Evidence for biosynthesis of preproinsulin in gut of rat.

Glucagon and other pancreatic peptides are made in the gut, but there is little evidence for the formation of insulin. The demonstration of insulin receptors on the mucosa of gut epithelium suggests that there may be an autocrine or paracrine role for insulin made in the gut. Such insulin may control cell division, the secretion of other peptides from the same or neighboring cells, or motility and absorption. To search for the ability of the gut to make insulin, sections of freshly excised segments of rat gut were treated with an antiserum against porcine insulin. Intracellular immunoreactivity appeared in glandular cells in the stomach and colon but not in the small intestine. Preproinsulin mRNA was detected in similar cells in the stomach and colon by in situ hybridization, using specific oligonucleotide probes. Rat preproinsulin 1 and 2 mRNAs were transcribed by reverse transcriptase to the corresponding cDNAs, which were then amplified by polymerase chain reaction, utilizing specific oligonucleotide primers. Restriction analysis confirmed the identity of rat preproinsulin 1 and 2 mRNA in the colon and rat preproinsulin 1 mRNA in the stomach. Neither was found in the small intestine. Base sequences of the cDNAs were identical to the coding regions of pancreatic rat preproinsulin 1 and 2 messages. These observations are strong evidence for the synthesis of preproinsulin in the gut of the rat.

Preproinsulin messenger ribonucleic acid in the rat adrenal gland.

Many studies support the concept of insulin synthesis in tissues other than the pancreas. Our previous investigations have demonstrated the presence of insulin immunoreactivity in the adrenal medulla of the rat. This immunoreactivity was found to be associated with the chromaffin granule. This study is directed at isolating the messenger ribonucleic acid that encodes for preproinsulin. Reverse transcription coupled with polymerase chain reaction was used. Complementary deoxyribonucleic acid (cDNA) was amplified, extracted and reamplified. It was then subjected to digestion with four different restriction endonuclease enzymes. Its resemblance to the corresponding cDNA that encodes for preproinsulin I in the rat was established. Our results suggest that insulin is synthesized in the rat adrenal gland for autocrine, paracrine, neuromodulation or local physiologic function.

Preproinsulin mRNA in the rat eye.

PURPOSE: The goal of this study was to extend the results of previous immunoassay, immunocytochemistry, and in situ hybridization studies showing the presence of insulin-related peptide in the rat retina by confirming the expression of insulin genes in the rat eye. METHODS: Total and poly(A)+ RNA were isolated from whole rat eyes, and separately from the retina, choroid, iris, lens, and vitreous. The poly(A)+ RNA was used for preparation of insulin-specific cDNA according to a coupled reverse transcription polymerase chain reaction (RT-PCR) protocol under high stringency conditions. Southern transfers, restriction fragment analyses, and nucleotide sequencing were used to characterize and identify the amplified cDNA products. RESULTS: Amplified cDNA fragments of 329 +/- 6 base pairs (bp) were derived from whole rat eye and rat retina poly(A)+ RNA, but not from other regions of the eye. Southern blots probed with preproinsulin-specific primer demonstrated homology with similar-sized cDNA from rat pancreas. Restriction digests with 10 restriction enzymes and direct nucleotide sequencing confirmed that the 329-bp cDNA was identical to the previously known coding sequence for rat pancreatic preproinsulin1 DNA. CONCLUSIONS: The identification of retinal preproinsulin1 mRNA was confirmed. This correlates with previous studies showing insulin immunoreactivity in rat eyes and in cultured retina, and verifies in situ hybridization evidence for the presence of insulin-related mRNA in retinal glial cells.

Human retinoblastoma Y79 cells contain both insulin-specific mRNA and insulin-binding sites.

We previously reported the presence of insulin-like immunoreactivity in cells from the human retinoblastoma Y79 cell line. In the present study, in situ DNA hybridization techniques were applied, using a human insulin cDNA probe to investigate whether the insulin-like activity is due to local synthesis of insulin. Our results suggest that Y79 cells contain mRNA for the synthesis of insulin or a homologous peptide. In addition, 125I-insulin binding autoradiographic studies show that these cells also contain specific insulin-binding sites. It is suggested that insulin may play an autocrine and/or paracrine role in the maintenance and metabolism of the Y79 retinoblastoma cells.

Insulin immunoreactivity in the fetal and neonatal rat retina.

The ganglion cell layer of pre- and postnatal rat retina is positive for insulin immunoreactivity. At birth the inner nuclear layer also stains for insulin. By 5 days after birth the layers characteristic of the mature retina are demonstrable. At this time the outer nuclear layer and both limiting membranes show insulin reactivity. The lens is positive for insulin at all stages studied and the retinal pigment and choroid layers are positive after birth. These observations suggest that insulin may be important in differentiation and/or maturation of the retina.

Immunocytochemical demonstration of insulin or insulin-like immunoreactivity in the rat prostate gland.

The indirect immunocytochemical technique was used in conjunction with anti-insulin antisera to localize insulin-like immunoreactivity in tissue sections and primary cell cultures from rat prostate gland. Positive immunostaining for insulin-like reactivity was demonstrated in the epithelium of the prostate gland. There appeared to be some variation in the intensity and localization of the immunoreactivity within different regions of the gland. Prostatic epithelial cells grown in culture in an insulin-free medium displayed strong cytoplasmic immunostaining when treated with anti-insulin antisera, while nuclear staining was absent. These results demonstrate that insulin-like immunoreactivity is present in the epithelium of the prostate gland and suggest that there may be some local insulin or insulin-like synthesis in this organ.

Demonstration of insulin-specific mRNA in cultured rat retinal glial cells.

A number of studies have recently demonstrated that insulin may be synthesized outside the pancreas. The present study was designed to investigate whether insulin-like activity exists in retinal glial cells and if so, whether it is due to local synthesis of insulin. Immunocytochemical techniques using insulin antisera were applied to cultured rat retinal glial cells, and insulin-like immunoreactivity was demonstrated in the cytoplasm of these cells. In situ DNA-RNA hybridization studies using 3H-labeled rat insulin cDNA indicated that the glial cells, particularly the Müller cells of the retina also contained the mRNA necessary for de novo synthesis of insulin or a closely homologous peptide. This peptide may be important in neuromodulation or regulation of metabolism of retinal cells and capillaries.

Evidence for insulin synthesis in normal mouse seminal vesicle based on in situ RNA-DNA hybridization.

Cultured seminal vesicle epithelial cells exhibited cytoplasmic immunoreactivity following treatment with anti-insulin antisera. In addition, these cultured epithelial cells were found, by in situ hybridization with a radiolabeled insulin complementary deoxyribonucleic acid (cDNA) probe, to contain an insulin or insulin-like messenger ribonucleic acid (mRNA). Autoradiograms of the hybridized cells exhibited heavy labeling over the cytoplasm and minimal distribution of grains over the nuclei and background areas. These observations indicate that cultured mouse seminal vesicle epithelium contains an insulin or insulin-like peptide as well as the mRNA that is required for its synthesis.

Detection of oncogene mRNA sequences in cultured cells by in situ hybridization.

The present study was undertaken to determine the feasibility of using in situ hybridization techniques to identify oncogene transcription in cultured cells. Following in situ hybridization with 32P-labeled v-src and v-Ha-ras DNA probes, src and Ha-ras related transcripts were identified in cell lines transfected with v-src and Ha-ras, respectively. In both the v-src and c-Ha-ras transfected cell lines, the number of silver grains over individual cells were significantly higher (p less than 0.001, t-test) than in a non-transfected, non-tumorigenic, rat esophageal epithelial cell line. There was a highly variable number of silver grains above individual cells. Significantly fewer silver grains were counted over cells that had been preincubated with either non-labeled v-src or v-Ha-ras DNA or that were pretreated with RNase A. Both oncogene transfected cell lines contained approximately 10 times more oncogene related mRNAs than non-transfected cells as judged by the numbers of silver grains over individual cells. Filter-hybridization analysis of the transfected and non-transfected cell lines confirmed that the expression of src and Ha-ras transcripts was higher in the transfected cell lines than in the non-transfected cell line. Therefore, the in situ hybridization technique would appear useful for the identification of oncogene transcripts in single cells and could potentially be applied to cytological preparations of human cells and to human tumor cells in culture.

Insulin or insulin-like peptides in the human pituitary.

Human pituitary cells and pancreatic islet beta cells from surgical and autopsy material showed positive immunoreactivity to anti-porcine insulin antisera demonstrating the presence in these tissues of insulin or insulin-like immunoreactive material. Glutaraldehyde-fixed, one micron epoxy sections were used and the polymerized resin was removed prior to staining with the peroxidase anti-peroxidase technique using guinea pig antisera to porcine insulin. Pancreatic beta cells served as the positive control, and appropriate negative controls were also utilized.

Immunocytochemical localization of insulin-like immunoreactivity in mouse seminal vesicle.

Insulin-like immunoreactivity was localized in tissue sections and cell cultures of mouse seminal vesicle using the indirect technique of immunocytochemistry. Seminal vesicles were cut into fragments, fixed in 2.5% glutaraldehyde, embedded in epoxy resin, sectioned at 1 micron, and transferred to glass slides. Epithelial cell cultures of seminal vesicle were grown on coverslips in Dulbecco's Minimal Essential Medium for 4-6 days and fixed in 2.5% glutaraldehyde. Sections (etched with sodium ethanolate) or coverslips were incubated in guinea pig antiporcine insulin antiserum, in antiserum immunoabsorbed with porcine insulin, or in normal guinea pig serum. For indirect immunocytochemistry, incubation with primary antiserum was followed by treatment with rabbit anti-guinea pig immunoglobulin (Ig) G conjugated to peroxidase, or with protein A and then rabbit peroxidase anti-peroxidase (PAP). Finally, treated samples were incubated in phenylenediamine-pyrocatechol-H2O2 substrate mixture for 6-8 min at room temperature. Specific immunoreactivity to insulin antisera was confined to the epithelium of the seminal vesicle in tissue sections. No staining occurred in subepithelial connective tissue. Specific immunoreactivity was also observed in the cytoplasm of cultured seminal vesicle epithelial cells.

Insulin-like immunoreactivity in human retinoblastoma Y79 cell line.

Cells from the Y79 human retinoblastoma cell line were examined by immunofluorescence immunocytochemistry using an antiserum against insulin. All the cells showed intense staining, indicating the presence of insulin-like immunoreactivity in these cells. Our observations suggest that insulin may play an important role in the metabolism of retinoblastoma cells and that it may be possible to use this cell line as an in vitro model for studies on the action of insulin in the metabolism of human retinal cells.

Detection of insulin synthesis in mammalian anterior pituitary cells by immunohistochemistry and demonstration of insulin-related transcripts by in situ RNA-DNA hybridization.

Insulin or highly homologous transcripts is shown to be synthesized in cultures of mammalian anterior pituitary cells using cloned insulin-specific cDNA probes and nucleic acid cytochemistry. The insulin-hybridizing cells are less abundant than the growth hormone-producing cells, occurring in the cultures at approximately one tenth the frequency. Immunocytochemistry demonstrates that insulin or insulin-like proteins is also synthesized by the cultured pituitary cells and that the insulin immunoreactivity is contained within secretory granules. It appears that many of these secretory granules are concentrated around the periphery of the cell, unlike the insulin-containing granules in pancreatic B-cells.

Glucagon-like immunoreactivity in mouse and rat retina.

Mouse and rat retinae were examined by the peroxidase-anti-peroxidase technique of immunocytochemistry using an antiserum against glucagon. The immunoreactivity was found in the cells of the ganglion cell layer and inner nuclear layer, including Müller cells. These observations may indicate that glucagon or a similar peptide is important in neuromodulation and/or metabolism of retinal cells.

The pentobarbital anesthetized dog: an animal model for assessing chemically induced changes in renal function and ultrastructure.

An experimental procedure was devised using the pentobarbital-anesthetized dog that could be used for the comprehensive evaluation of the renal effects of chemicals. After IV or renal arterial administration of 0.9% saline solution (vehicle), 12 renal function determinants were continuously monitored for periods of 2 and 6 hours. At the completion of the 2 or 6 hours of study, the kidneys of a number of dogs (usually between 1 and 7) in each vehicle-treated group were subjected to a modification of the intravascular perfusion-of-fixative technique to evaluate the ultrastructural status of the outer cortical, inner cortical, and outer medullary tissue. The remaining dogs (at least 3) in each vehicle-treated group were given a nonnephrotoxic, but maximally effective, diuretic dose of ethacrynic acid, which enabled an assessment of the functional integrity of the thick ascending limb of Henle's loop. Renal function and glomerular and tubular ultrastructure remained stable in the pentobarbital-anesthetized dog for up to 6 hours after administration of vehicle. Sustained infusion of inulin (included in the procedure to estimate glomerular filtration rate) throughout the duration of the experiments, and pentobarbital anesthesia of various durations did not alter the morphologic status of the canine nephron. The procedure used for the renal perfusion of fixative circumvented any manipulation of the kidneys before fixation and allowed for the acquisition of normal (unaltered) appearing tissue from all areas of the kidneys. The responses of pentobarbital-anesthetized dogs to ethacrynic acid administration were similar when given 2 and 6 hours after the vehicle administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemistry of mouse and human retina with antisera to insulin and S-100 protein.

Immunocytochemistry using peroxidase antiperoxidase (PAP) techniques showed insulin-like immunoreactivity in the human retina, and in the mouse retina and optic nerve. The immunoreaction product was seen in the inner nuclear, ganglion cell, outer and inner plexiform layers of the retinas, and in glial cell bodies of the optic nerve. A similar staining pattern using antiserum to S-100 protein, a marker for glial elements, was also seen in these tissues. This demonstrates that insulin or insulin-like immunoreactivity appears to be limited to glial cells of the retina and optic nerve. Our study suggests that the presence of insulin or a similar peptide in retina and optic nerve may be important for their normal function and metabolism.

Acute effects of alkylating agents on canine renal function and ultrastructure: high-dose ethacrynic acid vs. dihydroethacrynic acid and ticrynafen.

Ethacrynic acid (EA) is unique among diuretics in that it is both an avid alkylating agent and is actively secreted by renal proximal tubular cells. EA might therefore be expected to produce detrimental proximal tubular changes at elevated doses. Because of this possibility, we examined the renal effects of two relatively high doses of EA (i.e., 66 and 151 mumol/kg i.v.) and an equivalent high dose (i.e., 151 mumol/kg) of two nonalkylating relatives of EA [dihydro-EA (EA-H2) and ticrynafen]. Twelve renal function parameters were monitored in pentobarbital-anesthetized dogs for a period of 2 hr after administration of EA, EA-H2 and ticrynafen and renal tissue was acquired at the end of the 2 hr of study for light and electron microscopic evaluation. Both doses of EA produced a profound diuresis of similar magnitude. However, only the larger dose was associated with a concomitant reduction in the glomerular filtration rate, a downward trend in the renal blood flow, a proteinuric response in four of the seven dogs in the treatment group and a reproducible vacuolation of the initial portion of the proximal convoluted tubules (i.e., the S1 cells). EA-H2 induced a small, transient increase in the excretion rates of sodium, chloride and potassium, but failed to elicit a proteinuric response or alter proximal tubular ultrastructure. Ticrynafen, a far more efficacious diuretic agent than EA-H2, likewise failed to trigger a proteinuric response or changes in renal ultrastructure. The combination of acidic (anionic) and alkylating properties of EA is thought to be responsible for the proximal tubular effects observed in this study.

3H-methyl scopolamine binding to dispersed pancreatic acini.

Maximal amylase release occurred with 10(-5) M carbachol and slightly greater than half maximal response occurred with 3 x 10(-7) M carbachol in dispersed pancreatic acini. The preparation released more than 45% of its initial amylase content after 60 min of maximal carbachol stimulation. Electron microscopy revealed depletion of zymogen granules and the presence of secretory material in the ductules after carbachol stimulation. At 37 degrees C, maximal binding of methyl scopolamine occurred in about 45 min with 3 x 10(-10) M 3H-methyl scopolamine. The dissociation constant for 3H-methylscopolamine was 6.8 x 10(-10) M and saturation occurred at 109 pm/g protein. The I.C. 50 for 3H-methylscopolamine inhibition of carbachol-induced amylase secretion was 7 x 10(-10) M.

Fluorophosphate-sensitive esterases in mammalian liver: the radioautographic localization and measurement of fluorophosphate-reactive sites in adult rat liver.

Fluorophosphate-reactive (FPR) sites in the adult male rat liver, tentatively identified as esterase active centers, were localized and measured using the combined techniques of quantitative electron microscope radioautography and morphometric analysis with the light and electron microscope. The FPR sites were measured in liver which had been prefixed by perfusion with 1.5% glutaraldehyde and reacted with 10(-4) M tritium-labeled diisopropyl fluorophosphate (3H-DFP). Under the experimental conditions 64-67% of the esterase activity in fresh liver was retained for reaction with the 3H-DFP, which is known to bind irreversibly to the active sites of certain esterases. In light and electron microscope radioautographs the developed silver grains were concentrated over the cytoplasm of hepatocytes. A low concentration occurred over erythrocytes. All other areas in the liver had a concentration of grains resembling the background concentration. Quantitative measurements of grain density in the electron microscope radioautographs revealed the highest density, after correcting for radiation spread, in cytoplasmic granules (mainly cytolysomes). The grain densities over the rough and smooth endoplasmic reticulum and associated structures were also equal to or above the average hepatocyte grain density. Due to the large fractional volume of endoplasmic reticulum per hepatocyte (58% of cell volume) and the fraction of the liver occupied by hepatocytes (79% of liver volume) the majority of FPR sites in the liver occurred in the rough and smooth endoplasmic reticulum and associated structures. The average numbers of FPR sites were calculated per micrometer3 of hepatocyte (5.0 x 10(5) sites/micrometer3) and per unit volume of each significantly labeled organelle. In addition, the number of FPR sites per hepatocyte (2.5 X 10(9) sites/cell), per cm3 liver (4.1 X 10(17) sites/cm3) and in the total liver of an average 100 g male rate (2.2 X 10(18) sites/total liver) were also calculated.

Quantification of myocardial ischemia and infarction with 201thallium scintigraphy.

A quantitative method for the analysis of 201thallium myocardial scintigrams, developed in an experimental infarcted dog heart model, has been compared with two nonquantitative methods for interpretation of stress myocardial scintigrams in two groups of patients studied with coronary angiography: 11 with normal coronary arteries and 14 with coronary artery disease. Three independent observers interpreted scintigrams which were 1) not computer processed; 2) corrected for background activity in lungs and chest wall; and 3) processed by a computer method which uses a uniform threshold of counts determined from the dog model to define perfusion defects. Interobserver variability as well as sensitivity and specificity of detecting coronary disease were examined. In patients with coronary artery disease interobserver variability was improved by using the computer technique: observers agreed as to the existence of a perfusion defect in 93% of the scintigrams as compared to 55% and 81% for the unprocessed and background-subtracted images respectively. No false positive indications of coronary disease were obtained by any of the three techniques. Use of the computer method did not improve the sensitivity of detecting coronary disease, however--71% compared to 64% for unprocessed images and 79% for background-substracted images. The advantages of this quantitative computer method are increased consistency of interpretation and lack of false positive diagnoses of coronary disease. An improved sensitivity of detection may be gained by varying thallium count thresholds according to anatomic location in the heart.