Dopamine-synthesizing neurons include the putative H-cell homologue in the moth Manduca sexta.

The catecholamine dopamine (DA) plays a fundamental role in the regulation of behavior and neurodevelopment across animal species. Uncovering the embryonic origins of neurons that express DA opens a path for a deeper understanding of how DA expression is regulated and, in turn, how DA regulates the activities of the nervous system. In a well-established insect model, Manduca sexta, we identified the putative homologue of the embryonic grasshopper "H-cell" using intracellular techniques, laser scanning confocal microscopy, and immunohistochemistry. In both species, this neuron possesses four axons and has central projections resembling the letter H. The H-cell in grasshoppers is known to be derived from the midline precursor 3 cell (MP3) and to pioneer the pathways of the longitudinal connectives; in Drosophila, the H-cell is also known to be derived from MP3. In the current study, we demonstrate that the Manduca H-cell is immunoreactive to antibodies raised against DA and its rate-limiting synthetic enzyme, tyrosine hydroxylase (TH). In larvae and adults, one DA/TH-immunoreactive (-ir) H-cell per ganglion is present. In embryos, individual ganglia contain a single midline TH-ir cell body positioned along side its putative sibling. Such observations are consistent with the known secondary transformation (in grasshoppers) of only one of the two MP3 progeny during early development. Although a hallmark feature of invertebrate neurons is the fairly stereotypical position of neuronal somata, we found that the H-cell somata can "flip-flop" by 180 degrees between an anterior and posterior position. This variability appears to be random and is not restricted to any particular ganglion. Curiously, what is segment-specific is the absence of the DA/TH-ir H-cell in the metathoracic (T3) ganglion as well as the unique structure of the H-cell in the subesophageal ganglion. Because this is the first immunohistochemical study of DA neurons in Manduca, we have provided the distribution pattern and morphologies of dopaminergic neurons, in addition to the H-cells, within the ventral nerve cord during development.

Dexamethasone counteracts the effect of prolactin on islet function: implications for islet regulation in late pregnancy.

Islets undergo a number of up-regulatory changes to meet the increased demand for insulin during pregnancy, including increased insulin secretion and beta-cell proliferation. It has been shown that elevated lactogenic hormone is directly responsible for these changes, which occur in a phasic pattern, peaking on day 15 of pregnancy and returning to control levels by day 20 (term). As placental lactogen levels remain elevated through late gestation, it was of interest to determine whether glucocorticoids (which increase during late gestation) could counteract the effects of lactogens on insulin secretion, beta-cell proliferation, and apoptosis. We found that insulin secretion measured over 24 h in culture and acute secretion measured over 1 h in response to high glucose were increased at least 2-fold by PRL treatment after 6 days in culture. Dexamethasone (DEX) treatment had a significant inhibitory effect on secretion in a dose-dependent manner at concentrations greater than 1 nM. At 100 nM, a concentration equivalent to the plasma corticosteroid level during late pregnancy, DEX inhibited secretion to below control levels. The addition of DEX (>1 nM) inhibited secretion from PRL-treated islets to levels similar to those produced by DEX treatment alone. Bromodeoxyuridine (10 microM) staining for the final 24 h of a 6-day culture showed that PRL treatment increased cell proliferation 6-fold over the control level. DEX treatment alone (1-1000 nM) did not reduce cell division below the control level, but significantly inhibited the rate of division in PRL-treated islets. YoYo-1, an ultrasensitive fluorescent nucleic acid stain, was added (1 microM; 8 h) to the medium after 1-3 days of culture to examine cell death. Islets examined under confocal microscopy showed that DEX treatment (100 nM) increased the number of cells with apoptotic nuclear morphologies. This was quantified by counting the number of YoYo-labeled nuclei per islet under conventional epifluorescence microscopy. The numbers of YoYo-1-positive nuclei per islet in control and PRL-treated islets were not different after 3 days of culture. However, DEX treatment increased YoYo-1 labeling 7-fold over that in controls. DEX also increased YoYo-1 labeling in PRL-treated islets 3-fold over the control level. These data show that the increased plasma glucocorticoid levels found during the late stages of pregnancy could effectively reverse PRL-induced up-regulation of islet function by inhibiting insulin secretion and cell proliferation while increasing apoptosis.

Role of cAMP in upregulation of insulin secretion during the adaptation of islets of Langerhans to pregnancy.

Islets undergo a number of upregulatory changes to meet the increased demand for insulin during pregnancy, including an increase in glucose-stimulated insulin secretion with a reduction in the stimulation threshold. Treatment with the lactogenic hormone prolactin (PRL) in vitro has been shown to induce changes in islets similar to those observed during pregnancy. We examined cAMP production in islets treated with PRL to determine if changes in cAMP are involved in the upregulation of insulin secretion. Insulin secretion and cAMP concentrations were measured from islets in response to a suprathreshold (6.8 mmol/l) or high (16.8 mmol/l) glucose concentration in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Insulin secretion increased by 2.1-, 5.0-, and 5.9-fold at the suprathreshold glucose concentration and by 1.6-, 2.3-, and 2.9-fold at the higher glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. After a similar pattern, cAMP metabolism increased by 1.2-, 1.6-, and 2.1-fold at the suprathreshold glucose concentration and by 1.2-, 1.7-, and 2.2-fold at the high glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. The similar increases in insulin secretion and cAMP concentration suggest that changes in cAMP metabolism are involved in lactogen-induced upregulation of insulin secretion. To gain additional insight into the role of cAMP in the upregulation of islet function after lactogen treatment, we examined the relationship between changes in cAMP concentration and insulin secretion. Under all conditions (differing glucose concentrations and time periods), the increase in insulin release was directly proportional to the increase in cAMP. Thus increased glucose-stimulated insulin secretion from lactogen-treated islets could be accounted for by increased generation of cAMP and did not appear to require any further specific changes in intracellular processes mediated by cAMP. Because the PRL receptor is not directly involved in cAMP metabolism, the lactogen-induced increase in cAMP was most likely due to the increase in glucose metabolism that we have previously demonstrated in PRL-treated islets and in islets during pregnancy.

Adaptation of islets of Langerhans to pregnancy: beta-cell growth, enhanced insulin secretion and the role of lactogenic hormones.

Pregnancy is a unique event in the life span of islet beta-cells. Under the influence of pregnancy islet beta-cells undergo major long term up-regulatory structural and functional changes in response to the increased demand for insulin. Adaptive changes that occur in islets during normal pregnancy include: 1) increased glucose-stimulated insulin secretion with a lowered threshold for glucose-stimulated insulin secretion, 2) increased insulin synthesis, 3) increased beta-cell proliferation and islet volume, 4) increased gap-junctional coupling among beta-cells, 5) increased glucose metabolism, and 6) increased c-AMP metabolism. Of the islet changes that occur during pregnancy the increase in beta-cell division and enhanced glucose sensitivity of insulin secretion are most notable. The increase in beta-cell division leads to an increase in islet mass that contributes to the ability of islets to respond to the increased need for insulin. However, the increased glucose sensitivity of beta-cells is likely to be more important. The lowering of the threshold for glucose stimulated insulin secretion is the primary mechanism by which beta-cells can release significantly more insulin under normal blood glucose concentrations. Although the hormonal changes which occur during pregnancy are complex, it appears that lactogenic influences (either placental lactogen and/or prolactin) are sufficient to induce all of the up-regulatory changes that occur in islets during pregnancy. We have demonstrated that rat placental lactogens I and II are the hormones responsible for up-regulating islets during rodent pregnancy. Although most studies have been done using rodent islets, available evidence strongly suggests that human placental lactogen and/or human prolactin are the responsible lactogens for up-regulating islets during human pregnancy. A model for how lactogens up-regulate islets during pregnancy is proposed.

Novel mouse IgG-like immunoreactivity expressed by neurons in the moth Manduca sexta: developmental regulation and colocalization with crustacean cardioactive peptide.

Immunoglobulin-related molecules have been shown to play important roles in cell-cell recognition events during the development of both vertebrate and invertebrate nervous systems. In the moth, Manduca sexta, we report the presence of novel, mouse, immunoglobulin G (mIgG)-like immunoreactivity in a discrete population of identified neurosecretory neurons (the NS-Ls also known as the cell 27s) and interneurons (the IN-704s). A number of polyclonal anti-mIgG antibodies were used to immunostain these cells in wholemount. The mIgG-like-immunoreactive (IR) neurons were present during embryogenesis through the developing adult stages, but disappeared in the postemerged adult. Biochemical analysis of M. sexta ventral nerve cords revealed that the mIgG-like antigen is a membrane-associated 27-kDa protein which is likely responsible for the mIgG-like immunostaining observed. Unambiguous identification of the mIgG-like-IR neurons was based on neuronal morphology and our ability to demonstrate conclusively that these neurons expressed immunoreactivity to an antiserum against crustacean cardioactive peptide (CCAP). The NS-Ls and IN-704s were both shown to colocalize the CCAP and mIgG-like immunoreactivities. The mIgG-like and CCAP-IR neurons were identical to a subset of CCAP-IR neurons recently described by Davis et al. [(1993) J. Comp. Neurol., 338:612-627] in pupae. We found these CCAP-IR neurons, however, also to be present in larvae. The mIgG-like- and CCAP-IR neurons included the NS-L pair of the subesophageal maxillary neuromere, which projected anteriorly to the corpora cardiaca, and the NS-L of the labial neuromere whose axons projected out the dorsal nerve of the next posterior ganglion. The mIgG-like and CCAP-IR NS-Ls were also observed throughout the three thoracic ganglia, and all shared strikingly similar structural features. These cells exited out the dorsal nerve of the next posterior ganglion and eventually projected to the neurohemal release sites of the perivisceral organs. These neurons appear to be the homologues of the abdominal CCAP-IR NS-Ls, neurons that in the adult switch their neurotransmitter and release the neuropeptide bursicon. Our description of the distribution and developmental expression of this novel mIgG-like immunoreactivity may provide new insights into the regulation of neurotransmitter plasticity and/or recognition-signaling events involved in the embryonic and postembryonic assembly of the nervous system.

Evidence for the role of pancreatic acinar cells in the production of ornithine and guanidinoacetic acid by L-arginine:glycine amidinotransferase.

L-Arginine:glycine amidinotransferase (transamidinase) occurs at high concentrations in the kidney and the pancreas of rats. The cellular localization of transamidinase was investigated in fetal, neonatal, and adult rat pancreatic tissue using three indicators of the presence of transamidinase: (1) immunofluorescence microscopy, (2) in vitro enzymatic activity measurements on homogenates of whole pancreas and on isolated acinar and islet tissue from adult rats, and (3) ornithine production from perfused adult rat pancreas. The cellular localization of transamidinase was determined in fetal, neonatal, and adult rat pancreas, using a polyclonal guinea pig antibody made against a highly purified preparation of kidney transamidinase. Immunoreactive transamidinase was detected only in the pancreatic acinar cells. The cellular distribution of the immunostaining was compatible with the presence of transamidinase in mitochondria. The transamidinase enzymatic activity of whole pancreatic homogenates was 13.4 +/- 0.7 U/g wet weight (n = 11). In pancreata where islets had been isolated away from the acinar tissue, the transamidinase activity was similar to that of the whole pancreatic homogenates (16.8 +/- 2 U/g wet weight). Any transamidinase activity present in isolated islets was below the sensitivity of the assay. Transamidinase activity in the isolated perfused pancreas was determined by measuring the amount of ornithine released into the perfusate. The transamidinase activity of the perfused pancreas was 16.4 +/- 1.8 U/g pancreas and is an estimate of the physiological production capacity of the enzyme (270 +/- 29 nmol ornithine/min/g pancreas). These results indicate that transamidinase is present at high concentrations in the pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)

Innervation and vasculature of human sweat glands: an immunohistochemistry-laser scanning confocal fluorescence microscopy study.

Secreting tubules, nerves fibers, and blood vessels in human sweat glands (SGs) were fluorescently stained by immunohistochemical and lectin methods for examination with a laser scanning confocal microscope (LSCM). Using these techniques, the three-dimensional distribution of up to three substances within a single specimen was investigated by collecting a series of optical sections for each of three fluorophores. Each SG received several nerve fibers. These branched into delicate bands of one or more axons that ran longitudinal to the sweat tubule then encircled the tubule. A heavy complement of capillaries was interwoven among the sweat tubules. Sweat ducts were accompanied from the SG toward the skin surface by one or two longitudinally oriented nerve fibers and capillaries. Immunoreactive staining of nerves was heaviest with protein gene product 9.5 antibody, but triple labeling showed that immunoreactivity to calcitonin gene-related peptide, vasoactive intestinal polypeptide, and synaptophysin was also present in the same axons. Substance P-immunoreactive axons were sparse in SGs but were present in other areas of the skin. The techniques used have considerable potential in examination of human skin biopsies for diagnosis of disorders affecting the somatic and autonomic nervous systems.

Effect of tyrosine kinase inhibitors on islets of Langerhans: evidence for tyrosine kinases in the regulation of insulin secretion.

In order to determine if tyrosine kinase activation is involved in the changes in islet function, the effect of tyrosine kinase inhibitors on insulin secretion and islet cell proliferation was examined in cultured islets of Langerhans. When islets were exposed to 100 microM genistein or 2 microM herbimycin A, large 5- to 10-fold increases in insulin secretion were observed. The effect on insulin secretion was detected within 1 hr and was maintained for at least 4 days. The glucose sensitivity of islets exposed to genistein was dramatically increased as demonstrated by a shift of the glucose-dose response curve to lower glucose concentrations. In contrast, islet cell proliferation was dramatically reduced in the presence of these tyrosine kinase inhibitors in the absence or presence of PRL. These very large changes observed in islets suggest that tyrosine kinases may have important roles in the regulation of beta-cell function.

Regulation of islet beta-cell proliferation by prolactin in rat islets.

This study examined the effects of prolactin on beta-cell proliferation in pancreatic islet of Langerhans. Insulin secretion and beta-cell proliferation were significantly increased from neonatal rat islets cultured for 4 days in the presence of either 500 ng/ml ovine prolactin (oPRL) or rat prolactin (rPRL). These effects could be prevented by including anti-oPRL serum in the culture media. Although insulin secretion and beta-cell proliferation were slightly higher during the first 24 h of exposure to rPRL, maximal response was observed after 4 days for insulin secretion and 6-10 days for beta-cell proliferation. The initial mitogenic response of beta-cell to rPRL occurred by the limited recruitment of nondividing beta-cells into the cell cycle and by most daughter cells proceeding directly into additional cell division cycles. Subsequently, the maximal effect of rPRL on beta-cell proliferation was maintained by a higher rate of recruitment of previously nondividing beta-cells into cell cycle with only one fourth of the daughter cells continuing to divide. These observations are difficult to reconcile with the proposal that a limited pool of beta-cells capable of undergoing cell division exists in islets. Instead, these observations suggest that individual beta-cells are transiently re-entering the cell cycle and dividing infrequently in response to rPRL. In this case, the majority of the beta-cells would not be expected to be in an irreversible Go phase. We also demonstrated that the effects of rPRL on beta-cell proliferation occur at normal serum glucose concentrations and are affected by inhibitors of polyamine metabolism. Additional studies on the effects of rPRL on beta-cells should provide important information on the regulation of beta-cell proliferation during conditions of increased insulin demand.

Effects of steroid and lactogenic hormones on islets of Langerhans: a new hypothesis for the role of pregnancy steroids in the adaptation of islets to pregnancy.

Adaptive changes that occur in islets of Langerhans during pregnancy include enhanced insulin secretion, insulin synthesis, beta-cell proliferation, gap-junctional coupling among beta-cells, and glucose oxidation. We have determined that elevated lactogenic activity is directly responsible for these changes in beta-cell function. Recently, we showed that two of the principal adaptive characteristics (insulin secretion and beta-cell proliferation) of rat pregnancy peaked on day 15 and returned to control levels by day 20. As placental lactogen remains elevated during late gestation, it was of interest to determine whether pregnancy steroids could reverse the effects of lactogen on islets. In this study, rat islets were cultured with progesterone, estradiol, rat PRL (rPRL), or combinations of these hormones (progesterone and rPRL, estradiol and rPRL, or progesterone and estradiol and rPRL). Insulin secretion was examined for 8 days, and beta-cell proliferation by 2-bromo-5'-deoxyuridine (BrdU) incorporation on days 4 and 8. rPRL treatment resulted in a time-dependent increase in insulin secretion that was 3-fold greater than that from control islets by day 8. Progesterone and estradiol had minimal effects on insulin secretion. Estradiol had no effect on the increased insulin secretion observed with rPRL during the first 6 days and a small inhibitory effect on days 7 and 8. Although progesterone treatment had no effect on the increased insulin secretion induced by rPRL during the first 3 days, it subsequently resulted in a decline in insulin secretion to that from control islets. The combination of progesterone and estradiol was more effective than either steroid by itself in reversing the effects of rPRL on insulin secretion. Similar results were obtained in the BrdU labeling experiments: 1) a 7-fold increase in the number of BrdU-labeled nuclei per islet was observed after culture in the presence of rPRL; and 2) estradiol had a small inhibitory effect on the increased BrdU labeling observed with rPRL; however, 3) progesterone completely reversed the effect of rPRL on islet beta-cell division. These results demonstrate that progesterone counterregulates the effects of PRL on insulin secretion and islet beta-cell division. The temporal changes observed in islets in vitro under the influence of PRL and progesterone mimic those seen in islets during pregnancy. We conclude that progesterone, which increases in the later stages of gestation, is the primary hormone responsible for counteracting the stimulatory effects of elevated lactogenic activity on islets during late pregnancy.

Improvements for the anatomical characterization of insect neurons in whole mount: the use of cyanine-derived fluorophores and laser scanning confocal microscopy.

The optical sectioning capability of the laser scanning confocal microscope was utilized to image dye-filled neurons within whole-mounted insect ganglia. Specific pterothoracic interneurons, in the moth Manduca sexta, were retrogradely filled with Neurobiotin and subsequently visualized with a monoclonal anti-biotin conjugated with one of the following fluorophores: fluorescein, and the newly developed cyanines, Cy3.18 (Cy3) and Cy5.18 (Cy5). Overall, the Cy5 fluorophore was best suited for imaging insect neurons within ganglia. This new methodology allowed us to identify and characterize morphologically a collection of descending multisegmental interneurons with large or small diameter somata. A variety of larger molecular weight (10,000 daltons) tracers was also used to examine the possibility of nonselective filling of neurons with Neurobiotin, possibly through gap junctions. We also investigated the usefulness of Cy3 and Cy5 as fluorophores for transmitter immunostaining of neurons in whole mount. Neurons immunoreactive for serotonin and the neuropeptides, FMRFamide and SCPB, were imaged in the brain and the pterothoracic ganglion. The central projections of some of these immunoreactive neurons were imaged in their entirety.

Effect of homologous placental lactogens, prolactins, and growth hormones on islet B-cell division and insulin secretion in rat, mouse, and human islets: implication for placental lactogen regulation of islet function during pregnancy.

Up-regulation of maternal islet function is essential to accommodate the increased demand for insulin during pregnancy. Previously, we suggested that lactogenic activity regulates islet function during pregnancy. However, this hypothesis was based on the effect of homologous PRLs on islets, since the homologous placental lactogens (or islets) were unavailable. In this study we examine the direct effects of homologous placental lactogens (PL), PRL, and GH on insulin secretion and B-cell division in rat, mouse, and human islets in vitro. Neonatal rat islets were cultured for 8 days in the presence of 0-1000 ng/ml rat PL-I (rPL-I), rPRL, or rGH. Media were changed daily, and the insulin concentration was determined. rPL-I and rPRL (500 ng/ml) treatment resulted in a 2-fold increase in insulin secretion. rGH (1000 ng/ml) elicited a 30% increase in insulin secretion. Similarly, cell replication, as indicated by BrdU incorporation into B-cells, was increased 4-fold in the presence of rPL-I and rPRL. The ED50 for insulin secretion and 5'-bromo-2'-deoxyuridine (BrdU) incorporation was 70 ng/ml for rPL-I and 150 ng/ml for rPRL. Similarly, in adult rat islets, insulin secretion was increased 1.6-fold, and B-cell replication increased 3-fold in the presence of the lactogenic hormones. Neonatal mouse islets were cultured for 8 days in the presence of 500 ng/ml mouse (m) PL-I, mPL-II, mPRL, or mGH. mPL-I, mPL-II, and mPRL treatment resulted in a 2-fold increase in insulin secretion. mGH elicited a 30% increase in insulin secretion. BrdU incorporation into B-cells was increased 3-fold in the presence of mPL-I and mPRL and 2-fold in the presence of mPL-II. Adult human islets were cultured for 8 days in the presence of 1 microgram/ml human (h) PL, hPRL, or hGH. For human islets isolated from six pancreata obtained from females, hPL (138 +/- 10%), hPRL (133 +/- 9%), and hGH (117 +/- 3%) significantly increased insulin secretion compared to that from control islets. This study compares the direct effects among homologous PLs, PRLs, and GHs on insulin secretion and B-cell division in rat, mouse, and human islets. The results indicate that placental lactogen directly regulates islet function in several species and is probably the principal hormone responsible for the increased islet function observed during normal pregnancy.

Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red, and cyanine 3.18.

There are several red-emitting fluorophores available for immunofluorescence studies, including tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18; however, it is unclear which of these is best. The present study compared the brightness of these fluorophores to that of fluorescein. Staining was attempted using a primary antibody raised against serotonin and a secondary antibody that was conjugated with either fluorescein or one of the red fluorophores. The intensity of staining was determined densitometrically. It was found that a conjugate of cyanine 3.18 provided significantly brighter staining that conjugates of any of the other fluorophores, including fluorescein. It is concluded that cyanine 3.18 should be useful for multicolor fluorescence experiments and that it may be the brightest fluorophore available for single-color fluorescence immunocytochemistry.

Adaptation of islets of Langerhans to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion.

To elucidate the temporal profile of adaptive changes of the islets of Langerhans to the increased insulin demands of pregnancy, we have studied islet cell proliferation and insulin secretion during gestation in the rat. 5-Bromo-2'-deoxyuridine incorporation into dividing islet cells was significantly (P less than 0.05) increased over age-matched controls by day 10, rose continuously to a peak at day 14, and then returned to control levels by day 18. By day 20, cell division was significantly inhibited (P less than 0.05). The pattern of changes in insulin secretory profiles observed with perfused pancreata of pregnant animals was similar to that obtained for islet cell proliferation. Both the threshold of glucose-stimulated insulin secretion and the amount of above threshold insulin secretion began to diverge from controls by day 10. By day 12, the glucose-stimulation threshold was significantly decreased from 5.7 mM glucose to 3.3 mM (P less than 0.05), remained at this low level through day 15, and returned toward normal by day 20. Concomitant with the increased sensitivity of B cells to glucose, the above threshold insulin secretion was significantly increased by day 12 (P less than 0.05), peaked at day 15, and returned to control levels by day 20. This insulin secretory data demonstrates that the increased sensitivity of B cells to glucose is an important component of the adaptation of islets during pregnancy to the increased demand for insulin at physiological concentrations of plasma glucose. To correlate the above changes in islet cell proliferation and insulin secretion with levels of placental lactogen (PL), serum lactogenic hormone activity was measured by Nb2 lymphoma cell replication assays. This analysis revealed the expected biphasic pattern: a midpregnancy peak at day 12, followed by a nadir at day 14, and then continuously elevated levels until term. The bioassay data agreed with the known secretory profiles of rat (r) PL-I (midpregnancy) and rPL-II (late pregnancy). Our results provide the first systematic evaluation of changes in islet function during pregnancy in the rat. In addition, they provide evidence that rPL-I may be the critical hormonal signal which triggers the primary adaptive changes in islet function characteristic of pregnancy. The return to normal values of insulin secretion and inhibition of cell division observed at day 20 in the presence of high concentrations of rPL-II suggests that other inhibitory influences become dominant in the later stages of rat pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural and functional considerations of GABA in islets of Langerhans. Beta-cells and nerves.

gamma-Aminobutyric acid (GABA), a prominent inhibitory neurotransmitter, is present in high concentrations in beta-cells of islets of Langerhans. The GABA shunt enzymes, glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), have also been localized in islet beta-cells. With the recent demonstration that the 64,000-M, antigen associated with insulin-dependent diabetes mellitus is GAD, there is increased interest in understanding the role of GABA in islet function. Only a small component of beta-cell GABA is contained in insulin secretory granules, making it unlikely that GABA, coreleased with insulin, is physiologically significant. Our immunohistochemical study of GABA in beta-cells of intact islets indicates that GABA is associated with a vesicular compartment distinctly different from insulin secretory granules. Whether this compartment represents a releasable pool of GABA has yet to be determined. GAD in beta-cells is associated with a vesicular compartment, similar to the GABA vesicles. In addition, GAD is found in a unique extensive tubular cisternal complex (GAD complex). It is likely that the GABA-GAD vesicles are derived from this GAD-containing complex. Physiological studies on the effect of extracellular GABA on islet hormonal secretion have had variable results. Effects of GABA on insulin, glucagon, and somatostatin secretion have been proposed. The most compelling evidence for GABA regulation of islet hormone secretion comes from studies on somatostatin secretion, where it has an inhibitory effect. We present new evidence demonstrating the presence of GABAergic nerve cell bodies at the periphery of islets with numerous GABA-containing processes extending into the islet mantle. This close association between GABAergic neurons and islet alpha- and delta-cells strongly suggests that GABA inhibition of somatostatin and glucagon secretion is mediated by these neurons. Intracellular beta-cell GABAA and its metabolism may have a role in beta-cell function. New evidence indicates that GABA shunt activity is involved in regulation of insulin secretion. In addition, GABA or its metabolites may regulate proinsulin synthesis. These new observations provide insight into the complex nature of GABAergic neurons and beta-cell GABA in regulation of islet function.

Prominent expression of acidic fibroblast growth factor in motor and sensory neurons.

Several growth factors originally characterized and named for their action on a variety of cells have more recently been suggested to be importantly involved in the development and maintenance of the nervous system. Acidic fibroblast growth factor (aFGF) is a member of a family of seven structurally related polypeptide growth factors. The cells responsible for expression of aFGF in the nervous system of adult rats have been identified using an affinity-purified antibody to aFGF in immunohistochemical studies and synthetic oligonucleotide probes for in situ hybridization studies. High levels of aFGF expression were observed in motoneurons, primary sensory neurons, and retinal ganglion neurons. Glial cells did not express detectable amounts of aFGF. Confocal and electron microscopic analysis suggested that a large portion of aFGF immunoreactivity was associated with the cytoplasmic face of neuronal membranes, consistent with the hypothesis that aFGF is a sequestered growth factor.

Role of prolactin versus growth hormone on islet B-cell proliferation in vitro: implications for pregnancy.

This study investigated the effects of homologous rat PRL (rPRL) and rat GH (rGH) on islet growth as indicated by modifications in insulin secretion, islet cell proliferation, and islet volume with neonatal and adult rat islets in vitro. The number of proliferating cells was determined by immunocytochemical staining for 5-bromo-2'-deoxyuridine (BrdU) incorporated into replicating DNA during the final 24 h of culture. When neonatal rat islets were examined by laser scanning confocal microscopy, more than 90% of the BrdU-labeled nuclei were observed in B-cells with insulin immunoreactivity. In neonatal rat islets, rPRL was much more effective than rGH in increasing insulin secretion (3.7-fold vs. 1.4-fold) during the 10 days of culture. The number of BrdU-labeled nuclei per islet was increased from 2.9 +/- 0.4 (n = 77) in control islets to 47.3 +/- 2.7 (n = 95) with rPRL (16.3-fold) and 9.7 +/- 0.8 (n = 84) with rGH (3.3-fold). The effects of rPRL and rGH on both insulin secretion and BrdU labeling were approximately additive. After 10 days, an increased average islet volume was only observed with rPRL. When followed for 36 days, the total amount of islet tissue was unchanged for control islets (1.1-fold), more than doubled with rPRL (2.5-fold), and only slightly increased with rGH (1.4-fold). From the observed rates of islet cell proliferation and increases in islet volumes, doubling times of 23-24 days for rPRL and 89-91 days for rGH can be estimated. Of other proposed islet growth factors, cholecystokinin, epidermal growth factor, platelet-derived growth factor, and 2-aminoisobutyric acid, an increase in insulin secretion and islet cell proliferation was only observed with cholecystokinin in neonatal rat islets. However, both effects were less than 20% of those observed with rPRL. In adult rat islets, rPRL was also more effective than rGH in increasing insulin secretion (1.6-fold vs. 1.2-fold) during the 9 days of culture. The number of BrdU-labeled nuclei per islet was increased from 2.7 +/- 0.5 (n = 96) in control islets to 9.5 +/- 0.6 (n = 175) with rPRL (3.5-fold). In contrast to the neonatal islets, rGH had no effect on the number of BrdU-labeled nuclei per islet in adult rat islets (2.4 +/- 0.3, n = 194).(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of prolactin versus growth hormone on islet function and the importance of using homologous mammosomatotropic hormones.

The purpose of this study was to determine the effects of homologous rat PRL (rPRL) and rat GH (rGH) on islet B-cell function in neonatal and adult rat islets in vitro. In neonatal rat islets, exposure to rPRL for more than 24 h was necessary for a stimulatory effect on insulin secretion. By day 4, insulin secretion was 3.8-fold greater in the islets cultured with rPRL. rGH had a modest effect on insulin secretion, and this effect was additive with that of rPRL. Both rPRL and rGH increased islet insulin content and [3H]thymidine incorporation. After removal of rPRL, more than 24 h were necessary to detect a reversal in the level of insulin secretion. In addition, even after 5 days without rPRL the previously treated islets still had elevated levels of insulin secretion. In a dose-response study of rPRL on insulin secretion, a detectable effect was observed at 62.5 ng/ml, with a half-maximal effect of approximately 100 ng/ml. Glucose oxidation by neonatal islets was enhanced by rPRL treatment, but not by rGH treatment. In adult rat islets, exposure to rPRL, but not to rGH, enhanced insulin secretion. In contrast, when using heterologous human GH, results similar to those obtained with rPRL, but not rGH, were observed. The results from these experiments indicate that rPRL and rGH have both individual and shared regulatory effects on rat islets. However, it is rPRL and not rGH that has the primary influence on insulin secretion. When interpreting studies examining the effect of GH and PRL on islet function, it is important to consider whether homologous hormones are used.

Three-dimensional imaging of intact isolated islets of Langerhans with confocal microscopy.

We present a new technique for analyzing the three-dimensional structure of intact isolated islets of Langerhans. Adult rat and human islets were stained with whole-mount immunofluorescence techniques and optically sectioned with a confocal microscope. This has several advantages over traditional methods: 1) the technical difficulties in serial sectioning and handling the large numbers of sections are avoided, 2) optical sectioning by confocal microscopy gives improved resolution and strongly suppresses light from out-of-focus structures, and 3) entire islets can be rapidly imaged for the presence of positive staining. This new technique should facilitate the study of the three-dimensional structure of islets of Langerhans.