Immunocytochemical location of thyrotropin-releasing hormone (TRH) in the B-cells of adult hypothyroid rat pancreas

Thyrotropin-releasing hormone (TRH) is present in small quantities in the rat adult pancreas. As hypothyroidism increases dramatically the pancreatic content of this peptide, this model was used to localize TRH in the gland by immunocytochemistry. Immunocytochemical staining of semithin (0.5–1.0 μm) and thin (golden) sections was performed as well as antibody and method controls to check the specificity of the immunoperoxidase staining. At the light microscope level, a very faint TRH-like immunoreactivity was apparent in the pancreas of normal untreated animals. In hypothyroid rats, a strong TRH immunostaining was observed in the central portion of the islets of Langerhans. On the contrary, in previously hypothyroid rats made euthyroid, no TRH-like immunoreactivity was found. Serial sections alternately labelled with TRH and insulin antisera revealed the simultaneous occurrence of both immunoreactivities. In addition, the TRH immunoreactive cells were distinct from glucagon- or somatostatin-containing cells. At the electron microscope level, immunoreactive TRH was found over the secretory granules of insulin-containing cells. Hypothyroid animals offer therefore a suitable model for the study of TRH in the pancreas.     

Evidence that TRH controls prolactin release from rat lactotrophs by stimulating a calcium influx

Prolactin (PRL) release and intracellular free calcium concentration [Ca²⁺]_i were measured in two populations of normal rat lactotrophs (light and heavy fractions) in culture. Spontaneous PRL release of heavy fraction cells was more sensitive to dihydropyridines (DHPs; Bay K 8644 and nifedipine) when compared to the light fraction lactotrophs. The stimulatory effect of thyrotropin-releasing hormone (TRH) on PRL release from heavy fraction cells was inhibited by Cd²⁺ and mimicked by Bay K 8644. Indo-1 experiments revealed that TRH-increased [Ca²⁺]_i was reversibly inhibited by Cd²⁺. In a Ca²⁺-free EGTA-containing medium, TRH did not modify [Ca²⁺]_i.Abbreviations [Ca²⁺]_i intracellular free calcium concentration - DA dopamine - DHP dihydropyridine(s) - DMEM Dulbecco's Modified Eagle's Medium - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PRL prolactin - RIA radioimmunoassay - TRH thyrotropin-releasing hormone - VGCC voltage-gated calcium channel     

Cell swelling induced secretion of TRH by posterior pituitary,hypothalamic paraventricular nucleus and pancreatic islets: effect of L-canavanine

The aims of this study were to test if ethanol induces thyrotropin-releasing hormone (TRH) secretion in vitro from the posterior pituitary and hypothalamic explants by a mechanism involving cell swelling, and to characterize the pathway of stimulated secretion. Ethanol, at a concentration of 80 mM, stimulated the release of TRH from the posterior pituitary, the hypothalamic paraventricular nucleus, the median eminence, and the brain septum, when administered only in isosmolar but not in hyperosmolar medium. This indicates the involvement of a cell swelling-inducing mechanism. L-canavanine in a concentration of 3 mM, increased the basal and hyposmosis-induced TRH secretion from the posterior pituitary and the paraventricular nucleus, and both basal and ethanol-induced TRH secretion from isolated pancreatic islets. This indicates the presence of both constitutive and regulatory secretory pathways. Our results suggest that cell swelling induces exocytosis from clathrin coated granules.     

Anion secretion by the isolated rabbit pancreas

The isolated rabbit pancreas secretes a fluid containing chloride and bicarbonate in about equal concentrations. Replacement of bicarbonate by acetate, phosphate or isethionate, replacement of Na⁺ by Li⁺ and addition of ouabain to the bathing medium of the pancreas inhibit the secretion of fluid, chloride and bicarbonate in a similar fashion and by maximally 100%. Replacement of chloride by isethionate inhibits fluid secretion by maximally 50%, chloride secretion by 90% and bicarbonate secretion by 20%. It is concluded that fluid secretion is based on a Na⁺-gradient-dependent bicarbonate influx or proton efflux in the ductular cell, and that the secretion of chloride is secondary to that of bicarbonate.     

Atropine depresses release of neurotensin and its effect on the exocrine pancreas

In this study the effect of 10 and 20 μg · kg^?1 · h^?1 atropine sulfate on release and pancreatic effects of neurotensin was studied in 4 dogs. Neurotensin plasma levels rose significantly when a liquid fat preparation was infused intraduodenally. This rise was almost completely abolished by simultaneous infusion of atropine. Atropine further suppressed basal and fat-stimulated output of pancreatic volume, protein, and bicarbonate; it also reduced pancreatic secretion stimulated by an intravenous infusion of low doses (2.5 to 20 pmol · kg^?1 · min^?1) neurotensin. The effect of higher doses (80 and 240 pmol · kg^?1 · min^?1) of neurotensin was less affected.As neurotensin plasma levels in contrast to normal oral feeding did not rise after sham feeding, our findings suggest that release and action of neurotensin may at least in part be dependent on a cholinergic, non-cephalic mechanism.     

Nitric oxide and the pancreas: Morphological base and role in the control of the exocrine pancreatic secretion

The distribution of nitric oxide synthase in both neuronal and non-neuronal pancreatic tissues and the role of nitric oxide in the control of exocrine pancreatic secretion are reviewed in this article. Earlier reports based on in vivo studies suggested that nitric oxide can affect the secretory activity of the exocrine pancreas through changes in pancreatic blood flow. More recently, the employment of either nitric oxide synthase inhibitors or nitric oxide donors in in vitro preparations has provided evidence that nitric oxide can exert a direct action on this gland independently on its vascular effects. Most research in this area seems to indicate that modulation of exocrine pancreatic function by nitric oxide is exerted via activation of guanylate cyclase and generation of cGMP, although other pathways cannot be excluded. Experiments performed over the last year in our laboratory reveal a novel and interesting mechanism based on the ability of nitric oxide to control the release of endogenous neurotransmitter in the pancreas and, subsequently, the nerve-mediated enzyme secretion.     

Neurotensin interacts with carbachol, secretin, and caerulein in the stimulation of the exocrine pancreas of the rat in vitro

In the present investigation the effect of neurotensin on pancreatic secretion of isolated pancreatic lobules from the rat was examined. We found a dose- and time-dependent stimulation of amylase release beginning with a concentration of 10(-9) M neurotensin. This response was potentiated by the cholinergic agonist carbachol, the gastrointestinal peptide secretin, and the CCK analogue caerulein. As we found neurotensin-immunoreactive nerves within the pancreas and as neurotensin-like immunoreactivity is present in the circulation (found previously), neurotensin may well be a further peptide taking part in the regulation of exocrine pancreatic secretion either as a hormone or a neurotransmitter. Neurotensin would then cooperate with cholinergic mechanisms, secretin, and CCK.     

Angioarchitecture of the pancreas of the cat

Summary By the use of scanningand transmission electron microscopy, the possible sources of errors in interpretation of the microcirculation of the pancreas can be reduced in comparison to the classical India-ink injection method. Sphincter-like structures in the capillary wall of the cat pancreas are established by pericytes. These sphincters encircle the junctional zones between the endocrine and exocrine capillaries. They are assumed to be regulatory structures of blood flow and to regulate indirectly hormone secretion according to demand.This work was financially supported through the kindness of Eli Lilly GmbH, Bad Homburg, Bundesrepublik Deutschland     

Electron-microscopic cytochemistry of the catecholaminergic innervation of TRH neurons in the rat hypothalamus

Summary The catecholaminergic innervation of thyrotropin-releasing hormone (TRH) neurons was examined by use of a combined method of 5-hydroxydopamine (5-OHDA) uptake or autoradiography after intraventricular injection of ³H-noradrenaline (³H-NA) and immunocytochemistry for TRH in the same tissue sections at the electron-microscopic level.TRH-like immunoreactive nerve cell bodies were distributed abundantly in the parvocellular part of the paraventricular nucleus (PVN), in the suprachiasmatic preoptic nucleus and in the dorsomedial nucleus of the rat hypothalamus. In the PVN, a large number of immunonegative axon terminals were found to make synaptic contact with TRH-like immunoreactive cell bodies and fibers. In the combined autoradiography or 5-OHDA labeling with immunocytochemistry, axon terminals labeled with ³H-NA or 5-OHDA were found to form synaptic contacts with the TRH immunoreactive nerve cell bodies and fibers. These findings suggest that catecholamine-containing neurons, probably noradrenergic, may innervate TRH neurons to regulate TRH secretion via synapses with other unknown neurons in the rat PVN.This study was supported by grants from the Ministry of Education, Science and Culture, Japan     

The role of magnesium in regulating CCK-8-evoked secretory responses in the exocrine rat pancreas

This study investigates the effect of magnesium (Mg²⁺) on the secretory responses and the mobilization of calcium (Ca²⁺) and Mg²⁺ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10^–10 M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg²⁺ [Mg²⁺]_o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg²⁺]_o Similar effects of perturbation of [Mg²⁺]_o on amylase secretion and ⁴⁵Ca²⁺ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca²⁺ concentration [Ca²⁺]_i in zero and normal [Mg²⁺]_o compared to a much reduced response in elevated [Mg²⁺]_o Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB₂ cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca²⁺]_i. In magfura-2-loaded acinar cells CCK-8 (10^–8 M) stimulated an initial transient rise in intracellular free Mg²⁺ concentration [Mg²⁺]_i followed by a more prolonged and sustained decrease. This response was abolished when sodium Na⁺ was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg²⁺ resulted in an elevation in [Mg²⁺]_i. Upon stimulation with CCK-8, [Mg²⁺]_i. decreased only slightly compared with the response obtained in normal [Mg²⁺]_o. CCK-8 caused a net efflux of Mg²⁺ in pancreatic segments; this effect was abolished when extracellular sodium [Na⁺]_o was replaced with either NMDG or choline. The results indicate that Mg²⁺ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca²⁺ mobilization. Moreover, the movement of Mg²⁺ in pancreatic acinar cells is dependent upon extracellular Na⁺.     

TRH: Cardiovascular and sympathetic modulation in brain nuclei of the rat

The cardiovascular and sympathetic effects of TRH in discrete cardiovascular-related brain nuclei were studied. Microinjections of TRH were made into the nucleus preopticus medialis (POM) of conscious rats and the nucleus tractus solitarius (NTS) of pentobarbitone-anesthetized, artificially respired rats. POM injections (1 μl, 0.8–80 nM) elicited dose dependent pressor and tachycardic responses which were accompanied by increased levels of norepinephrine (NE) and epinephrine (EPI) in the plasma. These pressor/tachycardic effects of TRH were also elicited in adrenal demedullated (ADM-x) rats, but completely abolished in ADM-x rats pretreated with bretylium (30 mg/kg, IA). NTS injections (0.1 μl, 30 and 150 nM) had a short depressor effect on blood pressure (BP) and a delayed increase in heart rate (HR). From these findings we suggest that the POM, a central nucleus in the AV3V region, may be an important forebrain site for autonomic regulation by TRH, mediated through the sympathetic nervous system.     

Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas

Summary We have previously shown that inositol-1,4,5-trisphosphate (IP₃) releases Ca²⁺ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983,Nature (London) 306:67–69). This observation suggests that IP₃ might provide the missing link between activation of the muscarinic receptor and Ca²⁺ release from intracellular stores during stimulation. In order to localize the intracellular IP₃-sensitive calcium pool, IP₃-induced Ca²⁺ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl₂. IP₃-induced Ca²⁺ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000×g, sucrose-density centrifugation, or MgCl₂ precipitation there was a close correlation of IP₃-induced Ca²⁺ release with the endoplasmic reticulum markers ribonucleic acid (r=0.96, 1.00, 0.91, respectively) and NADPH cytochromec reductase (r=0.63, 0.98, 090, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochromec oxidase (r=–0.64) and glutamate dehydrogenase (r=–0.75) and with the plasma membrane markers (Na⁺+K⁺)-ATPase (r=–0.81) and alkaline phosphatase (r=–0.77) in all fractions analyzed. IP₃-induced Ca²⁺ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca²⁺ from endoplasmic reticulum in pancreatic acinar cells.     

Immunohistochemistry of opioid peptides in the guinea pig endocrine pancreas

Summary Previous immunochemical investigations have demonstrated various opioid peptides in the pancreas. However, controversies exist related to the cellular localization of these peptides in the endocrine pancreas. Therefore, the guinea pig endocrine pancreas was immunohistochemically investigated for the presence of opioid peptides derived from pro-dynorphin, pro-enkephalin or pro-opiomelanocortin. Immunoreactivities were demonstrated on serial semithin sections by the peroxidase anti-peroxidase technique. In routinely immunostained sections, immunoreactivities for dynorphin A and -neo-endorphin were localized in pancreatic enterochromaffin cells, but not in islet cells. Immunoreactivity for Met-enkephalin was confined exclusively to B-cells and was localized only in some secretory granules. However, pre-treatment of semi-thin sections with trypsin and carboxypeptidase B led to a marked increase of Met-enkephalin immunoreactivity in B-cells. In addition, immunoreactivities for Met-enkephalin-Arg-Gly-Leu and bovine adrenal medulla dodecapeptide could be demonstrated in B-and A-cells, and -endorphin immunoreactivity was localized in A-cells. In no case, however, were immunoreactivities detected for bovine adrenal medulla docosapeptide, peptide F, corticotropin, melanotropin or dynorphin 1–32. The immunohistochemical findings indicate that opioids of different peptide families are present in the guinea pig endocrine pancreas. Since several opioid peptides of the corresponding pro-hormones could be demonstrated in the reference organs but not in the pancreas, it is concluded that the biosynthetic pathways of the respective precursors are different from those in the adrenal medulla or in the pituitary.     

Differential motor and blood pressure effects of intrathecal oxytocin and TRH in the rat

While previous reports have immunocytochemically localized oxytocin and TRH in the spinal cord, the functional significance of these peptides is unclear. The present paper examined this issue and tested the effects of these peptides upon intrathecal administration. We found both peptides produced lasting motor and blood pressure changes. Oxytocin elicited prolonged jerks of the hindlimbs and the tail, while TRH produced an increase of hindlimb muscle tone and tail tremor. TRH in larger doses (5, 10 μg) also caused tail erections and whipping. The motor effects of both peptides were dose-dependent. Intrathecal oxytocin (0.75 or 1.5 IU) caused a transient drop in blood pressure followed by a rise, in 4 out of 7 rats. The other 3 only showed a hypertensive effect. In contrast, intrathecal TRH produced a rise in blood pressure in all the animals tested. These findings suggest that both oxytocin and TRH may play a role in the regulation of motor and automatic functioning at the spinal level.     

Insulin, glucagon and somatostatin localization in the pancreas of metamorphosed Xenopus laevis

The current study was designed to determine if insulin, glucagon and somatostatin-containing cells are present in the pancreas of adult Xenopus laevis. Localization methods utilized included cytochemical aldehyde fuchsin (AF) staining as well as the immunochemical peroxidase antiperoxidase (PAP) procedure for light microscopy. The results show numerous large clusters of AF-positive cells within a network of highly vascularized acinar tissue. PAP immunochemical localization with insulin antibody on adjacent sections demonstrates positive immunoreactivity to AF-positive cell groups and also the presence of immunoreactive insulin (IRI). Cells exhibiting this immunoreactivity are located in the central region of the islet-like structures. Serial sections not only show PAP immunoreactivity for IRI, but also for immunoreactive glucagon (IRG) and immunoreactive somatostatin (IRS) in the same islet-like structure. IRG and IRS-containing cells are situated around the periphery of the islet-like structures, surrounding the central core of IRI-containing cells. Antibody specificity was confirmed by homologous and heterologous antigen immuno-absorbance assays, as well as incubation of adjacent sections in preimmune sera. Based on this data we conclude that: the distribution of cells of the endocrine pancreas of metamorphosed Xenopus laevis is similar to that of many mammals and certain urodeles. Given the apparent specificity of the antigen-antibody reactions, it appears that Xenopus insulin, glucagon and somatostatin are structurally conserved.     

Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.     

Effect of proglumide, a cholecystokinin receptor antagonist, on caerulein-stimulated pancreatic enzyme secretion and pancreatic polypeptide release in the dog

In five conscious dogs we studied the effect of proglumide, a cholecystokinin (CCK) antagonist, on caerulein-stimulated pancreatic secretion and release of pancreatic polypeptide (PP). Graded doses of caerulein (15-240 ng/kg per h) were infused intravenously. Experiments were repeated with a fixed infusion of proglumide (40 mg/kg per h). Release of PP following increasing doses of caerulein was significantly inhibited by proglumide (P less than 0.01). However, proglumide did not significantly affect caerulein-stimulated pancreatic protein secretion. Proglumide might be useful in defining the physiological role of CCK.