The effects of age and streptozotocin diabetes on the sympathetic innervation in the rat penis.

The objective was to describe the changes in catecholamine levels, noradrenaline (NA) release and the ultrastructural and immunohistochemical changes in the sympathetic nerves in the penis of STZ-diabetic rats. Amines were measured using HPLC. Nerves were studied using immunocytochemistry for tyrosine hydroxylase, and electron microscopy. Diabetic animals were compared with age-matched controls. The concentration of penile NA increases at least 2.5-fold after about 10 weeks of hyperglycaemia, is maintained for over 40 weeks. The rate of release of NA in the diabetics also increases approximately by fourfold. Immunohistochemical staining for tyrosine hydroxylase showed either no change or an increase in the levels of the enzyme around the central arteries and the outer coverings of the corpus cavernosum. Cavernosal nerves show increased intensity of staining for tyrosine hydroxylase, and the presence of dilated nerve fibres and engorged endings. The axons of the dorsal nerve of the diabetic penis have a smaller cross-sectional area that is most marked in unmyelinated axons. In the diabetic penis, the nerve endings appear to contain significantly more NA than the controls, and the turnover of noradrenaline is increased substantially. There is immunocytochemical evidence of an increase in staining for tyrosine hydroxylase, suggesting an increase in synthetic activity. These results are discussed in relation to the existing literature on the role of amines in normal and disordered erectile function. In particular, the increased concentration and turnover of NA in the diabetic rat contrasts with the fall in NA in cavernosal blood described during normal erection in humans.

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.

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.

Peptidergic hormones and neuropeptides, and aminergic neurotransmitters of the pancreatic islets of the Houbara bustard (Chlamydotis undulata).

Immunoreactivity to insulin (Ins), somatostatin (Som), glucagon (Glu) and pancreatic polypeptide (PP) was found in 70%, 22%, 15% and 11% respectively of Houbara pancreatic endocrine islet cells. Whilst Ins occurred centrally and SOM was observed both in peripherally and centrally located islets, the other hormones were localised in peripheral islet cells; Som was also observed in neuronal cell bodies and nerve fibres. In addition, the islet cells contained substance P (SP) (65%) in the centre and vasoactive intestinal polypeptide (VIP) (2%) at the periphery. Immunoreactivity to choline acetyltransferase (ChAT), VIP and galanin (Gal) occurred in the walls of blood vessels located mainly at the periphery of islets. Occasionally, VIP and Gal immunoreactive varicose nerve terminals and ChAT immunoreactive cell bodies were also observed in the centre of islets. SP neuronal cell bodies were not observed but prominent SP immunoreactive varicose terminals were discernible in capillary walls within the islets. Neuropeptide Y (NPY) immunoreactive neurons were detected in neuronal cell bodies located mainly peripherally. Neuronal nitric oxide synthase (nNOS) immunoreactivity occurred in neuronal cell bodies and nerve fibres mainly at the periphery and also in centrally located islet endocrine cells. Immunoreactivity to tyrosine hydroxylase (TH) was similar in distribution to that of ChAT. In comparison with other avian species, the islets of the dorsal pancreatic lobe of the bustard contain all the peptidergic hormones normally present in the islets of other avian species, but are not segregated into dark A and light B cells. Many of the insulin containing cells also contained SP. The islets also contained several neuropeptides which are probably involved in their regulation.

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.

Evidence to suggest morphological and physiological alterations of lacrimal gland acini with ageing.

This study investigates changes in the morphology and physiology of lacrimal gland acinar cells with age. Changes in microstructural appearance of the acinar cells, the type and distribution of the different acini in glands and the secretory granules within the acini were examined in glands from animals of 3-5, 9, 12, 20, 24 and 28 month old rats. Differences in the secretory capacity of the acinar cells were also examined in animals of each age-group, with the exception of 28 months. The typical acini of young glands (3-5 months) were of the serous type. This was also true of 9 month glands, although there was a significant reduction in their overall distribution compared to young glands. The acini in the 12 month glands were predominantly of the seromucous type and appeared to be at the expense of the serous acini which were further significantly reduced compared to 3-5 and 9 month glands. This remained the prevalent acini type in 20 month glands, however by 24 months there was a significant increase in the occurrence of mucous acini and this time appeared to be at the expense of the seromucous acini which were significantly reduced in glands of this age-group. The predominant acinar cell in 28 month glands, like 24 month glands, was of the mucous variety. Qualitative EM studies revealed a progressive change in the secretory products of the lacrimal gland acini, strongly correlating to changes in acinar cell type. Typical acini of both 3-5 and 9 month glands contained numerous protein secretory granules. The seromucous acini also of these age groups contained both protein and mucous secretory granules, with the protein secretory granules in higher abundance. By 12 months the typical seromucous acini was packed with both protein and mucous secretory granules of equal proportions. However, by 20 months the predominant seromucous acini contained fewer protein secretory granules and elevated occurrence of mucous secretory granules. By 24 and 28 months the acini contained even fewer protein secretory granules and the typical acinar cell was of the mucous type containing exclusively mucous secretory granules. The secretory capacity of the acini was also altered with age. Maximum protein output in response to cholinergic stimulation resulted in an initial significant increase with ageing from 3-5 months to 9 and 12 months followed by a later significant age-dependent reduction in output. However, maximal peroxidase release from acinar cells of 3-5 and 9 month glands was the same. This was followed by a significant age-dependent reduction in peroxidase release. Furthermore, the concentrations required to evoke these responses differed with age. These results present evidence to suggest that acinar cells of the lacrimal gland undergo progressive alterations with age. The type of acini changing initially from serous to seromucous acini (intermediate phase) followed by a gradual transformation of the seromucous acini to mucous acini. This in turn changes the properties of the acini from protein producing and secreting acini to mucous producing and secreting acini. The results also suggest a reduction in the ability of the acini to synthesise proteins with age and altered responsiveness to cholinergic stimulation to secrete proteins. These findings may help in explaining the occurrence of altered protein/tear secretion with ageing.

Transmission of substances through the exocoelomic cavity after introduction into the vitelline circulation by cannulation: what route?

The morphology of the main vitelline vein and its tributaries which carry the embryotroph from the yolk sac into the rat embryo has been studied by electron microscopy after perfusing the conceptus with a solution of lanthanum nitrate in Karnovsky's fixative. The distribution of the contents of these vessels and the routes taken into and out of the various embryonic compartments have also been investigated. The vitelline vein and its tributaries are lined by a discontinuous endothelial layer, with no basement membrane or mural elements, and it is separated from the exocoelomic cavity by a continuous layer of squamous cells. In addition to the lumina of the vessels of the conceptus, lanthanum nitrate was observed in the mesenchymal space surrounding the yolk sac, the intercellular spaces between the yolk sac endodermal cells but not on their apical surfaces, the intercellular spaces between the cells lining the exocoelomic cavity, the exocoelomic cavity, the mesenchymal space around the umbilical vessels and the intercellular spaces between the ectodermal cells of the embryo. It has been demonstrated that substances enter the exocoelomic cavity mainly through the intercellular spaces of its lining cells via the mesenchymal space around the main vitelline vein and its tributaries. Whilst we were unable to demonstrate gaps in the endothelial lining of the umbilical vessels, it seems to be the likeliest explanation for the presence of lanthanum around its extravascular space. The significance of the distribution of the contents of he vitelline vasculature is discussed.

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.

Age-related changes in morphology and secretory responses of male rat lacrimal gland.

This study investigates the differences in the outward appearance and morphology of lacrimal glands, the morphology within the lacrimal acinar cells and the secretion of protein from acinar cells of young (3-5 months) and aged (20 and 24 months) male rats. The appearance of the glands, as seen by the naked eye, differed between the three age-groups. The lacrimal gland of young animals was a smooth pink tissue, while the tissue from aged animals appeared lobular and white in colour, thought to result from infiltration of fatty/connective tissue. Glands from 24 month old animals had a more pronounced lobular appearance than the glands from 20 month old animals. Light microscopy studies revealed that as the animals aged there was evidence of progressive morphological changes. These changes included thickening of the connective tissue sheath, chronic inflammation with increased infiltration by mast cells, patchy destruction of ductal and vascular tissues, enlargement of lacrimal ducts, luminal swelling of the acini, and changes in acinar type. Electron microscopy (EM) studies revealed the presence of 3 types of acini in the rat lacrimal gland: acini which contained only protein secretory granules (serous acini), acini which contained protein and mucous secretory granules (seromucous acini), and acini which contained only mucous secretory granules (mucous acini). In young glands the majority of acini were serous with a few seromucous acini and even fewer mucous acini. In aged glands there were significant reductions in serous acini (ANOVA; P < 0.01) when compared to the young glands. In 20-month-old glands, there were marked increases in the percentage occurrence of seromucous acini, while in 24 month old glands, there were large increases in the relative number of mucous acini. Qualitative EM studies demonstrated that the typical acini from young glands contained numerous protein secretory granules. Ageing was associated with a progressive loss of protein (serous) secretory granules. Furthermore, marked changes and patchy destruction of the endoplasmic reticulum and Golgi apparatus were observed in acini of glands from aged rats when compared to acini of glands from young rats. Measurement of total protein output from acini revealed a significant (Student's t-test, P < 0.05) decrease in protein secretion from aged glands compared to glands from young animals. These results suggest that not only is there considerable structural damage, chronic inflammation and mast cell infiltration to the lacrimal gland with ageing, but also possible redifferentiation of acini from serous to seromucous and then to mucous acini. Furthermore, the results also suggest a reduction or an inability of the acini to synthesise and to secrete protein from glands of aged animals compared to glands of young rats. All of these changes appear to occur more rapidly as the rats mature between 20 and 24 months. These findings provide a morphological basis to explain the phenomenon of reduced tear/protein secretion with ageing.

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.

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.

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.

Aspirin-alcohol interaction in the production of cleft palate and limb malformations in the TO mouse.

Our objective in the present study was to determine the effects of alcohol on stages when the limb buds and renal primordia develop in the TO mouse and to see if aspirin pretreatment would prevent these organ systems from being malformed as was shown by Randall et al. ('91) in the C57 mice. On one of days 9-12 of gestation, groups of TO mice were injected intraperitoneally (IP) with a single dose of 200 mg/kg of aspirin, or a proportionate volume of physiological saline. An hour later, half of the aspirin-treated animals received a single dose of 0.03 ml/g of freshly prepared 25% (v/v) solution of absolute alcohol and the other half received a proportionate volume of saline. Half of the saline-treated animals received a single dose of 0.03 ml/g of saline or a proportionate volume of alcohol solution. All animals were killed on day 18 of gestation. Alcohol significantly increased embryonic resorption and caused remarkable intrauterine growth retardation (IUGR). It also induced arched palate, cleft palate and deformities of the digits with haematomas in a modest number of embryos. Aspirin alone did not have any teratogenic effects. Pretreatment with aspirin significantly augmented alcohol-induced resorption, IUGR, cleft palate and digital malformations associated with haematomas. Chronological observations on the development of the treated limbs showed the occurrence of vascular stasis, haematomas, edema and cell death at early stages. Subsequently, digital rays were either destroyed (ectrodactyly) or remained hypoplastic (brachydactyly). It appears that limb development in the aspirin- and alcohol-treated TO mouse embryos is largely affected by vascular disruption. These data provide further evidence to our earlier observation that alcohol and aspirin interact in the production of malformations and that the teratogenic effects of alcohol in the TO mouse are possibly not mediated via treatment related prostaglandin elevation.

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.

Post-mortem changes in the normal rat carotid body: possible implications for human histopathology.

The carotid bodies in experimental animals contain only one variety of type I cells whilst in the human organ three varieties of this cell type have been described. We have examined the effects of post-mortem change on the structure of the type I cell of the rat carotid body. When the organ is examined immediately after death of the animal all of the type I cells exhibit similar morphology. With increasing delay in fixation of the tissue the type I cells undergo autolytic changes. Within 2 h of death the nuclei become hyperchromatic and the cytoplasm exhibits an increasing eosinophilia. In carotid bodies fixed 4 h post-mortem a further type I cell variant is seen in which the nucleus lacks a chromatin pattern and become pyknotic. We believe that previous descriptions of three varieties of type I cells in the human carotid body are based upon a description of post-mortem change. Furthermore, in any study of this highly oxygen dependent tissue it is essential that due account be taken of the delay between death and fixation.