Blockage of Ca-activated Cl conductance by furosemide in rat lacrimal glands

Single cells isolated from rat lacrimal glands were studied with the tight-seal whole-cell recording technique. It was found that furosemide (1 mM, applied externally) selectively blocked one part of the electrical response elicited by muscarinic agonists. This component of the response had been shown in a previous work (Marty et al. 1984) to be due to Ca-dependent Cl channels.The action of furosemide was further studied on cells which were dialysed with a high-Ca, high-Na solution, and which mainly displayed the Ca-dependent Cl conductance. In these experiments, furosemide (1 mM) was again found to depress the Ca-dependent Cl current.The present findings offer an explanation for previous reports that furosemide blocks ion fluxes and electrolyte secretion in exocrine glands without necessarily involving the neutral Na-K-Cl carrier usually assumed to be affected by the drug.     

Effects of chronic and in utero treatment with diuretics on mouse exocrine cells studied by X-ray microanalysis.

In an attempt to develop an animal model for the disease cystic fibrosis, mice were chronically treated with diuretics. In addition, pregnant mice were treated with diuretics and the effect of this treatment in utero on the newborn mice was studied. Pancreas and submandibular gland acinar cells were investigated by X-ray microanalysis and transmission electron microscopy. Long-term treatment with furosemide (up to 13 months) caused transient changes in the elemental content of the pancreatic acinar cells: a decrease in chloride and sulfur, and an increase in phosphorus, potassium and magnesium. All changes normalized with prolonged treatment. Some morphological changes were found in the zymogen granules. Treatment with amiloride or furosemide in utero caused a decrease in cellular sodium and chloride levels, indicative of inhibition of transepithelial ion and fluid transport. Also treatment of adult animals for two months with amiloride caused lower intracellular sodium and chloride levels. In adult animals only minor effects of diuretic treatment on submandibular gland acinar cells were noted. In utero treatment with amiloride caused an increase in sodium and chloride content indicative of cell damage.     

Effects of chronic corticosterone treatment on the morphology of rat neuromuscular junctions

This study examined the effects of chronic corticosterone (CORT) treatment on the morphology of two physiologically different muscles. Nerve terminals from the slow twitch soleus (SOL) and fast twitch extensor digitorum longus (EDL) of Fischer 344 rats were stained using the zinc iodide osmium (ZIO) technique. Nerve terminal area, perimeter, and longitudinal extent length were measured using computer-aided morphometry. Slow and fast muscle fibers from animals which received 5-10 mg CORT per day for 3 months were atrophied compared with controls. Treated animals failed to gain weight during the study, while controls gained 37%. Adrenal weights in treated animals were 30% less than controls, after correction for body weight. Morphological parameters for SOL nerve terminals were generally larger in the CORT group, while EDL nerve terminals from the CORT group did not differ significantly from controls. Soleus nerve terminal area was 43% greater, perimeter 14% longer, and longitudinal extent length 18% longer than the control nerve terminals. This study demonstrates a greater effect of CORT treatment on slow twitch muscle than has been demonstrated in previous studies. Changes in the nerve terminal morphology of the SOL were also greater than in previous studies and suggest that a functional adaptation or remodelling may occur following CORT treatment to maintain the neuromuscular interface during the enhanced catabolic effects of the steroid. These steroid-induced stress changes are similar in some respects to those observed in aging and disuse studies of the vertebrate neuromuscular junction. This suggests that glucocorticoid hormones may play an etiological role in the homeostasis of the neuromuscular junction in response to various stimuli.     

Effects of chronic corticostorone treatment on the morphology of rat neuromuscular junctions

This study examined the effects of chronic corticosterone (CORT) treatment on the morphology of two physiologically different muscles. Nerve terminals from the slow twitch soleus (SOL) and fast twitch extensor digitorum longus (EDL) of Fischer 344 rats were stained using the zinc iodide osmium (ZIO) technique. Nerve terminal area, perimeter, and logitudinal extent legth were measured using computer-aided morphometry. Slow and fast muscle fibers from animals which received 5–10 mg CORT per day for 3 months were atrophied compared with controls. Treated animals failed to gain weight during the study, while controls gained 37%. Adrenal weights in treated animals were 30% less than controls, after correction for body weight. Morphological parameters for SOL nerve terminals were generally larger in the CORT group, while EDL nerve terminals from the CORT group did not differ significantly from controls. Soleus nerve terminal area was 43% greater, perimeter 14% longer, and logitudinal extent length 18% longer than the control nerve terminals. This study demonstrates a greater effect of CORT treatment on slow twitch muscle than has been demonstrated in previous studies. Changes in the nerve terminal morphology of the SOL were also greater than in previous studies and suggest that a functional adaptation or remodelling may occur following CORT treatment to maintain the neuromuscular interface during the enhanced catabolic effects of the steroid. These steroid-induced stress changes are similar in some respects to those observed in aging and disuse studies of the vertebrate neuromuscular junction. This suggests that glucocorticoid hormones may play an etiological role in the homeostasis of the neuromuscular junction in response to various stimuli.     

Morphology of the exocrine glands of the frog skin

Frog skin contains three distinct types of exocrine glands: granular (poison), mucous, and seromucous. The granular gland forms a syncytial secretory compartment within the acinus, which is surrounded by smooth muscle cells. The mucous and seromucous glands are easily identifiable as distinct glands. The serous and mucous secretory cells are arranged in a semilunar configuration opposite the ductal end and are filled with granules. Within the acinus, located at the ductal pole of the gland, are distinct groups of cells with few or no granules in the cytoplasm. In both the mucous and seromucous gland there is a cell type with abundant mitochondria; the one in the mucous gland is located in the region adjacent to the secretory cells. The duct of these glands is two-layered, with the individual cells appearing morphologically similar to the layers of the skin epithelium as the duct traverses the skin. The duct appears to be patent throughout its length. The morphological heterogeneity and distinct distribution of the cell types within the gland acinus may be indicative of a functional heterogeneity that allows the production of distinctly different types of secretion from the same gland type, depending on the type of stimulus.     

The B-cell system of human mucosae and exocrine glands

Summary: The mucosae and exocrine glands harbour the largest activated B-cell system of the body, amounting to some 80–90% of all immunoglobulins (Ig)-producing cells. The major product of these immunocytes is polymeric (p)IgA (mainly dimers) with associated J chain. Both pIgA and pentameric IgM contain a binding site for the polymeric Ig receptor (pIgR), or secretory component (SC), which is a requirement for their active external transport through secretory epithelia. The pIgR/SC binding site depends on covalent incorporation of the J chain into the quaternary structure of the polymers when they are produced by the local immunocytes. This important differentiation characteristic appears to be sufficient functional justification for the J chain to be expressed also by most B cells terminating at secretory effector sites with IgD or IgG production; they probably represent a ‘spin-off’ from sequential downstream C_H switching on its way to pIgA expression, thus apparently reflecting a maturational stage of effector B-cell clones compatible with homing to these sites. Observations in IgA-deficient individuals suggest that the magnitude of this homing is fairly well maintained even when the differentiation pathway to IgA is blocked. Certain microenvironmental elements such as specific cytokines and dendritic cells appear to be required for induction of IgA synthesis, but it remains virtually unknown why this isotype normally is such a dominating product of local immunocytes and why they have such a high level of J chain expression. Also, despite the recent identification of some important requirements in terms of adhesion molecules (e.g. integrin α4β7 and MAdCAM-1) that explain the “gut-seeking” properties of enterically induced B cells, the origin of regionalized homing of B cells to secretory effector sites outside the gut remains elusive. Moreover, little is known about immune regulation underlying the striking disparity of both the class (IgD, IgM) and subclass (IgA1, IgA2, IgGI, IgG2) production patterns shown by local iinmttnocytes in various regions of the body, although the topical microbiota and other environmental stimuli might be important. Rational design of local vaccines will depend on better knowledge of both inductive and migratory properties of human mucosal B cells.     

Histochemical localization of prostaglandin synthetase in human exocrine glands

Prostaglandin synthetase activity is histochemically detected in the epithelial cells of the human prostate, seminal vesicle, and deferential ampulla. The addition of the specific inhibitor indomethacin to the incubating medium strongly reduces the staining reaction. The presence of exogenous substrate in the medium is not required for the reaction, since identical results are observed following incubation with and without arachidonic acid. The presence of the enzyme appears to be related to the secretion of prostaglandins into the seminal fluid, but there is a possibility that some prostaglandins influence the metabolism of the secreting cells themselves. The localization of prostaglandin synthetase in the ductal epithelia of human salivary glands is related to a possible regulatory role of the prostaglandins upon the reabsorptive activity of the ductal cells.     

Effects of intrapancreatic neuronal activation on cholecystokinin-induced exocrine secretion of isolated perfused rat pancreas

 The role of intrapancreatic neurons in the action of cholecystokinin (CCK) on pancreatic exocrine secretion of the totally isolated, perfused rat pancreas was investigated. Intrapancreatic neurons were activated by applying electrical field stimulation (EFS) to the isolated pancreas for 45 min. When applying EFS, spontaneous pancreatic secretions of fluid and amylase increased until the second 15-min period of EFS and then decreased during the third 15-min period. Atropine (2 μM) notably reduced the EFS-evoked pancreatic secretions of fluid and amylase. The CCK-induced (10 pM) pancreatic secretions of fluid and amylase elevated further in the first 15-min period of EFS and then gradually resumed to the levels observed during application of CCK alone in the third 15-min period of EFS. However, the CCK-induced pancreatic secretions remained elevated even in the third 15-min period of EFS when an action of endogenous somatostatin was inhibited by cyclo-(7-aminoheptanonyl-Phe-d-Trp-Lys-Thr[BZL]) (10 nM) or pertussis toxin (200 ng/ml). EFS further elevated spontaneous exocrine secretion by the cysteamine-treated (300 mg/kg) pancreas, but this was markedly reduced, to normal levels, by infusing somatostatin (100 pM). EFS increased the numbers of immunoreactive somatostatin cells in the Langerhans’ islets. The results indicate that intrapancreatic neuronal activation influences CCK-induced pancreatic secretions in a dual-phase pattern in the rat: an increase during the early phase and a decrease during the late phase. Endogenous somatostatin released from the islets appears to inhibit the enhancing effect of neuronal activation on CCK-induced pancreatic secretion. Of the intrapancreatic neurons, the cholinergic ones appear to predominate in EFS’s effects on CCK-induced pancreatic secretion. Received: 21 April 1998 / Received after revision: 3 November 1998 / Accepted: 16 November 1998     

Microvascular changes in aged rat forebrain. Effects of chronic nimodipine treatment

In the present study the effects of long-term treatment with the 1,4-dihydropyridine calcium antagonist nimodipine on ultrastructural alterations of the microvascular morphology were examined in the frontoparietal cortex, entorhinal cortex and CA1 of the hippocampus in the aged rat. Qualitative observations of cerebral microvasculature of aged (30 months) Wistar rats revealed the presence of microvascular fibrosis, membranous inclusions within the basement membrane and basement membrane thickenings. In several cortical regions the percentage of aberrant microvessels was significantly reduced in the nimodipine-treated rats. The observed microvascular anomalies were classified into five distinct categories of which microvascular fibrosis type II, defined as collagen deposits up to 1 micron within the microvascular basement membrane, showed the strongest reduction in the nimodipine-treated cases. The decrement of the percentage of aberrant microvessels and the relative occurrence of several classes of microvascular deviations showed some variation in the various brain regions examined and was most pronounced in frontoparietal cortex layer III. These results may provide a morphological basis for the improved motor and cognitive performance in aged rats after long-term oral nimodipine administration.     

Effects of chronic stressors or corticosterone treatment on the septohippocampal cholinergic system of the rat

The effects of prolonged (2 months) corticosterone (CORT) treatment on several cholinergic markers of various brain areas were compared to the effects of prolonged intermittent exposure to stress. CORT, but not stress, caused a significant reduction in the number of acetylcholinesterase-stained neurons in the medial septal area. Neither treatment resulted in any hippocampal pyramidal cell loss. It is concluded that a time-dependent degeneration of the septohippocampal cholinergic system follows 2 months of CORT administration but not chronic intermittent stress of this duration.     

Configuration of myoepithelial cells in various exocrine glands of guinea pigs

To study the configuration of myoepithelial cells, we isolated glandular endpieces of various guinea pig glands by collagenase, and visualized the myoepithelial cells by immunohistochemistry for actin, or by Bodipy-phallacidin, under both a light microscope and laser scanning confocal microscopes. In parotid and mandibular glands, the glandular acini were small (about 20–30 m diameter) and spherical, and each acinus had one or two myoepithelial cells attached that were stellate in shape (central cell body and four to six thin processes). Most of the basal surface of the glandular cells was not covered by myoepithelial cells, and processes often extended to the neighboring acinus. The tubular glandular endpieces of the major sublingual gland, which secretes a mucous substance, were almost fully encircled by bandlike myoepithelial cells (about 3–6 m wide). Although there were many differences between the lacrimal gland and the Harderian gland (e.g., the secretory product of the lacrimal gland was mucous, and glandular lumina were narrow; the Harderian gland secreted lipids and showed wide lumina), the outer contours of both glandular endpieces were the same (about 50–100 m diameter, ellipsoid or spherical in shape). In both glands, 5–20 stellate myoepithelial cells were attached onto a glandular endpiece, and their arrangement had a lacy appearance. Actin filaments in myoepithelial cells aggregated and formed bundles in the broad processes and cell bodies. The bundles ran across the cell body, but there was no point where the bundles converged. In the arborization, some distal processes reversed their direction. We conclude that the configuration of myoepithelial cells depends on the outer contour of the glandular endpieces rather than on the secretory material or luminal width. The variety of myoepithelial cell configurations in the different exocrine glands we examined suggests that it is quite difficult to assign to myoepithelial cells the general role of expelling secretory products from glandular lumina. These cells seem to maintain the contour of the glandular endpieces, serving as the exoskeleton of the endpieces.     

Effects of furosemide,acetazolamide, and mannitol on medullary collecting-duct function in the rat kidney

The microcatheterization technique was used to study transport of fluid, sodium, and potassium in the medullary collecting duct in rats (232–357 g) before and during administration of diuretic drugs. Series I received furosemide (1.5 mg initial dose + 1.5 mg/h), Series II acetazolamide (0.75 mg +0.75 mg/h), and Series III and IV mannitol at 0.3 g+0.3 g/h, and 0.3 g+1.0 g/h, respectively. All diuretics caused diuresis, natriuresis, and kaliuresis. The renal response to furosemide and acetazolamide was associated with increased hematocrit and decreased filtration rate, indicating depletion of blood volume. No such effect was seen in Series III and IV. In the collecting duct furosemide completely abolished the normal reabsorption of sodium and fluid and initiated net secretion of potassium. Partial inhibition of salt and water transport in the collecting duct was observed with acetazolamide and the low dose of mannitol (III). Both treatments resulted in potassium secretion. In Series IV the high rate of mannitol infusion was associated with complete inhibition of salt and water reabsorption from the medullary collecting system similar to that of Series I. The greater excretory response in this group compared to the furosemide series was due to increased delivery of tubular load to the collecting duct. It is concluded that a major site of action of furosemide is in the medullary duct, resulting in quantitative inhibition of salt and water reabsorption from this nephron segment. The partial transport inhibition during acetazolamide or modest mannitol diuresis can be explained by the presence of poorly absorbable solute in duct fluid. The mechanism of inhibition of reabsorption after the high rate of mannitol infusion remains undetermined.     

The effect of reserpine treatment on choline acetyltransferase activity in rat submaxillary glands

Rats were treated daily with a low dose of reserpine (0.1 mg kg-1) injected subcutaneously for 3 weeks. In the submaxillary glands the noradrenaline content was reduced by about 95%. The total activity of the acetylcholine-synthesizing enzyme choline acetyltransferase remained unchanged. However, the activity of this enzyme was found to be increased when the reserpine treatment was followed by surgical sympathetic denervation and the glands were analysed 3 weeks post-operatively. The enzyme activity also increased in the glands when the surgical sympathetic denervation was performed on the day of the start of the reserpine treatment. The lack of effect of reserpine on choline acetyltransferase activity in the glands seems to exclude the possibility that it is the depletion of neuronal noradrenaline stores that initiates the events giving rise to increases in choline acetyltransferase activity after sympathetic denervation.     

Carbonic anhydrase isoenzymes in the rat kidney. Effects of chronic acetazolamide treatment

Biochemical, immunocytochemical and histochemical methods were used to study the effect of chronic acetazolamide treatment on carbonic anhydrase (CA) isoenzymes in the rat kidney. Male inbred rats (Lew/Mol) were treated with 15 mg kg-1 day-1 acetazolamide s.c. by Alzet minipump during 2-9 weeks; some animals had a drug-free period of 1-4 weeks before being killed. The renal content of CA II was higher in the acetazolamide-treated rats than in the controls, 178 +/- 10 vs 144 +/- 4.8 micrograms enzyme protein g-1 tissue (mean +/- SE). The distribution of CA isoenzymes did not change during or after chronic acetazolamide treatment. Thus, only CA II was detected in the kidney tubules by immunofluorescence using specific antisera against CA I, CA II and CA III. All animals showed a similar staining pattern, with intense cytoplasmic CA II staining in intercalated cells of collecting ducts, moderate staining in descending thin limbs of Henle, and weak cytoplasmic staining in proximal tubules and chief cells of collecting ducts. All animals also showed histochemical staining of cell membranes in proximal and distal tubules and thick limbs of Henle, suggesting the presence of a membrane-bound isoenzyme (CA IV). The only difference noted by histochemistry and immunocytochemistry was that the intercalated cells appeared bulkier and protruded more markedly into the tubular lumen in treated than in untreated animals. The functional importance of this finding is unclear. The observed changes in CA cannot alone explain why the effect of acetazolamide, in causing loss of bicarbonate and sodium, is self-limited on continued administration.     

Effect of furosemide on the lipid abnormalities in chronic renal failure

The effect of furosemide on the lipoprotein profile and the activities of postherapin plasma lipases were studied in 12 patients with chronic renal failure. The concentrations of serum total, VLDL and LDL triglycerides were significantly higher and the concentration of HDL triglyceride was significantly lower in the patients with renal failure than in healthy controls. HDL cholesterol was significantly lower in the patients than in the controls. The activity of postherapin lipoprotein lipase was significantly lower in the patients than in the controls. The introduction of furosemide induced a significant increase in the concentrations of VLDL cholesterol and VLDL triglyceride. These changes were reversed when the drug treatment was discontinued. Postherapin lipase activities were not further altered by furosemide.     

Effects of furosemide on allergic asthmatic responses in mice

Background The syndrome of allergic asthma features reversible bronchoconstriction, airway inflammation and hyperresponsiveness as well as airway remodelling, including goblet cell hyperplasia. Managing severe asthma is still a clinical challenge. Numerous studies report that furosemide, an inhibitor of Na⁺–K⁺–Cl^? cotransporter (NKCC) reduces airway hyperresponsiveness (AHR) in asthmatic patients. However, the mechanism by which furosemide exerts anti‐asthmatic action remains unclear. Objective This study sought to investigate the cellular profile of NKCC1 expression in the lung and examine the effects of furosemide on several outcome measurements in a mouse model of allergic asthma. Methods Mice were sensitized and challenged with ovalbumin (OVA). Before challenge, the OVA‐sensitized mice were treated with furosemide (4.0 mg/kg/day, via daily intraperitoneal injection for 5 days). Outcome measurements in naïve, OVA‐exposure, furosemide‐treated naïve and furosemide‐treated OVA‐exposed mice included the slope of the relationship between inhaled methacholine (MCh) concentration and respiratory system resistance (Slope·R_RS), bronchoalveolar lavage (BAL) cell counts and immunohistochemical and immunoblotting assays of lung tissues. Results NKCC1 immunoreactivity was observed in airway epithelial cells (AECs) and alveolar type II (ATII) cells of the control mice. OVA exposure enhanced the expression of NKCC1 in AECs and ATII cells, and increased the infiltration of NKCC1‐expressing T lymphocytes in the lung. NKCC1 immunoreactivity was not detected in the airway smooth muscle (ASM) cells. Furosemide treatment reduced the Slope·R_RS in both naïve and OVA‐exposed mice by about 50%. Furosemide treatment also increased T lymphocyte infiltration to the lung in OVA‐exposed mice by approximately 53%, but had no effect on pulmonary goblet cell hyperplasia. Conclusions and Clinical Relevance Furosemide decreases basal airway responsiveness, thereby reducing the extent of allergen‐induced AHR. However, the same treatment also increases T lymphocytes infiltration in the course of allergic asthma. Further studies are necessary to address the usefulness of furosemide in the clinical treatment of asthma. Cite this as: S. Wang, Y.‐Y. Xiang, R. Ellis, J. Wattie, M. Feng, M. D. Inman and W.‐Y. Lu, Clinical & Experimental Allergy, 2011 (41) 1456–1467.     

LFA-1 expression on exocrine glands as a potential novel marker of malignant disease.

The lymphocyte function associated antigen 1 has been found only on leukocytes and lymphoid tissues; however, the expression of lymphocyte function associated antigen 1 on nonhematopoietic cells has not been reported previously. In this study, immunohistochemical expression of lymphocyte function associated antigen 1 was examined on various tissues from 35 patients with malignant diseases and 36 patients with benign diseases including benign tumors. The expression of lymphocyte function associated antigen 1 was found on various exocrine tissues (eg, gastric glands, bronchial epithelium, alveolar epithelium, duodenal glands, bile ducts, pancreatic acini, and salivary glands) uninvolved by tumor in patients with malignant diseases. Localization of lymphocyte function associated antigen 1 was limited to the exocrine glands and differed from tissue-infiltrating leukocytes. The expression of lymphocyte function associated antigen 1 on exocrine tissues was confirmed in all 35 cases of malignant diseases that were examined. These included a wide spectrum of carcinomas and hematopoietic tumors. In contrast, none of the 36 cases with benign diseases examined expressed lymphocyte function associated antigen 1 on their exocrine glands. These results indicate a strong correlation between lymphocyte function associated antigen 1 expression on exocrine glands and malignant disease. The expression of lymphocyte function associated antigen 1 on nonhematopoietic cells was further confirmed in nonhematopoietic cell lines. Two of 19 nonhematopoietic cell lines (MKN45 and PANC-1; exocrine gland cell lines) examined expressed lymphocyte function associated antigen 1 on both cell surface and cytoplasm. These results suggested that immunohistochemically defined lymphocyte function associated antigen 1 molecules on nontumorous exocrine gland cells are a potential marker for the presence of malignant diseases.