[Direct genotype analysis in congenital myotonic dystrophy with an unusual family anamnesis]. / Direkte Genotypanalyse bei Kongenitaler Myotoner Dystrophie (CMD) mit ungewöhnlicher Familienanamnese.

We report a case of congenital myotonic dystrophy (CMD) in which not only the mother but also the paternal family is affected by myotonic dystrophy (DM). Clinical symptoms consisted of poor spontaneous movements, typical facial appearance, respiratory insufficiency attributable to diaphragmatic weakness, feeding difficulties due to impaired gastrointestinal tract motility and poor sucking, joint contractures and thin ribs. She died at 7 month of age, still ventilated, from aspiration pneumonia. By employing molecular genetic methods we were able to show that the affected child was not homozygous for the DM gene.

Activation of basolateral Cl- channels in the rat colonic epithelium during regulatory volume decrease.

Exposure to a hypotonic medium caused an increase in the diameter of isolated crypts from the rat colon. The increase in cell volume was only transient and lasted about 7 min. Despite of the continuous presence of the hypotonic medium, cell volume decreased again. This regulatory volume decrease (RVD) was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), a Cl- channel blocker, and by Ba2+, a K+ channel blocker. Cell-attached patch-clamp recordings revealed that the RVD was associated with the activation of previously silent basolateral channels. These channels were identified after excision of the patch as Cl- channels (28 pS) and as K+ channels (45-60 pS). The RVD was dependent on the presence of external Ca2+. The phospholipase A2 inhibitor, quinacrine, and the lipoxygenase blocker, nordihydroguaiaretic acid, inhibited RVD, while indomethacin had no effect. In Ussing chamber experiments an exposure to hypotonic media caused an initial, transient increase in tissue conductance (Gt), followed by a prolonged decrease in short-circuit current (Isc) and the potential difference (V). The height of the electrical response was dependent on the decrease in the osmolarity in a range from 20 mosmol l-1 to 90 mosmol l-1. The increase in Gt was blocked by NPPB and Ba2+, whereas the decrease in Isc or V was inhibited by NPPB but enhanced by Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of neuronally mediated secretion in rat colonic mucosa by prostaglandin D2.

The effect of prostaglandin D2 (PGD2) on ion transport across the mucosa of the descending colon was studied in rats. PGD2 dose-dependently decreased baseline short-circuit current of mucosa-submucosal preparations mounted either in the Ussing chamber or mounted as an everted sac. However, with the everted sac technique, the tissue was about 1000 times more sensitive to PGD2. Concomitant with the decrease in short-circuit current, PGD2 increased the mucosal-to-serosal fluxes of sodium and chloride and decreased the serosal-to-mucosal flux of chloride. PGD2 inhibited the secretory action of the PGI2 analogue iloprost, PGD2 alpha, and neurotensin. The action of these secretagogues was dependent on the presence of the submucosal plexus. In contrast, PGD2 had no effect on the increase in short-circuit current caused by PGD2, substance P, or serotonin, the actions of which were not dependent on the presence of the submucosal plexus. The results indicate that the action site of the antisecretory mechanism of PGD2 is localized in the secretomotor neurons.

Direct and indirect actions of HgCl2 and methyl mercury chloride on permeability and chloride secretion across the rat colonic mucosa.

The actions of two mercury compounds, the inorganic HgCl2 and the organic methyl mercury chloride (MeHg), and of CdCl2 on ion transport across the rat colon were studied with the Ussing chamber and the everted sac method. The mercury compounds (5-50 microM), but not CdCl2, administered to the luminal side, induced a large, concentration-dependent increase of tissue conductance (Gt). The transepithelial movement of the extracellular marker, mannitol, was enhanced in the presence of the mercury compounds, indicating that they cause an increase in the permeability of the epithelium. Morphological studies revealed that at least for HgCl2 this increase of the permeability was associated with a loosening of the tight junctions, severe alterations of the enterocytes, and a loss of the continuity of the epithelium. After washing out HgCl2, cells neighboring the altered enterocytes developed lateral processes and in this way restored the continuity of the epithelial layer. In parallel, the mercury compounds induced an increase of short-circuit current (Isc), which is indicative of an induction of Cl- secretion. The increase of Isc, but not that of Gt, was suppressed by indomethacin and, in the case of MeHg, also by tetrodotoxin. These results suggest that MeHg and HgCl2 induce Cl- secretion by an indirect effect on the epithelium, which is mediated by prostaglandins.

Chloride secretion induced by mercury and cadmium: action sites and mechanisms.

The actions of two mercury compounds, HgCl2 and methyl mercury chloride (MeHg), and of CdCl2 on the epithelium of the rat colon were studied with the whole-cell patch-clamp technique and the Ussing chamber. MeHg (50 microM) induced an increase of membrane outward current (I(out)) in enterocytes of isolated crypts patched from the basolateral side. This action was inhibited by a Cl- channel blocker and a K+ channel blocker, indicating an increase of both the Cl- and the K+ conductance. In contrast, HgCl2 (50 microM) did not affect I(out), whereas CdCl2 (50 microM) decreased it slightly. In mucosal preparations all three compounds induced a concentration-dependent increase in short-circuit current (Isc) when administered to the serosal, i.e., contraluminal side. Sensitivity to chloride transport blockers and anion replacement experiments revealed that the increase in Isc represented Cl- secretion. In contrast to the actions of luminally applied mercury compounds, the increase of tissue conductance (Gt) was only small. Tetrodotoxin and indomethacin suppressed the effect of the metal compounds on Isc and Gt, while atropine diminished it only partly. This indicates that the secretory action of these heavy metals has not only a direct effect on epithelial cells but is also mediated by prostaglandins and cholinergic and noncholinergic neurons.

Characterization of the antisecretory action of prostaglandin D2 in the rat colon.

Previous studies have shown that prostaglandin D2 (PGD2) inhibits neuronally mediated secretion in the rat colon. This antisecretory action of PGD2 was further characterized by the use of a prostaglandin D receptor blocker. Prostaglandin D2 inhibited the neuronally mediated short-circuit current evoked by prostaglandin I2, which represents Cl- secretion. The concentration-response curve for the inhibition by PGD2 was shifted to the right in the presence of the prostaglandin D receptor blocker, AH 6809. AH 6809 had no effect on the short-circuit current response induced by prostaglandin E2 or iloprost, a stable prostaglandin I2 analogue, suggesting an interaction of the blocker with receptors specific for PGD2. A direct interaction of PGD2 with enteric neurones was studied by determining its effect on acetylcholine release from enteric neurones preloaded with [3H]choline. Prostaglandin D2 suppressed 3H release induced by electric field stimulation. It had, however, no effect on the release induced by depolarization with potassium. The results suggest that the inhibitory action of PGD2 on enteric cholinergic neurones is mediated by prostaglandin D receptors.

Histo- and immunocytochemical characterization of the neurons of the mucosal plexus in the rat colon.

The mucosal plexus of the rat colon descendens is constituted of a network of nerves that, in contrast to most other segments of the digestive tract, contains also ganglia. The ganglia, consisting of neurons and glial cells, are located in the basal part of the lamina propria at distances between 100 and 1,200 microns. They are not vascularized. The neurons in these ganglia were characterized by means of: (1) the histochemical demonstration of acetylcholinesterase (AChE) activity, (2) the immunocytochemical identification of neurofilament proteins (NFP; 200 kD) and (3) their ultrastructure. The glial cells, which were AChE negative, could be distinguished from the neurons by differences in size and chromatin pattern. All neurons of the mucosal plexus reveal AChE activity in the perikaryon, but only parts of the axons are AChE positive. NFP-like immunoreactivity was detected in the perikarya but only in a minor part of the axons. These findings confirm previous light-microscopical observations and add new evidence for the existence of neurons (ganglia) in the mucosal plexus of the rat colon.

Electron microscopy of the mucosal plexus of the rat colon.

The ultrastructure of neurons, glial cells and axons of the mucosal plexus of the rat colon descendens was studied. Serial semithin sections and a re-embedding technique were used in order to localize the ganglia. The ganglia are free of blood vessels and connective tissue. The ratio of neurons to glial cells is approximately 1. Ganglia and nerve strands are enclosed by a basement membrane, without a well-defined perineural connective tissue. The neurons show a structure similar to other enteric plexus. Synaptic contacts were observed frequently in the neuropil, where nerve endings and varicosities show a diverse outfit in vesicles. The glial cells, which contain immunocytochemically detectable glial fibrillary protein, possess the same ultrastructural attributes in the intra- and extraganglionic localizations. In the nerves, axonic profiles and varicosities appear in close relation with glial cells or their processes. The distance between the nerves and their target cells, i.e. the enterocytes, is 0.5 microns or more with interposed basement membranes and fibroblasts.

Calcium- and cyclic-AMP-mediated secretory responses in isolated colonic crypts.

Whole-cell recordings were performed at isolated crypts from the distal colon of the rat. Enterocytes in intact crypts, patched from the basolateral side, exhibited a gradient in the resting zero-current potential. Along the axis of the crypt, the highest potentials were measured in the ground region, the lowest in the surface region. The cholinergic agonist, carbachol, induced a hyperpolarization and an increase of the outward current in both the middle and the ground cells of intact crypts. This effect could be prevented by Ba2+ or by the intracellular Ca2+ antagonist, 8-(N, N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8). Its action, however, was not dependent on the presence of external Ca2+. Both ground cells and the cells in the middle part of the crypt responded to forskolin, an activator of the adenylate cyclase, with a depolarization. In the middle part of the crypt, the depolarization induced by forskolin was associated with an increase of the outward current. It could be blocked by the Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, indicating an increase of Cl- conductance. In contrast, the forskolin-induced depolarization in the ground part of the crypt was associated with a decrease of the outward current. This effect could be prevented by Ba2+, indicating a decrease of a potassium conductance. The changes in outward current could be prevented by the presence of an inhibitor of protein kinase A in the pipette solution. In conclusion, these results suggest that carbachol, an agonist acting on the Ca2+ pathway, indirectly causes Cl- secretion by an increase of the driving force, i.e. the membrane potential. Only the activation of cyclic AMP synthesis by forskolin is able to increase Cl- conductance in the rat colon. The latter response seems to be dependent on the state of differentiation of the enterocytes.

Segment-specific effects of the heat-stable enterotoxin of E. coli on electrolyte transport in the rat colon.

The heat-stable enterotoxin of E. coli (STa) induced an increase in short-circuit current (Isc) in the rat colon. The maximal increase in Isc was about three times larger in the proximal than the distal colon. The action of STa was mimicked by 8-Br-cyclic GMP. Unidirectional flux measurements revealed that STa decreased Na+ and Cl- absorption in the distal colon, while it decreased Na+ absorption and activated Cl- secretion in the proximal colon. In the distal, but not in the proximal colon, indomethacin inhibited the action of STa and of 8-Br-cyclic GMP. Inhibition by indomethacin could be overcome by addition of prostaglandin E2 or forskolin, but not by addition of a non-hydrolysable analogue of cyclic AMP, suggesting an action of STa on cyclic AMP hydrolysis. Amrinone and trequinsin, two inhibitors of cyclic GMP-inhibited phosphodiesterases, mimicked the action of STa on Isc and inhibited the response to a subsequent administration of the toxin indicating the modulation of a cyclic GMP-inhibited phosphodiesterase by STa in the distal colon. The results give evidence for different intracellular action sites of STa in the two parts of the rat colon.

Phospholipase C-induced anion secretion and its interaction with carbachol in the rat colonic mucosa.

Phospholipase C (PLC) from clostridium perfringens induced a biphasic increase in short-circuit current (Isc) in the rat colon. The Isc rose rapidly to a transient peak, before it increased again to a plateau lasting for several hours. Ion replacement experiments and sensitivity to furosemide or a Cl- channel blocker indicated that PLC induced Cl- secretion. The first peak was suppressed by indomethacin, indicating mediation by prostaglandins. In contrast, the second phase was only partially sensitive to the cyclooxygenase blocker. The long-time action of PLC was dependent on intra- and extracellular Ca2+, although PLC did not induce an increase in the intracellular Ca2+ concentration of the enterocytes. The effect of PLC was blocked by the protein kinase inhibitor, staurosporine. Carbachol, when added during the second phase of the PLC response, induced a 'paradox' change in Isc: a rapid, transient increase in Isc was followed by a long-lasting decrease. This inhibition of the PLC response was more pronounced after elevation of the external Ca2+ concentration. A Ca2+ ionophore, ionomycin, and a Ca2+ channel activator, BAY K 8644, also inhibited the PLC response. The results suggest dual dependence of the action of PLC on the intracellular Ca2+ concentration.

Segmental heterogeneity of the rat colon in the response to activators of secretion on the cAMP-, the cGMP- and the Ca(2+)-pathway.

The electrolyte transport was compared in proximal and distal segments of the rat colon under control conditions and after induction of secretion on the cAMP-, the cGMP- and the Ca(2+)-pathway. Baseline short-circuit current was decreased by indomethacin and tetrodotoxin in the distal colon, indicating a spontaneous production of neuronally acting prostaglandins. In contrast, baseline short-circuit current in the proximal colon was decreased only by indomethacin, but not by tetrodotoxin. Unidirectional flux measurements revealed that in the distal colon sodium and chloride were absorbed, while the proximal colon secreted chloride. A morphological comparison between the distal and proximal epithelium revealed that the zonulae occludentes and the microvilli were longer in the distal colon. The size of the Golgi apparatus was several times larger in the crypt than in the surface region without differences between proximal and distal colon. Distal segments were more sensitive to an activator of the Ca(2+)-pathway, carbachol, or activators of the cAMP-pathway such as forskolin and a cAMP-analogue. In contrast, the activation of the cGMP-pathway by a cGMP-analogue or by the heat-stable enterotoxin of E. coli (STa) was more effective in the proximal colon. The results give evidence for a segmental specificity with regard to the intracellular pathways responsible for the activation of secretion.

Phospholipase A2 and mediation of the activation of short-circuit current in the rat colonic mucosa.

Melittin (0.5-2 micrograms ml-1) increased the short-circuit current (ISC) in mucosa-submucosa and mucosa preparations of the rat colon descendens in a dose-dependent manner. In the preparation with the submucosal plexus, quinacrine and indomethacin completely blocked the effect of melittin, indicating activation of phospholipase A2 and production of prostaglandins induced by the drug. The melittin response was also partially sensitive to the lipoxygenase inhibitor, nordihydroguaiaretic acid. Complete inhibition by tetrodotoxin and atropine gives evidence for the involvement of cholinergic neurons in the mediation of the response induced by melittin. In contrast, in the preparation without the submucosal plexus the effect of melittin was only partially inhibited by quinacrine, indomethacin, or by neuronal blockers, suggesting direct interactions of melittin with the epithelium in addition. The effect of melittin resembles to the action of bradykinin, which is neuronally mediated and quinacrine-sensitive in the mucosa-submucosa preparation, and quinacrine-resistant and not neuronally mediated in the mucosa preparation. In the mucosa-submucosa preparation, the melittin response is even partially sensitive to the bradykinin receptor antagonist [D-Phe7]-bradykinin. The results provide evidence for the presence of a quinacrine-sensitive phospholipase A2 in the preparation with and that without the submucosa.

Antisecretory effect of prostaglandin D2 in rat colon in vitro: action sites.

The effect of prostaglandin D2 (PGD2) on colonic ion transport was studied in the Ussing chamber. PGD2 (10(-6) M) decreased baseline short-circuit current (Isc) in two preparations of rat colon descendens, a mucosa-submucosa preparation with and a mucosa preparation without the submucosal plexus. In both preparations, PGD2 inhibited the neuronally mediated secretory responses to electric field stimulation, the sea anemone toxin ATX II, and different cholinergic agents. Unidirectional flux measurements revealed that PGD2 diminished the secretagogue-induced increase in the serosal-to-mucosal flux of Cl- and thereby inhibited net Cl- secretion. PGD2, however, had no effect on the adenosine 3',5'-cyclic monophosphate-mediated response to forskolin or vasoactive intestinal peptide or on guanosine 3',5'-cyclic monophosphate-mediated secretion induced by the heat-stable enterotoxin of Escherichia coli. The PGD2 also blocked the increase in Isc evoked by two neuronally acting inflammatory mediators, i.e., bradykinin and PGI2 in the mucosa-submucosa preparation, but had no effect on the response to PGE2. Consequently, PGD2 exerts an indirect antisecretory effect caused by an inhibition of enteric secretomotor neurons of both the submucosal and the mucosal plexus.

Carbocyclic thromboxane A2 inhibits Cl- absorption in the rat colon.

The effect of carbocyclic thromboxane A2 (CTXA2) on short-circuit current (Isc) was studied in two preparations of the rat colon descendens, one with and one without the submucosal plexus. In both preparations, CTXA2 (10(-7)-5 x 10(-6) mol.l-1) increased Isc concentration-dependently. Its action was not inhibited by the neurotoxin, tetrodotoxin, or by indomethacin, indicating a direct action on the epithelium. The increase in Isc was dependent on the presence of Cl- and HCO3- anions in the medium, but it was not affected by inhibitors of Cl-secretion such as furosemide or a Cl- channel blocker. Measurements of the unidirectional fluxes of Na+ and Cl-revealed that the dominant action of CTXA2 was an inhibition of the mucosa to serosa flux of Cl-. The action of CTXA2 was prevented by pretreatment with the thromboxane receptor blocker, SK&F 88046. It was also inhibited by TMB-8, a substance preventing the release of Ca2+ from intracellular stores, but its effect was not dependent on the presence of extracellular Ca2+. The results indicate that thromboxanes can modulate Cl-transport through the colonic epithelium by a mechanism dependent on intracellular Ca2+.

The epithelial basal lamina of the isolated colonic mucosa: scanning and transmission electron microscopy.

The morphology of the rat colonic basement membrane was studied under control conditions and after induction of secretion by distension in the Ussing chamber. The basement membrane was visualized by removing the epithelium with the aid of Ca2+ chelation combined with vibration. Specimens with and without metallic coating were studied with SEM and TEM, respectively. The surface of the basement membrane in the surface region of the epithelium, i.e. between the mouths of the crypts, is rough. In contrast, the deeper parts of the crypts have a rather smooth basal lamina, which is finely pleated. Fenestrations of the basal lamina with a diameter of 0.5-1 microns were found frequently at the surface region, but less frequently in the crypts. Fenestrations with diameters larger than 2 microns were never observed. After the distension experiments and long-time incubation in vitro (5 h), the size of the fenestrations remained unchanged, however, their number increased compared to the controls.

Storage of glycogen in rat colonic epithelium during induction of secretion and absorption in vitro.

Changes induced in the ultrastructure of the epithelium of the rat colon descendens by long-term electric field stimulation (EFS) in an Ussing chamber were investigated. The anion secretion, which was induced by EFS and was measured by the short-circuit current, fell continuously during a 5 h stimulation. At the end of the stimulation period, small particles were observed in the epithelium; these did not appear in unstimulated control tissue. They were localized predominantly in the apical part of the cell. By staining with periodic acid-thiosemicarbazide-silver proteinate and because of their sensitivity to alpha-amylase, they were identified as glycogen deposits. This storage of glycogen was time-dependent and was first visible after an EFS of 2 h. It did not appear if glucose was substituted in the bathing solution by sodium butyrate. Glycogen particles were also observed after addition of forskolin, which in contrast to EFS causes a high secretory activity that is stable over several hours. The surface cells contained significantly more glycogen than the crypt cells when secretion was stimulated by EFS or forskolin. The formation of glycogen during EFS was not prevented by tetrodotoxin (TTX). In contrast, TTX itself, which causes maximal absorptive activity by blocking secretomotor neurons, induced the appearance of glycogen in the enterocytes without EFS. However, in the presence of TTX, the amount of glycogen was the same in surface and crypt cells. The results demonstrate that the capacity to synthesize and store glycogen, which has up to now only been observed in embryonic or tumor epithelial cells, is still present in adult colonic mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular and paracellular magnesium transport across the terminal ileum of the rat and its interaction with the calcium transport.

Concentration and voltage dependence of unidirectional magnesium fluxes across the stripped mucosa of the rat terminal ileum were measured in an Ussing chamber. The mucosa-to-serosa magnesium flux exhibited a curvilinear concentration dependence, whereas serosa-to-mucosa flux of magnesium was linearly related to magnesium concentration between 0.25 and 5 mM. At low concentrations magnesium was absorbed, whereas at the magnesium concentration of 5 mM the serosa-to-mucosa magnesium flux was higher than the mucosa-to-serosa flux, resulting in magnesium secretion. Only the mucosa-to-serosa flux of magnesium had a voltage-independent (i.e., nondiffusive) cellular component. Due to the high capacity of this cellular mucosa-to-serosa transport of magnesium, which was about 7.5 times greater than that of calcium, absorption of magnesium was performed in the terminal ileum in contrast to calcium, which was secreted under the same conditions. However, magnesium serosa-to-mucosa flux was totally voltage dependent (i.e., diffusive) and probably restricted to the paracellular pathway. The diffusive serosa-to-mucosa flux of magnesium was about two times greater than the diffusive fraction of the mucosa-to-serosa transport of magnesium. The prevalence of the diffusive serosa-to-mucosa flux of magnesium over that from mucosa to serosa, responsible for magnesium secretion observed at the magnesium concentration of 5 mM, may be explained by an "anomalous solvent drag effect." Voltage clamp experiments showed that magnesium had no effect on the cellular mucosa-to-serosa transport of calcium. However, it decreased the diffusive calcium flux in this direction. 1 alpha,25-dihydroxyvitamin D3 did not influence the unidirectional or net magnesium transport but increased the calcium flux in both directions to the same degree. In conclusion, magnesium is absorbed in the terminal ileum at least partially by a cellular, vitamin D3-insensitive process that is different from the calcium transport mechanism.

Distension-induced secretion in the rat colon: mediation by prostaglandins and submucosal neurons.

Distension of the rat colon descendens in vitro by a hydrostatic gradient induced an increase in short-circuit current (Isc). In a mucosa-submucosa preparation containing the plexus submucosus, the increase in Isc was biphasic with a half-time of about 200 s for the spontaneous returning to the baseline. The time course was monophasic in a mucosa preparation without the plexus submucosus. The increase in Isc in the mucosa-submucosa preparation was inhibited by an inhibitor of phospholipase A2, quinacrine, and by indomethacin, tetrodotoxin or atropine; each of these compounds also abolished the second phase of the response. In contrast, only indomethacin was effective in reducing the increase in Isc in the mucosa preparation. In both preparations the response to distension was inhibited by scilliroside, by replacement of Cl- with gluconate, and by administration of frusemide or the chloride channel blocker, anthracene-9-carboxylic acid. The results indicate that distension induces chloride secretion by causing the release of prostaglandins, which act indirectly, i.e. mediated by the submucosal plexus, and directly at the epithelium.

The valency state of absorbed iron appearing in the portal blood and ceruloplasmin substitution.

(1) Attempts to determine the redox-state of the absorbed iron, which appeared in the portal blood when the free iron-binding capacity was previously saturated, indicate that about 30-90% of this iron was in the ferrous state. This effect was particularly prominent after luminal administration of ferrous iron, but was also seen when iron was given in the ferric state. (2) Total iron absorption is significantly higher in ceruloplasmin-substituted copper-deficient animals as compared to copper-deficient controls. (3) The appearance rate of absorbed iron in the portal blood of copper-deficient animals increased several times immediately after the intravenous infusion of ceruloplasmin. (5) The distribution of absorbed iron was changed due to the ceruloplasmin substitution: it was increased in the reticulocytes (+66%), plasma (+400%) and the body (+112%), whereas in the liver it was decreased by about 78%. (5) In iron-deficient rats intravenously injected ceruloplasmin did not increase iron absorption. (6) The conclusion was drawn that, as for the entrance into the mucosa from the luminal side, also for the release at the contraluminal side into the portal blood, the ferrous state of iron is favoured and that ceruloplasmin accelerates the release into the portal blood by catalyzing the oxidation of ferrous iron due to its high Fe(II): oxygen oxidoreductase (EC 1.16.3.1) activity.