The Sternberg-Reed cell: Mononuclear,multinucleated or multilobated?

Summary The relationship between mononuclear, multinucleated and multilobated Sternberg-Reed cells was studied by reconstructions done on serial sections 1–2 m thick. The 21 cells so studied showed the following combinations: 7 were binucleated, but two nuclei were composed of two parts at certain levels; these nuclei we propose to call composite. 8 cells were mononuclear and had composite nuclei and 6 cells were mononuclear and had single nuclei.     

Freeze-fracture study of the mechanoreceptive digital corpuscles of mice

Summary The freeze-fracture replication technique was used to study the mechanoreceptive digital corpuscles in toe pads of mice. The axon terminal plasmalemma had intramembranous particles (IMPs) at a density of 2367 ± 517 m^–2 (mean ±s.e.m.) in the P-face and 84 ± 4 m^–2 in the E-face. Particles were 10 ± 1.8 nm in diameter in the P-face and 10 ± 1.5 nm (mean ±s.d.) in the E-face. Particle-rich and particle-free areas were noted in the P-face. The lamellar cell plasmalemma had IMPs at a density of 3359 ± 224 m^–2 in the P-face and 265 ± 95 m^–2 in the E-face. Particles were 10 ± 1.4 nm in diameter in the P-face and 10 ± 1.6 nm in the E-face. Non-terminal unmyelinated fibres in the connective tissue compartment of toe pads were also examined: the P-faces of the axolemma and Schwann cell plasmalemma had IMPs at a density of 1356 ± 283 m^–2 and 1514 ± 514 m^–2, respectively, while the E-face of these membranes had only a few particles. Particles were 9 ± 1.2 nm and 10 ± 1.6 nm in diameter in the P-faces of axon and Schwann cell plasmalemmata, respectively.The results show that the IMPs in terminal axolemma and in lamellar cell plasmalemma have a much higher density than those of non-terminal axons or Schwann cells in myelinated and unmyelinated fibres. In addition, IMPs in the terminal axolemma are larger than those in non-terminal axolemma except for the nodal axolemma. It can be said that plasmalemmata of both the axon terminals and lamellar cells of digital corpuscles are specialized in terms of IMPs, suggesting that they have specific physiological properties in mechanoreceptive functions including mechano-electric transduction.     

Electron microscopic studies on replicating form of defective SV 40 virus DNA

Summary Replicating circular molecules of SV 40 DNA were isolated from SV 40 infected VERO cells. Preparations from dilute and undiluted passaged SV 40 virus stocks were shown to give different frequency distributions of contour length of replicating DNA molecules. The mean length of RF-DNA was 1.26±0.21 and 1.57±0.18 for the undiluted and dilute passaged SV 40 stocks, respectively. In undiluted passaged virus stocks, small replicating molecules with a contour length less than 1.0 were noted, which were not detected in dilute passage stocks. The presence of shortened replicating forms of DNA in the undiluted passaged virus stocks would indicate that the DNA of defective SV 40 virus can replicate.     

Synthesis and release of platelet-activating factor and eicosanoids in human endothelial cells induced by different agonists

Production of platelet-activating factor (PAF) and eicosanoids by human umbilical vein endothelial cells (HUVEC) after stimulation with different agonists has been studied. Significant amounts of PAF were measured in the cellular fraction after treatment with thrombin (2 NIHu/ml), calcium ionophore A23187 (2 M) and histamine (100 M) (110.3±14.3, 80.7±19.2 and 119.2±22.4 pg/10⁵ cells, respectively). Only thrombin caused a partial release of PAF into the supernatant. IL-1 (0.1 nM), TNF (1 nM), arachidonic acid (10 M) and endothelin (0.1 M) were not able to induce any PAF synthesis. High levels of 6-keto-PGF₁ were found after stimulation with thrombin and calcium ionophore A23187 (8641±2575 and 6715±3340 pg/10⁵ cells, respectively). Cytokines IL-1 and TNF were also able to stimulate PGI₂ synthesis, although to a lesser extent. PGE₂ production increased after treatment with thrombin and calcium ionophore A23187 three- and two-fold, respectively. Our results confirm that stimulated HUVEC are able to synthesize PAF and eicosanoids simultaneously, the relative amounts depending upon the agonist used. None of the agonists studied showed any significant effect on 15-HETE production.     

A morphologic study of FL cells infected with para-influenza 3 virus

Summary The sequential changes in FL cells infected with para-influenza 3 virus under conditions in which persistent infection develops were studied by light microscopy, specific immuno-fluorescence and electron microscopy. Hemadsorption in this system was also examined by electron microscopy. The giant multinucleated cell, the characteristic initial cytopathic effect, was shown to be truly syncytial. Specific immunofluorescence appeared in syncytia in the nuclei and at the nuclear membrane; later when the giant cells had broken down and proliferating pleomorphic, spindle-shaped and rounded cells appeared, fluorescence was most pronounced at the plasma membranes. Virus particles which arise from the plasma membranes were first seen along the plasma membrane of otherwise unchanged cells just outside the syncytia. There was extensive virus release at the borders of spindle-shaped cells and in the region of virus release the cytoplasm was finely filamentous. The virus particles ranged in size from 100 to 160 m. They were seen to have a limiting membrane with an inner structure consisting of several dense, round profiles. There was also an outer amorphous, ill-defined layer. Definite changes in the ultrastructure of the nucleus were infrequent but small granules in the nuclear matrix with diameters of 11 to 17 m were of special interest. Both virus-and cyto-hemadsorption were seen but the pattern differed from that seen with influenza virus.This study was aided by grant P 145 B from The American Cancer Society, Inc. and by grant H-3493 (Cl) from The National Heart Institute, National Institutes of Health.Deceased 1960.     

Specializations of plasma membranes in Pacinian corpuscles: Implications for mechano-electric transduction

Summary Pacinian corpuscles of cat mesentery were studied with freeze-fracture and thin sectioning methods after chemical fixation.Intramembranous particles (IMPs) exhibit differences in both density and pattern of distribution between the axolemma of the smooth short axis (x-axis) region and that of the axonal spine region of the long axis (y-axis) of the axon terminal. The axolemma of thex-axis has IMPs at a density of 2687±581 per m² (mean±S.E.M.), and these particles are 9.0±1.7 nm (mean ±S.D.) in diameter. In contrast, the axolemma of they-axis has a higher density of IMPs (3607±612 per m²) which are larger (diameter, 10.0±1.7 nm). The particle distribution is not homogeneous inx-axis membranes as there are small patchy areas devoid of particles scattered throughout the entire surface. The E-face of the axolemma has a low density of IMPs ( 200 perm² in bothx- andy-axes). However, IMPs in the E-face are smaller (9nm) in thex-axis than in they-axis (10nm).The inner core lamellar cells have IMPs at a density of 3276±739 per m² and 553±169 per m² in the P- and E-faces, respectively. The particles are about 10 nm in diameter in both faces. Many gap junctions occur between lamellar cells especially near the clefts, suggesting that hemilamellae of each inner core half are kept at the same electrotonic potential.The outer core lamellar cells have IMPs at a density of 2239±403 per m² and 536±123 per m² in their P- and E-faces, respectively. The particles are approximately 10 nm in diameter in both faces. A noteworthy finding is that tight junctions are prominent at cell-to-cell appositions within individual lamellae, especially in the first and second (or sometimes third) innermost lamellae of the outer core. These tight junctions are considered to be a barrier to the leakage of fluid and/or ions between interlamellar spaces as well as between inner and outer cores.An intermediate cell layer is identified between the inner and outer cores. The connective tissue space of this cell layer corresponds to the endoneurium, indicating that intermediate layer cells are comparable to endoneurial fibroblasts. These cells exhibit a low density of IMPs (658±119 per m² in the P-face) and particles are about 9 nm in diameter.The above findings indicate that the plasmalemmata of the axon terminal of the inner and outer core cells are specialized in terms of content and distribution of IMPs. The difference in axolemmal IMPs between thex-axis and they-axis suggests that there is some separation of function in components of mechano-electric transduction between these two regions. In addition it can be assumed that the hemilamellae of each inner core half constitute an electrotonically coupled environment on each side of the axon terminal abutting thex-axis. Furthermore, the fluid of the inner core is completely segregated and probably different in composition from that of the outer core.     

Diarrhea, CD4+ cell counts and opportunistic protozoa in Indian HIV-infected patients

The aim of this study was to examine the relationships among diarrhea, CD4⁺ cell counts and opportunistic protozoa in HIV-infected patients in North India. In a retrospective study, blood and stool samples of 200 HIV-infected patients from March 2001 until 2003, submitted to the AIDS division of National Institute of Communicable Diseases (NICD), were analyzed. Each patient was examined for opportunistic protozoa, HIV-1 status and CD4⁺ cell counts, and screened for diarrheal symptoms. The rate of diarrhea was 38% in the stool examination. In HIV-infected patients in the groups CD4⁺ > 500 cells/L, 200 cells/L < CD4⁺ < 500 cells/L and in the AIDS patients CD4⁺ < 200 cells/L, diarrhea was 14.7, 29.8 and 56.1%, respectively. It is clear that the diarrhea in the AIDS patients was significant compared with the two former groups (P < 0.0005). In the AIDS patients CD4⁺ < 200 cells/L with diarrhea, Cryptosporidium infection was, at 56.5%, the highest and statistically significant compared with the other parasites (P = 0.037). Microsporidium was detected in 30.4% of the AIDS patients. Diarrhea was common and most strongly associated in patients with low CD4⁺ cell counts. The data stress the importance of opportunistic protozoa in the HIV-infected patients, and that opportunistic protozoa should be expected in HIV-infected patients with low CD4⁺ and diarrhea.     

Shear Stress Induction of C-type Natriuretic Peptide (CNP) in Endothelial Cells is Independent of NO Autocrine Signaling

C-type natriuretic peptide (CNP) is secreted by endothelial cells and has vasodilatory and antiproliferative activity against smooth muscle cells. Using defined laminar shear stress exposures of cultured bovine aortic endothelial cells, we investigated the regulation of CNP gene by PhosphorImaging the ratio of CNP mRNA to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. A 6 h exposure to arterial shear stress of 25 dyn/cm² caused a marked elevation (10.5 ± 6.2-fold; n=10, p < 0.001) of CNP/GAPDH mRNA ratio compared to stationary controls. Arterial shear stress was 2.6 times more potent than a venous level of shear stress of 4 dyn/cm² in elevating the CNP/GAPDH mRNA ratio. After 6 h, CNP secretion by shear stressed BAEC was elevated over stationary controls by 3.1-fold (n=5, p < 0.001) to a level of 34 ± 7.5pg/cm² BAEC. Shear stress elevated CNP mRNA in the presence of L-NAME (400 M) indicating that autocrine signaling through shear-induced NO production or guanylate cyclase activation was not involved. Similarly, the tyrosine kinase inhibitor genistein (10 M), which can also block shear-induced NO production, had no effect on CNP mRNA induction by shear stress in BAEC. The intracellular calcium chelator BAPTA/AM (5 M) attenuated the shear stress-induced CNP mRNA expression by 71%. Interestingly, dexamethasone (1 M) potentiated by 2-fold the shear stress enhancement of CNP mRNA. Shear stress was a more potent inducer of CNP than either phorbol myristrate acetate or lipopolysaccharide. Hemodynamic shear stress may be an important physiological regulator of CNP expression with consequent effects on vasodilation and regulation of intimal hyperplasia. © 1999 Biomedical Engineering Society. PAC99: 8717-d, 8719Tt     

Tubular transport processes in proximal tubules of hypothyroid rats. Lack of relationship between thyroidal dependent rise of isotonic fluid reabsorption and Na+−K+-ATPase activity

Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T₃) for 4 days resulted in normal free T₃ and TSH levels. FT₃ levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T₃/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT₃ levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J _v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10^–2 nl·mm^–1·s^–1 in euthyroid animals). 1 day after T₃ (10 g/kg b.w./day) injectionJ _v was increased significantly to 2.05±0.20 10^–2 nl·mm^–1·s^–1 and continued to increase during 4 days of T₃ reconstitution.When 50 g T₃/kg b.w./day was used,J _v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10^–2 nl·mm^–1·s^–1 after 4 days.J _v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T₃ was unchanged over a 4 day course with either high or low doses of T₃.Na⁺–K⁺-ATPase activity was found to be 2.91±0.16 M P_i/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T₃ resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ _v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T₃ on Na⁺–K⁺-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na⁺ permeability is suggested.     

Physikalische Eigenschaften des Australia-SH-Antigens (Au-SH-Antigen)

Zusammenfassung Die Komplementbindungsreaktion weist ein Au-SH-Partikel nach, das sich weder in Dichte, Sedimentation, chromatographischem Verhalten, noch in seiner Morphologie von dem Partikel unterscheidet, das im Immundiffusionstest bestimmt wird. Zwischen beiden serologischen Tests besteht außerdem eine sehr gute Korrelation der Antigentiter. Die Sedimentationskonstante des Au-SH-Antigens beträgt 40 ±7 S, seine Dichte 1.16 g/cm³. Aus beiden physikalischen Konstanten berechnet sich ein mittlerer Durchmesser von 21.2 ±2 m. Dieser Wert entspricht der elektronenoptisch ermittelten Größe des Au-SH-Antigens. Beide Bestimmungsmethoden liefern eine Größenvariation, die sich von 14 bis 27 m erstreckt.

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Physical properties of Australia-SH-antigen

Summary The complement fixation test detects an Australia-SH-antigen particle which physically (density, sedimentation, chromatographic behaviour on DEAE-cellulose) and morphologically is identical to that particle which is detected by immunodiffusion. Between both serological tests there is an excellent correlation of antigen titers. The sedimentation coefficient of Australia-SH-antigen is 40 ±7S, its density in sucrose 1.16 g/cm³. From these both physical constants the average diameter of Australia-SH-antigen can be calculated to 21.2 ±2 m, a value which corresponds to the seize of Australia-SH-antigen estimated by electron microscopy. The seize of the particles varies between 14 and 27 m.

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Prof. Dr. med. R.Haas zum 60. Geburtstag.

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Die Untersuchungen wurden von der Deutschen Forschungsgemeinschaft unterstützt.     

The thyroid cell monolayer in culture

When cultured on collagen coated nitrocellulose filters, thyroid epithelial cells form morphologically and functionally polarized monolayers. The bioelectric parameters of these monolayers were measured after mounting in Ussing chambers; transepithelial potential (V _ab), short circuit current (I _sc) and transepithelial resistance were respectively 12±1 mV (apical side negative), 3.8±0.2 A cm^–2 and 3250±214 cm² (mean±SEM,n=75). Eighty two percent of the short circuit current was related to sodium absorption as shown by inhibition by apical amiloride (K _m=0.2 M) and by basal ouabain (K _1/2=0.3 M). Amphotericin B (5–25 g/ml) added to the apical bath increasedI _sc suggesting an apical rate-limiting step. Step by step replacement of choline by Na⁺ in a Na⁺-free medium resulted in a progressive increase inV _ab andI _sc with half maximal effect at 20±1 mM Na⁺. Thyrotropin (TSH) increasedI _sc andV _ab in a biphasic way with a transient maximum after 5 min and a plateau after 20 min (about four times the basal level at 100 U/ml TSH). This increase in sodium transport was also inhibited by apical amiloride. Thus, in culture, the thyroid cell monolayer behaves as a tight sodium absorbing epithelium controlled by TSH, with a rate limiting apical sodium channel as the entry mechanism and a basolateral Na⁺, K⁺-ATPase as the electromotive force.     

ATPase activity in macula densa cells of the rabbit kidney

Na-K- and Mg-activated ATPase activities were determined in maculae densae and glomeruli dissected from both superficial and juxtamedullary nephrons of normal rabbits, using an ultramicro method including a cycling reaction. Activities were expressed as P_i generated per macula densa or per glomerulus and normalized for tissue volume. Results indicate that the mean volume of superficial and juxtamedullary macula densa samples was not statistically different, while glomeruli from deep nephrons had sample volumes that were 29% larger than those from superficial nephrons (P<0.001). Correcting for volume both superficial and juxtamedullary macula densa samples had an Na-K-ATPase activity of 0.37±0.21 fmol · h^–1 · (m³)^–1. Mg-ATPase activity in both pools was also similar [0.41±0.07 and 0.52±0.1 fmol · h^–1 · (m³)^–1]. Na-K-ATPase activity in macula densa cells is estimated to be about 1/40th the activity of surrounding cortical thick ascending limb cells. Total glomerular ATPase per unit volume was significantly higher in glomeruli from superficial than from deep nephrons [0.41±0.04 vs. 0.28±0.04 fmol · h^–1 · (m³)^–1 P<0.05]. There was no statistically significant activity of Na-K-ATPase in either superficial or deep glomeruli. These results suggest that in contrast to previous reports, the macula densa contains Na-K-ATPase, but at a low level relative to surrounding tubular cells. Further, in normal rabbits, this activity is invariant in superficial and juxtamedullary samples.     

Sodium current in single myocardial mouse cells

Morphologically intact single myocardial cells of the adult mouse show a length of 132±20 m, a width of 21±5 , and a height of 10±4 m (all mean ± SD) and are brick-like in shape. A one suction pipette method is used for voltage clamp of those single cells. The determined time constant of capacitive current =35±14 s is very short. Series resistancer _s, membrane resistancer _m, and membrane capacityc _m are calculated to be 192±48 k, 6.1±1.1 M, and 186±92 pF (all mean ± SD), respectively. Assuming the specific unit membrane capacitance of 1 F/cm², a total membrane area of 1.86×10^–4 cm² is determined yielding a specific membrane resistanceR _m of 1,134 cm². Settling time of voltage clamp is 30 s. TTX-block of sodium current is described by 1:1 binding with aK _D value of 1.4×10^–6M. Using a reduced extracellular sodium concentration the maximum Na current is between 25 and 40 nA at voltages between –40 and –30 mV. Currents of between +20 and +30 mV reverse in an outward direction. Inward currents are approximated by a m³h model. The time constant of activation decreases from 0.7 ms at –60 mV to 0.12 ms at +20 mV. The time constant of inactivation falls from 9.1 ms at –60 mV to 0.6 ms at +20 mV.Steady state inactivationh is characterized by the half maximum valueV _H=–76.1±4.3 mV and the slope parameters=–6.3±1.1 mV (mean ± SD). A prepulse duration of 500 ms is essential for real steady state inactivation. Steady state activationm and inactivationh overlap each other defining a maximum window current at –65 mV.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

Neuromuscular transmission in experimental autoimmune myasthenia gravis (EAMG)

Chronic experimental autoimmune myasthenia gravis (EAMG) was induced in rats by immunization with acetylcholine receptor (AChR) purified from the electroplax of Torpedo californica. 35–40 days after immunization, serum anti-AChR antibody titers were about 40 nM. At this stage, electrophysiology was performed on isolated M. omohyoideus muscle-preparations from myasthenic and from normal (control) rats.For the study of the equilibrium interaction between acetylcholine (ACh) and AChR, dose-response curves were obtained by quantitative ionophoretic application of ACh to voltage-clamped end-plates. Analysis of dose-response curves yielded the following parameters: maximum end-plate conductance per unit surfaceg _max (EAMG)=10.3±1.1 nS/m²,g _max (normal)=20.2±1.8 nS/m²; apparent dissociation constant K (EAMG)=96±5 M, K (normal)=58±6 M; Hill-coefficient n_H (EAMG)=2.3±0.1, n_H (normal)=2.3±0.1. Single channel properties were derived from an analysis of ACh-induced end-plate current noise: the mean single channel conductance was (EAMG)=29.1±2.2 pS, (normal)=27.6±1.8 pS and the mean channel life-time (EAMG)=1.39±0.09 ms, (normal)=1.32±0.08 ms (T=22°C).The electrophysiological data are interpreted as follows: (1) At myasthenic end-plates there is a 50–60% reduction of functioning AChR (decrease ofg _max). A total number of about 2×10⁶ (1×10⁶) channels per end-plate was calculated for control (myasthenic) rats. (2) The affinity of AChR for ACh is reduced and/or there is an impediment of the conformational change from the closed- to the open-channel configuration (increase of K). (3) Single channel properties are essentially unaffected.This work was supported by the Deutsche Forschungsgemeinschaft SFB 38, project N and We 667/6     

Quantitative electromicroscope study of the oculomotor parasympathetic neurons projecting to the ciliary ganglion in cats: comparison of the synaptic (axo-somatic and axo-proximal dendritic) organization of anterior-dorsal and ventral cell groups

The synaptic organization of the oculomotor parasympathetic preganglionic neurons (OPNs), labeled retrogradely after a horseradish peroxidase (HRP) injection into the ciliary ganglion, was studied in cats by electron microscopy. We divided the OPNs into two groups, anterior-dorsal (ADG) and ventral (VG) cell groups, based upon physiological studies in cats suggesting that accomodation-related OPNs are predominantly located anterior and dorsal to the somatic nuclei of the oculomotor nuclear complex (i.e., the anteromedian and Edinger Westphal nuclei, and the ventral central gray area), while pupillo-constriction-related OPNs are predominantly located ventral to the somatic nuclei (i.e., the ventral tegmental area). The synaptic organization of these two groups was quantitatively compared, using a nested analysis of variance to determine statistical significance (P<0.05). Partial reconstructions of the labeled somata and proximal dendrites were made from tracings of electron micrographs of every 2nd section in serial ultrathin sections that included the nucleolus or were adjacent to sections that included the nucleolus. The mean number of boutons of apposition on a reconstructed labeled soma of VG was significantly greater than that of ADG (mean ±SD; ADG, 5.3±3.3; VG, 8.6±3.2). The mean synaptic density on a VG soma was significantly greater than on an ADG soma (mean±SD; ADG, 3.74±2.11 counts/100 (m²; VG, 6.30±1.99 counts/100 m²). The mean synaptic covering ratio on a VG soma was significantly greater than on an ADG soma (mean±SD; ADG, 5.21±2.91%; VG, 10.14±3.76%). The mean estimated number of boutons of apposition on a VG soma was significantly greater than on an ADG soma (mean±SD: ADG, 53±36; VG, 100±48). Boutons were classified on the basis of the shape of their synaptic vesicles as S-type (containing spherical clear synaptic vesicles) or P-type (containing both flattened and spherical clear synaptic vesicles). The mean S-type/S+P-type bouton ratio on a VG soma was significantly greater than on an ADG soma (mean±SD; ADG, 0.31±0.20; VG, 0.67±0.18). The differences demonstrated in this study reinforce, morphologically, the assumption of functional localization of OPNs, and further allow us to estimate the relative characteristics of the synaptic organization of accommodation-related OPNs and pupillo-constriction-related OPNs.     

Calmodulin in mast cells and its role in histamine secretion

Calmodulin content and distribution in rat peritoneal mast cells was determined by radioimmunoassay. Isolated pure mast cells were disrupted by sonication and the total calmodulin content and its distribution were determined. Calmodulin bound to the membranes was released by heating with 0.1% Lubrol PX to 95°C for 5 min. The total calmodulin content of mast cells was found to be 160±14 ng/10⁶ cells (9.4±0.82 pmoles/10⁶ cells). The highest amount (68%) was present in the supernatant representing the cytosol. The next highest amount (26%) was found in the composite fraction consisting of mitochondria, endoplasmic reticulum, Golgi vesicles and plasma membrane (100,000g pellet). The mast cell granules contained 4% of the total calmodulin.Trifluoperazine (TFP) was used as an antagonist to explore the role of calmodulin in histamine secretion. At 10 M concentration, TFP caused a negligible spontaneous histamine release by its membrane effect. TFP (10 M) inhibited histamine release by all the three secretagogues used, but the degree of inhibition varied: 60% with antigen, 40% with compound 48/80 and 20% with ionophore A23187. It is suggested that the TFP effect is due to calmodulin-antagonism and interference with the activation of enzymes, essential to the secretory process.     

Architecture and synaptic relationships in the intermediolateral nucleus of the thoracic spinal cord of the rat: HRP labelling, catecholamine histochemistry and electron microscopic studies

Summary The organization of the intermediolateral nucleus (IML) of the thoracic spinal cord was examined using glyoxylic acid-induced fluorescence histochemistry, retrograde horseradish peroxidase (HRP) labelling and electron microscopy. In serial sections of T2, it was found that the distribution of catecholamine nerve terminals was intimately related to the neuronal perikarya of IML. Potassium permanganate fixation and 5-hydroxydopamine treatment revealed small dense-cored vesicles in axon varicosities with or without synaptic specializations. A gelatinous region, composed of small diameter dendrites and unmyelinated axons, formed a narrow longitudinal bundle in the centre of the nucleus. The population of the axon varicosities in the IML was 0.17 ± 0.02/m² in 75 nm sections. The average size of the axon varicosities with flat synaptic vesicles was 1.44 ± 0.05 m² and that of varicosities with spherical vesicles was 0.97 ± 0.02 m². After HRP injection into the superior cervical ganglion, ipsilateral IML neurons were labelled in T1–T3 segments of the spinal cord. Axon varicosities with flat and others with spherical synaptic vesicles synapsed on the dendrites labelled by HRP. Among axon varicosities synapsing on the preganglionic sympathetic neurons, 74.8 ± 7.1% at axo-somatic synapses and 46.0 ± 6.7% at synapses on proximal dendrites contained flat synaptic vesicles.     

Insulin enhances l-dopa renal proximal tubule uptake: a regulatory mechanism impaired in insulin resistance

A stimulatory role for insulin in the uptake of neutral amino acids has been reported for a variety of tissues. Here we examine the effect of insulin on l-dopa uptake by proximal tubule cells (PT cells) isolated from control and fructose-fed rats (FR-rats, 10% w/v fructose solution in tap water), a model of insulin resistance. Insulin (200 U/ml) increased l-dopa uptake into PT cells by about 50% (705±186 vs.1117±140 pmol l-dopa/mg protein per minute) (p<0.05). The higher uptake correlated with a 40% increase in the number of high-affinity l-dopa transport sites (l-dopa 0.2 M) (0.59±0.05 vs. 0.82±0.09 pmol l-dopa/mg protein per minute), without changing their affinity. The effect of insulin was not modified by ouabain (1 mM), nocodazole (1–10 M) or colchicine (50–100 M), whereas it was abolished by cytochalasin D or latrunculin B (both 1 M). This suggests that the process is independent of Na⁺,K⁺-ATPase activity or the microtubule network but that it requires the integrity of the actin cytoskeleton. l-dopa transport by the low-affinity transport sites (l-dopa 5 M) was not affected by insulin, neither was the effect of insulin observed in PT cells isolated from FR-rats. In line with this, FR-rats showed lower renal l-dopa reabsorption as compared to control animals (81±4 vs. 51±9%). Taken together, our results support the involvement of insulin in the multifactorial regulation of renal l-dopa reabsorption.     

Taurocholate depolarizes rat hepatocytes in primary culture by increasing cell membrane Na+ conductance

Rat hepatocytes in primary culture were impaled with conventional microelectrodes. Addition of 5–100 mol/l taurocholate led to a slowly developing depolarization that was maximal at 50 mol/l (10.5±1.5 mV, n=15) and not reversible. The effect was Na⁺ dependent and decreased in cells preincubated with 1 mol/l taurocholate. Increasing external K⁺ tenfold depolarized the cells by 12.3±2.3 mV under control conditions and by 6.3±1.2 mV with 50 mol/l taurocholate present (n=7). Depolarization by 1 mmol/l Ba²⁺ was 7.6±0.8 mV and 6.0±0.7 mV (n=9) before and after addition of taurocholate, respectively. Cable analysis and Na⁺ substitution experiments reveal that this apparent decrease in K⁺ conductance reflects an actual increase in Na⁺ conductance: in the presence of taurocholate, specific cell membrane resistance decreased from 2.8 to 2.3 k · cm² · Na⁺ substitution by 95% depolarized cell membranes by 8.9±2.9 mV (n=9), probably due to indirect effects on K⁺ conductance via changes in cell pH. With taurocholate present, the same manoeuvre changed membrane voltages by –0.8±2.6 mV. When Na⁺ concentration was restored to 100% from solutions containing 5% Na⁺, cells hyperpolarized by 3.5±3.6 mV (n=7) under control conditions and depolarized by 4.4±2.9 mV in the presence of taurocholate, respectively. In Cl^– substitution experiments, there was no evidence for changes in Cl^– conductance by taurocholate. These results show that taurocholate-induced membrane depolarization is due to an increase in Na⁺ conductance probably via uptake of the bile acid.