peptides extended at the C-terminus of pro-S-(1-10) to characterize N-terminal processing of rat pro-S. Specifically, we have 1) examined the relative abundance of pro-S-(1-10)-like immunoactivity [pro-S-(1-10)] in different somatostatin tissues as an index of tissue-specific N-terminal processing, 2) compared the concentrations of pro-S N- and C-terminal immunoreactive peptides, 3) used HPLC and region-specific RIAs directed against both the N- and C-terminal segments of pro-S to identify and characterize novel N-terminal peptides, 4) studied the tissue distribution and release of the N-terminal peptides; and 5) characterized and quantified a 7-kDa molecule equivalent to pro-S without the C-terminal S-28 sequence. Acetic acid (1 M)-pepstatin extracts of hypothalamus, cerebral cortex, antrum, jejunal mucosa, and pancreas were fractionated by reverse phase and gel permeation HPLCs. Whole tissue extracts as well as the column effluent were monitored by region-specific RIAs using antibodies against pro-S-(1-10), S-28-(1-12), and S-14. Other than the pancreas, all S-producing tissues were rich in pro-S-(1-10) LI. Its concentration was 1- to 4-fold lower than those of S-14 LI and S-28-(1-12) LI. Tissue pro-S-(1-10) LI was heterogeneous, consisting of at least eight molecular forms with respective mol wt of 1,000 (1 kDa), 1,500 (1.5 kDa), 2,500 (2.5 kDa), 3,500 (3.5 kDa), 4,500 (4.5 kDa), 7,000 (7 kDa), 8,000 (8 kDa), and 10,000 (10 kDa). Based on the simultaneous presence or absence of C-terminal immunoreactivity, the 10-kDa form corresponded to pro-S, 8 kDa to pro-S-(1-76), and 7 kDa to pro-S without the S-28 sequence. The predominant N-terminal forms corresponded to 1 kDa [pro-S-(1-10)] and 7 kDa. The 1-, 1.5-, 2.5-, and 7-kDa forms were identified as secretion products in portal blood or in medium from cultured 1027 B2 islet somatostatin cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Prosomatostatin is cleaved at dibasic and monobasic sites to produce somatostatin-14 and somatostatin-28 respectively. The mammalian pro-protein convertases comprising furin, PACE4 and PC1-6 have recently been identified and are believed to mediate endoproteolysis of prohormone precursors such as prosomatostatin. Furin is membrane bound, localized to the Golgi and mediates constitutive processing. PC1 and PC2 are soluble and are expressed solely in endocrine and neuroendocrine tissues suggesting a key role in prohormone processing. We have investigated the endogenous and heterologous synthesis and processing of rat prosomatostatin in 1027B2 rat islet somatostatinoma cells and in constitutive (COS-7, PC-12) and regulated (AtT-20, GH3/GH4C1) secretory cells. We have correlated processing efficiency with: secretion through the constitutive or regulated pathways; endogenous expression of furin, PC1 and PC2; and expression or overexpression of furin, PC1 and PC2. Pulse-chase studies showed that prosomatostatin is rapidly and independently processed to somatostatin-14 and somatostatin-28. Furin is capable of monobasic processing of prosomatostatin and is a candidate somatostatin-28 convertase. PC1 and PC2 both effect dibasic processing of prosomatostatin and qualify as putative somatostatin-14 convertases. PC1 is active in constitutive and regulated secretory cells, has a broader specificity and is overall more potent than PC2. Efficient processing of prosomatostatin begins in a Golgi or pre Golgi compartment. It requires the milieu of the secretory cell but not the secretory granule.