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D G Baskin

Publications and source records attributed to D G Baskin.

At least 109 records · Page 6Linked to original sources
PubMed1986

An inexpensive microcomputer digital imaging system for densitometry: quantitative autoradiography of insulin receptors with 125I and LKB Ultrofilm.

This article describes a video digitizing system designed for measuring film optical density. The system, which is based on a 6-bit (64 gray level) digitizer, solid state video camera, and Apple II microcomputer, digitizes a rectangular area selected by the operator and converts the gray level values into preselected standard units. In order to develop autoradiographic standard curves for quantitative autoradiography with 125I-insulin, liver slices labeled with 125I-insulin and plastic sections containing known amounts of tritium were apposed to the same sheet of LKB Ultrofilm for exposures of 1-7 days. The results indicate that 3H plastic standards can be used to calibrate QAR of 125I-labeled ligands with LKB Ultrofilm. The Apple system was also used to measure binding of 125I-insulin to the external plexiform layer (EPL) in slices of the rat olfactory bulb. Results suggest that the EPL has two binding sites for insulin, a high affinity site with Kd = 1.0 X 10(-8) M and a low affinity site having a Kd = 1.4 X 10(-5) M.

Animals↗
PubMed1986

Ontogenetic changes in vasopressin binding site distribution in rat brain: an autoradiographic study.

Binding sites for [3H]vasopressin were identified in brains of neonatal (days 0-23) and adult (90 day) Long-Evans and Brattleboro rats, using a technique of in vitro receptor autoradiography. Results indicate that the regional distribution of binding sites for [3H]vasopressin in the brain changes markedly during postnatal development. Binding sites in the septum proliferated slowly to attain adult distribution in the dorsal and lateral septum, while in other regions, such as the caudate, hippocampus and cingulate gyrus, intense labeling appeared early in development but disappeared by adulthood. In the amygdala, binding did not change during postnatal development. Binding sites in the septum appeared before vasopressinergic fibers are present. Also, binding was present for vasopressin in regions which have not yet been reported to receive vasopressinergic innervation. Therefore, it is proposed that the presence of binding sites for vasopressin is independent of the presence of vasopressin itself. This hypothesis is supported by labeling in the Brattleboro rat which was comparable to that in the Long-Evans.

Amygdala↗
PubMed1986

Identification of binding sites for an insulin-like growth factor (IGF-I) in the median eminence of the rat brain by quantitative autoradiography.

The microanatomical location of IGF-I binding in the rat brain was determined by in vitro autoradiography with slide-mounted sections of frozen brain. Sections incubated in 0.1 nM [125I]-iodo-IGF-I produced a dense grain concentration in regions of the autoradiographic image corresponding to the external palisade zone of the median eminence; other hypothalamic regions were not so heavily labeled. This reaction was significantly reduced in the presence of 100 nM IGF-I. Measurement of binding by computer digital image analysis of autoradiographic images showed that specific binding for IGF-I in the median eminence was 41.3 +/- 8 X 10(-3) fmol/mm2 (mean +/- SEM); nonspecific binding was 11.9 +/- 1.8 X 10(-3) fmol/mm2. In contrast, specific binding to other hypothalamic regions was uniformly lower. In a separate experiment, 1000 nM unlabeled insulin was added. Without insulin, specific binding was 23 +/- 0.9 X 10(-3) fmol/mm2; nonspecific binding was 8 +/- 0.5 X 10(-3) fmol/mm2. In the presence of 1000 nM unlabeled insulin, specific binding for [125I]-iodo-IGF-I was 23 +/- 1 X 10(-3) fmol/mm2. The results suggest that a high concentration of receptors for an IGF-I-like molecule is present in the median eminence.

Animals↗
PubMed1986

Quantitative autoradiographic evidence for insulin receptors in the choroid plexus of the rat brain.

Techniques of in vitro receptor autoradiography were used to visualize binding of 125I-insulin on slices of frozen rat brain. Slide-mounted sections of frozen rat brain were incubated in 0.05 nM porcine 125I-monoiodoinsulin, alone or mixed with 1 microM unlabeled porcine insulin, ribonuclease, or glucagon, for 2 h at 22 degrees C. The labeled brain slices were apposed to LKB Ultrofilm to generate autoradiograms. The method permitted equal access of labeled insulin to both sides of the blood-brain barrier and localization of insulin binding sites in small anatomic regions. Quantitative estimates of specific iodoinsulin binding were made by computer digital image densitometry of the autoradiographic film images. High concentrations of specific binding sites for iodoinsulin were present in the choroid plexus of the lateral (26.9 +/- 2.0 X 10(-3) fmol/mm2), fourth (18.3 +/- 3.0 X 10(-3) fmol/mm2), and third (13.2 +/- 1.5 X 10(-3) fmol/mm2) ventricles (insulin binding is expressed per unit area of autoradiographic image). Binding to the third ventricular choroid plexus was similar to the concentrations observed for liver slices and the external plexiform layer of the olfactory bulb. Specific binding of iodoinsulin in the cingulate cortex and other surrounding regions was less than in choroid plexus. Ribonuclease or glucagon had no measurable effect on binding when mixed with labeled insulin. The results support the hypothesis that the choroid plexus has a high density of receptors for insulin, and suggests that the choroid plexus may be a target of CSF insulin action and/or a site of insulin transport into the CSF.

Animals↗
PubMed1986

Physiological and morphological changes in islet B cells following treatment of the guinea pig with alloxan.

Alloxan injections (200 mg/kg i.v.) into guinea pigs led to a significant decline of serum insulin levels and a marked inhibition of normal weight gain during the first 96 hr following treatment. Both of these parameters tended to normalize between 96 and 120 hr after treatment. However, neither hyperglycemia nor glycosuria occurred at any time. Volume densities of islet B cells (= volume density of islet cells immunostained for insulin per islet volume density) decreased and pyknotic nuclei appeared in the islets during the initial 48 hr after alloxan treatment. Islet B-cell volume density then returned to normal over the ensuing 24 hr, and remained normal up to 120 hr following administration of alloxan. In summary, after high doses of alloxan were given guinea pigs, serum insulin levels were reduced below control levels within 24 hr; this was associated with changes in body weight which paralleled changes in insulin levels more closely than did levels of blood or urine glucose; the volume density of immunostained B cells initially decreased and then returned to control levels by 72 hr following alloxan injection. The alloxan-treated guinea pig may represent a unique model for studying the restoration of insulinogenic capacity in insulinopenic diabetes.

Alloxan↗
PubMed1985

Genetically obese Zucker rats have abnormally low brain insulin content.

The concentration of immunoreactive insulin (IRI) extracted from the olfactory bulb, hypothalamus, hippocampus, cerebral cortex, amygdala, midbrain, and hindbrain was significantly lower in obese (fa/fa) and heterozygous (Fa/fa) Zucker rats in comparison to lean (Fa/Fa) Zucker rats. This deficit in brain IRI content was most severe in the hypothalamus and olfactory bulb and was independent of severe obesity since the marked reduction of brain IRI content was also found in heterozygous rats which possessed only one copy of the fa allele. These results demonstrate that in the 2-3 month-old female Zucker rat, the fa allele is associated with defective regulation of insulin in the brain.

Animals↗
PubMed1985

Brain and liver insulin binding is decreased in Zucker rats carrying the 'fa' gene.

Insulin binding was measured in membrane particles prepared from the liver and several brain regions of 4-month-old female Zucker fa/fa (obese), Fa/fa (heterozygous), and Fa/Fa (lean) rats. High affinity insulin binding was decreased in the olfactory bulb of fatty (0.23 pmol bound/mg protein) and heterozygous (0.16 pmol/mg) rats compared with that in the lean controls (0.64 pmol/mg). Total binding was not changed in the cerebral cortex or hypothalamus. High affinity insulin binding was also decreased in the liver of both fatty (0.44 +/- 0.22 pmol/mg; P less than 0.01) and heterozygous (0.75 +/- 0.35 pmol/mg) animals compared with that in the lean rats (2.10 +/- 1.55 pmol/mg). This decreased binding is probably not due to down-regulation of receptors in the heterozygous rats, as they do not exhibit the hyperinsulinemia observed in the fatty rats. Rather, our findings suggest that there is a gene-related alteration in insulin binding in the Zucker rat, as low binding was observed in rats carrying either one (Fa/fa) or two (fa/fa) doses of the gene. We postulate that this central defect in insulin binding may contribute to inadequate perception of a central insulin feedback signal and to the hyperphagia observed in the obese rats.

Animals↗
PubMed1984

Immunocytochemical identification of cells containing insulin, glucagon, somatostatin, and pancreatic polypeptide in the islets of Langerhans of the guinea pig pancreas with light and electron microscopy.

Cell types containing insulin, glucagon, somatostatin, and pancreatic polypeptide were identified in guinea pig islets with light and electron microscopic immunoperoxidase staining. Cells containing immunostainable insulin (B cells) are located throughout the islets, including the islet periphery, and contain irregularly shaped granules (350-550 nm). Granule contents are of variable opacity and are often fragmented but not crystalloid. Cells containing immunoreactive glucagon (A cells) are found in the interior of islets and contain numerous spheroid electron-opaque granules (250-350 nm). Cells containing immunoreactive somatostatin (D cells) have elongate, axonlike processes that end adjacent to islet capillaries. D cells, which are very numerous and distributed uniformly throughout the islet parenchyma, contain small spheroid granules (150-25 nm) of pale electron opacity. Cells with immunoreactive pancreatic polypeptide (F cells) are rare in islets but numerous among the exocrine parenchyma. F cells contain pale spheroid granules (100-200 nm). Morphological criteria are reliable indicators for A cells and B cells, but D cells and F cells require immunostaining for positive identification.

Animals↗
PubMed1984

Immunocytochemical localization of Na+,K+ adenosine triphosphatase in the rat retina.

Immunocytochemical localization of Na+,K+-adenosine triphosphatase (ATPase) in fixed sections of rat retina was carried out using polyclonal antibodies to the highly purified enzyme and peroxidase-antiperoxidase and immunofluorescence techniques. In the photoreceptor layer of the rat retina, Na+,K+-ATPase immunoreactive sites were associated primarily with inner segments and only background activity was associated with the outer segments. This localization supports the proposed distribution of Na+,K+-ATPase in photoreceptor cells suggested by physiological studies. The Na+,K+-ATPase immunoreactive sites were also localized in the inner and outer plexiform layers, which are rich in synaptic contacts. The reticular pattern of immunostaining in those regions suggests that Na+,K+-ATPase immunoreactivity is associated with neuronal processes in those layers.

Animals↗
PubMed1984

Localization and characterization of vasopressin-binding sites in the amygdala of the rat brain.

Putative receptors for arginine-vasopressin (AVP) in rat brain were localized and characterized using autoradiographic and radioligand-binding techniques. When brain slices were incubated with tritium-labeled vasopressin, an intense concentration of binding sites was identified which included the medial aspects of the amygdala. Binding of labeled AVP to membrane preparations from this brain region was saturable, specific, reversible, and of high affinity. An antagonist of the peripheral pressor activity of vasopressin was found to be a potent competitor for binding to this site.

Amygdala↗
PubMed1983

Cytotoxic effects of alloxan treatment in vitro on monolayer cultures of neonatal rat pancreas.

Monolayer cultures of neonatal rat pancreas were exposed briefly (5 min) to alloxan and were then maintained for several days (up to 14 days) to examine the long-term effects of alloxan exposure on B cells. Alloxan (0-10 mM) caused a dose-dependent reduction in rates of insulin release during the first 24 h after treatment. This reduction persisted throughout the period of observation. Rates of glucagon release were unaltered by all concentrations of alloxan tested. Spontaneously decomposed alloxan had little or no effect on rates of insulin release. The simultaneous presence of high concentrations of glucose during exposure of cells to alloxan exerted a rapid protective effect against alloxan toxicity that was both glucose and alloxan dose dependent. Alloxan treatment of pancreatic monolayer cultures may allow for a comparison of those actions of alloxan that are directly on and specific for islet B cells. Furthermore, alloxan-treated cultures may represent a unique in vitro system for studying the physiology and biochemistry of other islet cell types in cultures in which B cells and insulin are reduced, as well as for studying glucose interactions with the B cell.

Alloxan↗
PubMed1983

Regional concentrations of insulin in the rat brain.

Recent evidence that insulin receptors are concentrated in the hypothalamus and olfactory bulb suggests that insulin may have an important regulatory function in these regions. This hypothesis would be supported by finding that insulin itself is concentrated in the hypothalamus and olfactory bulb. Therefore, we extracted the hypothalamus and olfactory bulb, as well as the amygdala, hippocampus, cerebral cortex, hindbrain, midbrain, and whole brains, of fasted male Wistar rats and measured immunoreactive insulin (IRI). Recovery of insulin added to extraction volumes of 500-1000 microliters was 90-100%, whereas recovery of insulin from tissue extracts was 63%. Mean IRI concentrations were relatively uniform throughout the brain (0.19 ng/g wet wt; uncorrected for recovery) and were significantly lower than plasma levels (1.03 ng/ml). Nevertheless, IRI concentrations were significantly higher in hypothalamus (0.39 +/- 0.02 ng/g; P less than 0.01) and olfactory bulb (0.37 +/- 0.02 ng/g; P less than 0.05) compared to those in other brain regions sampled.

Animals↗
PubMed1983

Immunocytochemical detection of insulin in rat hypothalamus and its possible uptake from cerebrospinal fluid.

Insulin-like immunoreactivity (IRI) was detected in the rat hypothalamus, particularly in the paraventricular, periventricular, supraoptic, suprachiasmatic, arcuate, and lateral hypothalamic nuclei. The immunostainable IRI was diffusely distributed in comparison to the neuronal concentrations of immunostainable vasopressin in the periventricular nucleus, or of IRI in islet B cells, suggesting that immunostainable IRI in the hypothalamus is not concentrated in neuronal perikarya. To determine if insulin in cerebrospinal fluid (CSF) may be a source of some insulin in brain tissue, [125I]iodoinsulin was stereotaxically injected into a lateral cerebral ventricle, and the uptake of radioactivity into periventricular hypothalamus was localized by both quantitative autoradiography of paraffin-embedded brain sections and by measuring the radioactivity present in microdissected brain regions. In brains that received lateral ventricular injections of labeled insulin, the concentration of radioactivity in the periventricular region of the hypothalamus, as revealed by autoradiographic grains, was significantly greater than that in the periventricular region of brains that received lateral ventricular injections of labeled insulin mixed with an equimolar excess of an unlabeled peptide (insulin, ribonuclease, or both together). The highest levels of radioactivity detected in both autoradiographic and microdissection procedures were in regions nearest to the third ventricle, suggesting that insulin in the lateral ventricles has access to the periventricular neuropile in the hypothalamus. The staining pattern of immunostainable insulin in the hypothalamus along with the distribution of radioactivity after CSF injection of labeled insulin are consistent with the hypothesis that insulin is taken up into brain from the CSF.

Animals↗
PubMed1983

Immunoreactive insulin levels are elevated in the cerebrospinal fluid of genetically obese Zucker rats.

Immunoreactive insulin (IRI) concentrations were measured in plasma and cerebrospinal fluid (CSF) of four-month old genetically obese Zucker rats, their heterozygote lean littermates, and age-matched normal-weight Wistar rats. Basal plasma IRI was 201 + 35 microU/ml (means +/- SEM) in the obese animals and was significantly elevated compared to both lean Zucker rats (18 +/- 2.4 microU/ml, P less than 0.001) and Wistar rats (12 +/- 2.4 microU/ml, P less than 0.001). The mean CSF IRI concentration of fasted obese Zucker rats was 1.59 +/- 0.19 microU/ml; this was significantly higher than the CSF IRI level of either fasted Zucker lean rats (0.31 +/- 0.08 microU/ml, P less than 0.001) or Wistar rats (0.34 +/- 0.12 microU/ml, P less than 0.001). Plasma and CSF IRI concentrations were increased in free-feeding as compared with fasted animals. These data provide evidence that endogenous CSF insulin is derived from circulating plasma insulin in the rat and suggest that the hyperphagia and obesity of the Zucker fatty rat are not due to an inability of circulating insulin to gain access to the CSF.

Animals↗
PubMed1982

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney.

To determine if rat kidney Na+, K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na+, K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na+, K+-ATPase and rat kidney microsomes were treated with antiserum (1:200), followed by [125I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the alpha-subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the beta-subunit. When the Na+, K+-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na+, K+-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na+, K+-ATPase concentration in the rat kidney.

Animals↗
PubMed1982

Electron microscopic immunoperoxidase staining of insulin using 4-chloro-1-naphthol after osmium fixation.

Ultrastructural localization of insulin in B cells of guinea pig pancreas was accomplished after osmium fixation with an immunoperoxidase procedure that utilized 4-chloro-1-naphthol (CN) in the substrate solution. The principal features of this protocol were: a) osmium tetroxide postfixation; b) omission of hydrogen peroxide "etching"; c) use of CN instead of diaminobenzidine in the substrate solution; d) elimination of osmium tetroxide after the substrate reaction; e) uranyl acetate and lead citrate counterstaining. This procedure produces intense specific staining with low background using highly dilute antiserum, and appears to be useful for postembedding immunoperoxidase staining of a variety of peptide antigens in osmium-fixed tissue.

Animals↗
PubMed1981

Immunocytochemical detection of B cells in the guinea pig pancreas using guinea pig antiserum to porcine insulin.

The purpose of this study was to determine if antisera to an heterologous insulin could replace specific anti-guinea pig insulin serum for immunoperoxidase localization of guinea pig B cells. We found that antisera to porcine insulin was effective for this purpose even though it was produced by guinea pigs. The immunostaining results obtained with anti-porcine insulin sera and anti-guinea pig insulin serum were identical, by both light and electron microscopic immunoperoxidase staining. Control experiments supported the conclusion that the guinea pig antisera to porcine insulin contained antibodies that reacted with guinea pig insulin.

Animals↗