Biochemical studies on the release of catecholamines from the rat carotid body in vitro.

The effects of hypoxia and carbachol on the release of newly synthesized catecholamines from superfused rat carotid bodies have been examined. Hypoxic superfusion medium was found to evoke catecholamine release which was dependent on the extracellular calcium concentration and was reduced by nitrendipine and atropine. Superfusion with the muscarinic agonist, carbachol, stimulated catecholamine release independently of the oxygen tension of the medium. The effect of carbachol on catecholamine release was abolished by atropine, suggesting that it was mediated by activation of cholinergic receptors of the muscarinic type. Both hypoxia and carbachol stimulated the release of 45Ca from carotid bodies prelabelled with 45Ca. The release of 45Ca with either stimulus was reduced by atropine and nitrendipine. These results suggest that although extracellular calcium plays an important role in the exocytotic secretory process of the carotid body, the mobilization of intracellular calcium pools may also contribute to the secretory response.

Serotonin immunoreactivity in the carotid body of adult humans.

The distribution of serotonin immunoreactivity (-IR) was studied in adult human carotid bodies, obtained at post-mortem, using both the peroxidase-antiperoxidase method on paraffin sections and a double-labelling immunofluorescence on frozen sections. Antibodies against synaptophysin and protein gene product (PGP) 9.5 were used for identification of serotonin-IR cells. Serotonin-IR was demonstrable in the carotid bodies of adult humans and it was coexpressed mostly with synaptophysin or PGP 9.5 in type I cells. Some serotonin immunopositive type I cells were located in close proximity to capillaries. Serotonin-IR was also observed in a few endothelial cells.

Acute hypoxic ventilation, carotid body cell division, and dopamine content during early hypoxia in rats.

In a previous study, we showed that the acute hypoxic ventilatory response was blunted in anesthetized chronically hypoxic rats and was restored by blockade of the dopamine D2 receptor with domperidone. We now report observations made during 1-8 days of exposure to 10% O2 on the acute hypoxic ventilatory response and the effect of domperidone and relate them to dopamine content and cellular proliferation in the carotid body. Hypoxic exposure caused a parallel shift in the hypoxic response curve to higher levels of ventilation and arterial oxygen saturation. The greatest response occurred on day 1 and was unaffected by domperidone: dopamine content diminished and mitotic activity increased. By 8 days, hypoxic ventilation approached normal and was significantly augmented by domperidone; in the carotid body, dopamine levels had risen above the control level and mitoses had diminished. Thus the increase in ventilation was inversely related to carotid body dopamine content, which was depressed. The possibility of a causal relationship is discussed.

Distribution of vasoactive intestinal polypeptide, neuropeptide-Y and substance P and their effects on insulin secretion from the in vitro pancreas of normal and diabetic rats.

This study examined the pattern of distribution of vasoactive intestinal polypeptide (VIP), neuropeptide-Y (NPY) and substance P (SP) in the pancreas of diabetic rat to determine whether there are changes in the number and pattern of distribution of these neuropeptides after the onset of diabetes. Moreover, the effect of VIP, NPY and SP on insulin secretion from the pancreas of normal and diabetic rats was also examined. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (i.p.) (60 mg kg body weight(-1)). Four weeks after the induction of DM, diabetic (n = 6) and normal (n = 6) rats were anesthetized with chloral hydrate and their pancreases removed and processed for immunohistochemistry and insulin secretion. The number of insulin-positive cells in the islets of Langerhans was reduced while that of VIP and NPY increased significantly after the onset of diabetes. The pattern of distribution of VIP, NPY and SP in the nerves innervating the pancreas was similar in both normal and diabetic rats. VIP-evoked large and significant (P < 0.02) increases in insulin secretion from the pancreas of normal and diabetic rats. NPY also induced a marked (P < 0.005) increase in insulin release from pancreatic tissue fragments of normal rat. Stimulation of pancreatic tissue fragments of diabetic rat with NPY resulted in a slight but not significant increase in insulin release. SP induced a large and significant (P < 0.005) increase in insulin secretion from the pancreas of normal rat but inhibited insulin secretion significantly (P < 0.03) from isolated pancreas of diabetic rat. In summary, VIP and NPY can stimulate insulin secretion from the pancreas after the onset of diabetes. The stimulatory effect of SP on insulin secretion is reversed to inhibitory in diabetic rats.

Immunohistochemical identification and effects of atrial natriuretic peptide, pancreastatin, leucine-enkephalin, and galanin in the porcine pancreas.

This study demonstrates the presence and distribution of atrial natriuretic peptide (ANP) pancreastatin (PST), leucineenkephalin (Leu-ENK), galanin (GAL), and insulin in the pig pancreas. The effects of PST, ANP, Leu-ENK, and GAL on protein and amylase secretion were also investigated to determine their functional role in the control of pancreatic secretion. PST-immunoreactive cells were observed in the islet of Langerhans and in the wall of the ducts. Leu-ENK-immunopositive cells were observed in both the endo-and exocrine pancreas. It is colocalized with insulin in the islet of Langerhans. ANP immunoreactivity was discernible in nerve fibers and cells of the exocrine pancreas. GAL-immunopositive cells were observed in close association with insulin-positive cells in the islets of Langerhans and in the exocrine pancreas. Stimulation of isolated pancreatic segments with either ANP or Leu-ENK resulted in increased protein secretion and amylase output. The Leu-ENK-evoked amylase secretion was antagonized by naloxone. Pancreastatin was effective at all concentrations, but low concentration had more marked secretory effects whereas GAL failed to evoke any significant increases in either protein or amylase secretion. The results of the study have demonstrated a close association of peptidergic fibers with the secretory cells of the pancreas. The nerve fibers can release peptides that in turn can stimulate protein and amylase secretion.

Interactions of islet hormones with acetylcholine in the isolated rat pancreas.

This study investigates the effects of the islet hormones, insulin (INS), glucagon (GLU) and somatostatin (SOM) on acetylcholine (ACh)-evoked amylase secretion and calcium (Ca2+) mobilization in the isolated rat pancreas. Stimulation of pancreatic segments and acini with either INS, GLU or SOM resulted in small increases of amylase output compared to much large increases in enzyme output with ACh. Combinations of the peptide hormones with ACh resulted in enhanced secretory responses compared to the effects obtained with either ACh or each of the islet hormone alone. Genistein, the tyrosine kinase inhibitor, evoked a decrease in amylase output from pancreatic segments. It had no effect on the ACh evoke secretory response but it markedly inhibited the potentiation of the islet hormones with ACh. In pancreatic acinar cells either INS, GLU or SOM elicited moderate increases in amylase output compared to much larger responses with ACh. Furthermore, the islet hormones failed to potentiate the secretory effect of ACh in pancreatic acini. In fura-2 AM loaded acinar cells both INS and GLU evoked small increases in intracellular free calcium concentration [Ca2+]i compared to a much larger elevation with ACh. Both INS and GLU enhanced the ACh-evoked [Ca2+]i. Genistein elicited a decrease in [Ca2+]i both in the absence and presence of both INS and GLU. It also decreased the rise in [Ca2+]i resulting from the combined presence of ACh with both INS and GLU. SOM had no significant effect on the ACh-induced [Ca2+]i. When genistein was combined with ACh and SOM there was a decrease in [Ca2+]i compared to the response obtained with SOM and ACh alone. The results indicate that both tyrosine kinase and cellular Ca2+ seem to be the intracellular mediators associated with the enhanced secretory responses obtained with a combination of the islet hormones with ACh. Finally, our results using immunohistochemical techniques confirm the presence of INS-, GLU- SOM- and ACh-immunoreactive cells in the endocrine and neural elements of the rat pancreas.

Innervation of the pancreas of the one-humped camel (Camelus dromedarius) by neuropeptide-Y, galanin, calcitonin-gene-related-peptide, atrial natriuretic peptide and cholecystokinin.

The distribution of neuropeptide-Y (NPY), galanin (GAL), cholecystokinin-8 (CCK-8), atrial natriuretic peptide (ANP) and calcitonin gene-related peptide (CGRP) in the pancreas of the camel was investigated using immunohistochemical techniques. NPY-immunoreactive neurons were observed in the pancreatic ganglia and also in the interacinar regions of the exocrine pancreas. NPY was discernible in fine varicose nerve fibres ending on NPY-negative cells and in the walls of blood vessels. ANP immunoreactivity was observed in nerve fibres situated on the basolateral surfaces of the acinar cells. CCK-8, GAL and CGRP immunoreactivity were observed in neurons and varicose nerve fibres in the walls of blood vessels. Of all the neuropeptides investigated, only NPY appeared to be densely distributed in the pancreas of the camel. It is concluded that the pattern of distribution of these neuropeptides in the camel pancreas is similar to those observed in the pancreata of other mammals.

Direct biochemical and neuropharmacological identification of dopamine D2-receptors in the rabbit carotid body.

Dopamine D2-receptors were directly identified in receptor binding assays with washed particulate preparations of rabbit carotid body using the selective ligand, [3H]domperidone. High affinity, saturable specific binding of [3H]domperidone was clearly demonstrable and chronic section of the sinus nerve resulted in a 32% decrease in the labelling of the dopamine D2-sites. Adenylate cyclase activity was also detected in rabbit carotid body homogenates and although this enzyme was stimulated 4-fold by 10 mM sodium fluoride, neither dopamine nor isoprenaline significantly altered basal activity. On the other hand, in the intact carotid body incubated in vitro, 10(-5) M isoprenaline increased the basal cyclic AMP content 6-fold, though dopamine was again ineffective. The effect of various selective dopamine receptor antagonists and agonists was also studied on chemoreceptor afferent discharge. The results confirm that depression of 'spontaneous' chemosensory discharge is the predominant effect of dopamine (0.01-100 micrograms) in rabbits. The 'selective' D2-agonist, LY 141865, proved very effective (ID50 3.3 nmol) and was equipotent with dopamine (ID50 4.2), whereas, the D1-agonist, SK & F 38393, was very ineffective (ID50 150). The D2-antagonists domperidone and (-)-sulpiride produced a dose-related decrease in the chemodepressant responses to dopamine and LY 141865. However, there was no evidence for any appreciable excitatory action of either of these agonists after blockade of their chemo-depressant effects. The D2-antagonists variably affected the spontaneous activity, there being an increase in discharge on average, whereas responses to hypoxia, cyanide and CO2 were reduced. The present results from biochemical and neuropharmacological studies, provide strong evidence for the presence of functional dopamine D2-receptors in the rabbit carotid body, and suggest that the receptor involved in dopamine-induced depression of chemosensory discharge is of D2-type.

Catecholamines in the carotid body of several mammalian species: effects of surgical and chemical sympathectomy.

The catecholamine content of the carotid body of several mammalian species has been assayed using high performance liquid chromatography coupled to electrochemical detection and radioenzymatic assays. Although there were strain differences in the content of catecholamines in the carotid body of the rat, noradrenaline was equal to or exceeded the dopamine level in this species. No apparent differences were found in carotid bodies of animals killed by cervical dislocation or those dissected from anaesthetized animals. Noradrenaline concentrations were found to be substantially higher than those of dopamine in the cat and guinea-pig carotid body, though dopamine was the predominant amine in the rabbit and ferret. Unilateral superior cervical ganglionectomy or chemical sympathectomy with 6-hydroxydopamine substantially depleted noradrenaline without influencing dopamine in the rat carotid body. A marked selective reduction in noradrenaline was also observed in the rabbit and guinea-pig following ganglionectomy, though similar procedures in the cat failed to alter the levels of either catecholamine in the carotid body. The present data highlights the marked species variation in catecholamine content and the contribution to the latter by sympathetic innervation to this organ. This information will be useful in determining the species specificity regarding the relative roles of dopamine and noradrenaline in the modulation of chemoreceptor afferent discharge.

Effects of Momordica charantia fruit juice on islet morphology in the pancreas of the streptozotocin-diabetic rat.

An investigation was made of the effect of Momordica charantia fruit juice on the distribution and number of alpha, beta and delta cells in the pancreas of streptozotocin (STZ)-induced diabetic rats using immunohistochemical methods. The results indicated that there was a significant (Student's t-test, P < 0.004) increase in the number of beta cells in M. charantia-treated animals when compared with untreated diabetics, however, their number was still significantly less than that obtained for normal rats. There was also a significant (P < 0.006) increase in the number of delta cells in STZ-diabetic rats compared to non-diabetic rats. This increase in the number of delta cells was not affected by M. charantia treatment. The number of alpha cells did not change significantly in M. charantia-treated rats when compared with untreated diabetic rats. Our results suggest that oral feeding of M. charantia fruit juice may have a role in the renewal of beta cells in STZ-diabetic rats or alternately may permit the recovery of partially destroyed beta cells.

Peptidergic and aminergic neurotransmitters of the exocrine pancreas of the Houbara bustard (Chlamydotis undulata).

The immunochemical distribution of peptidergic and aminergic neurotransmitters in the exocrine pancreas of the Houbara bustard, Chlamydotis undulata, was determined. Immunoreactivity to choline acetyltransferase (ChAT), vasoactive intestinal polypeptide (VIP), and galanin (Gal) occurred mainly as varicose terminals in the walls of capillaries around the acini and arterioles within the connective tissue. Neuronal cell bodies immunoreactive to ChAT were infrequently observed. Neuropeptide Y (NPY), pancreatic polypeptide (PP), and somatostatin (Som) were observed mainly in intra-acinar cell bodies but nerve fibers immunoreactive to these neuropeptides were also seen along the basal surfaces of the acini. Immunoreactivity to NPY and PP was also discernible in cells of the pancreatic ducts. In addition, NPY occurred as varicose terminals in vessels around the ducts. SP occurred rarely in interacinar ganglia. The distribution of tyrosine hydroxylase (TH) was similar to that of ChAT and, in addition, the occasional TH immunoreactive intra-acinar neuronal cell body was observed. Neuronal nitric oxide synthase (nNOS) occurred in neuronal cell bodies among the acinar cells as well as nerve fibers along the bases of the acini. The potential roles of these peptidergic and aminergic neurotransmitters in the neurohormonal control of pancreatic secretion are discussed.

Distribution of neurotransmitters and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues.

The distribution of adrenergic, cholinergic and amino acid neurotransmitters and/or their enzymes were examined in both the normal and diabetic pancreatic tissues in rat using immunohistochemistry to determine whether changes in the pattern of distribution of nerves containing these neurotransmitters will occur as a result of diabetes mellitus. In addition to this, the effect of noradrenaline (NA), adrenaline (ADR), acetylcholine (ACh) and gamma-amino butyric acid (GABA) on glucagon secretion from the isolated normal and diabetic pancreatic tissues was also investigated. Pancreatic fragments from the tail end of normal and diabetic rats were removed and incubated with different concentrations (10(-8)-10(-4) M) of these neurotransmitters. Glucagon secretion into the supernatant was later determined by radioimmunoassay. NA at 10(-6) M evoked a three-fold increase in glucagon secretion from normal pancreatic tissue fragments. In diabetic pancreatic tissue, NA at 10(-6) M was able to increase glucagon secretion 1.5 times the value obtained from diabetic basal. ADR (10(-8) M) increased glucagon secretion slightly but not significantly in normal pancreatic tissue. ADR inhibited glucagon secretion from diabetic pancreas at all concentrations. ACh (10(-8) M) induced a five-fold increase in glucagon secretion from normal pancreatic tissue. In a similar way, ACh evoked a two-fold increase in glucagon secretion from diabetic pancreas at 10(-4) M. In normal pancreatic tissue, GABA produced a slight but not significant increase in glucagon secretion at 10(-4) M. In contrast to this it inhibited glucagon secretion from diabetic pancreatic tissue fragments at all concentrations. In summary, tyrosine hydroxylase- and choline acetyltransferase-positive nerves are equally well distributed in both normal and diabetic rat pancreas. There was an increase in the number of glucagon positive cells and a decrease in the number of GABA-positive cells in diabetic pancreas. NA and ACh have a potent stimulatory effect on glucagon secretion from normal pancreatic tissue fragments, whereas ADR and GABA produced a small but not significant increase in glucagon secretion from normal pancreas. NA and GABA stimulated glucagon secretion from diabetic pancreas. In contrast, ADR and ACh inhibited glucagon secretion from diabetic pancreas. Neurotransmitters vary in their ability to provoke glucagon secretion from either normal or diabetic pancreas.