Cytodifferentiation and synaptogenesis in the neostriatum of fetal and neonatal rhesus monkeys

Summary Cytodifferentiation and synaptogenesis in the neostriatum (caudate nucleus and putamen) were analyzed by the Golgi impregnation method and electron microscopy in 14 fetuses and 8 postnatal rhesus monkeys. During the second fetal month the neostriatum consists primarily of simple, mostly bipolar, immature cells and a small number of undefined profiles ending with growth cones. The first morphologically defined synapses appear in the putamen at embryonic day 60 (E60) and in the head of the caudate nucleus at E65. Synaptic density in both structures is less than one per 1000/m² of neuropil at this stage; synapses are characterized by asymmetric junctions between axonal profiles and immature dendritic shafts, accumulation of an intermembrane web and aggregation of round clear vesicles in presynaptic profiles. During the third fetal month neuronal cell bodies and glial cells enlarge, and axonal and dendritic processes in Golgi preparations become more complex. Although the basic morphology of synapses remains unchanged, their density increases to 9/1000 m² in the putamen and 3.7/1000 m² in the caudate. During the fourth fetal month the four principal cell classes of the neostriatum emerge. Spines on the shafts of dendrites are followed closely by the appearance of axospinous synapses. Synaptic density in the putamen is still significantly higher (10.1/1000 m²) than in the caudate (5.4/1000 m²), but by the end of the fifth fetal month (E150) it is the same (80/1000 m²) in both structures. A dramatic increase in synaptic density to 125/1000 m² occurs before term (E165) with the emergence of the first asymmetric synapses as well is symmetric synapses with flat or pleomorphic vesicles that terminate predominately on dendritic shafts. Synaptic density continues to increase after birth, reaching a plateau of approximately 190/1000 m² at the end of the first postnatal month. Throughout postnatal development the proportions of symmetric and asymmetric synapses on the smooth dendritic shafts undergo systematic fluctuations which may reflect the ingrowth of various afferents as well as local cytological differentiation including the formation of cellular compartments.     

Quantitative estimation of the area of luminal and basolateral membranes of rat parotid acinar cells: some physiological applications

Knowledge of luminal and basolateral acinar cell membrane areas of the secretory endpieces is a prerequisite for a detailed quantitative analysis of the ion transport involved in secretion of the primary saliva. In the present study, these areas were estimated in rat parotid acinar cells using standard stereological methods. A total of 480 micrographs — obtained by random sampling from eight glands from four rats — were analysed at a final magnification of 40000x. Expressed per unit cell volume, the area of the luminal acinar cell membrane was: 0.125 m² · m^–3 (SEM=0.027 m² · m^–3, n=4 animals) and the area of the basolateral membrane was: 1.54 m² · m^–3 (SEM=0.085 m² · m^–3, n=4 animals). These figures make it possible to perform a synthesis based upon different categories of experimental data, e.g. on ion fluxes, membrane potentials and single-channel conductances. Thus, we have estimated the density of open, low-conductance Cl^– channels in the luminal membrane — which are not readily accessible for direct, patch-clamp analysis — to be approximately 18 channels per m² in the stimulated state.     

Ultrastructure of the primate carotid body: a morphometric study of the glomus cells and nerve endings in the monkey (Macaca fascicularis)

Summary The carotid body of the monkey (Macaca fascicularis) was studied at both the light and electron microscopic levels in an effort to provide a detailed quantitative characterization of this chemoreceptor organ in the primate. Structurally, the monkey carotid body was organized into lobules of from three to eight glomus cells (in section) and their ensheathing supporting cells. Interspersed among the lobules was abundant connective tissue stroma, fibroblasts and mast cells. Fenestrated capillaries, small arterioles and venules also permeated the organ. Each supporting cell partially ensheathed about three glomus cells and could be easily differentiated from glomus cells by their darker cytoplasmic staining, lack of dense-core vesicles and angular nuclear profile. Glomus cells exhibited an intense catecholamine histofluorescence and contained abundant dense-core vesicles. On the basis of dense-core vesicle size, shape and numerical density, four types of glomus cells were identified. The most common type (62% of all glomus cells) contained vesicles with an average diameter of 219 nm and a density of 8 vesicles per m² of cytoplasm. The second type possessed larger vesicles (264 nm in diameter) and accounted for about 14% of all glomus cells. A third type of glomus cell contained smaller (167 nm) and fewer (5 vesicles per m²) dense-core vesicles. The fourth type of glomus cell contained pleomorphic-shaped vesicles with a maximal diameter of 232 nm. Each of these last two types accounted for about 12% of all glomus cells. All four types of glomus cells were innervated, averaging 1.43 nerve endings per glomus cell (in sections). Nerve endings were primarily of the bouton-like variety averaging 2 m² in sectional area and containing 34.3 clear-core synaptic vesicles (average size 73.5 nm in diameter) per m² of cytoplasm. Of the 57 nerve endings examined in single sections, 16% displayed junctions typical of synaptic specializations and most of these were presynaptic to glomus cells. Glomus cell-glomus cell synapses were not observed. Based on these quantitative observations and on previous studies of carotid body cytoarchitecture in other laboratory species, it appears that the primate organ most closely resembles the cat carotid body, although several differences exist.     

Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K⁺ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K⁺ channel blocker barium (10 nmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mol/l), by cobalt (100 mol/l), zinc (100 mol/l), gadolinium (100 mol/l), verapamil (10 mol/l) and diltiazem (10 mol/l), but are abolished in the presence of 100 mol/l lanthanum, 1 mmol/l cadmium, 10 mol/l nifedipine, 25 mol/l SK & F 96365 and 200 mol/l TMB-8. Stimulation of calcium entry by 10 mol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.     

Positive cooperativity in activation of the cardiac muscarinic K+ channel by intracellular GTP

Concentration-dependent effects of intracellular GTP on activation of the muscarinic K⁺ channel were examined in inside-out patches of cardiac atrial myocytes. The pipette solution contained 0.1 M ACh. GTP (0.01–30 M) and 0.5 mM MgCl₂ were applied to the inside side of the patch membrane. K⁺ channels were activated with GTP concentration above 0.1 M. Channel activation reached a maximal value with 1–3 M GTP. It decreased at GTP concentrations larger than 3 M, probably due to desensitization. The dependence of the open probability of the channel on intracellular GTP showed a sigmoidal relationship with a Hill coefficient of around 3. A positive cooperative effect of intracellular GTP on the K⁺ channel may play an important role in amplifying the signal from the membrane receptor to the K⁺ channel.     

Functional expression and properties of the human skeletal muscle sodium channel

Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the-subunit of the adult human skeletal muscle Na⁺ channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injectedXenopus oocytes and from transiently transfected tsA201 cells. The Na⁺ currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line. The inactivation kinetics of Na⁺ currents in oocytes, during a depolarization, were fitted by a weighted sum of two decaying exponentials. The time constant of the fast component was comparable to that of the single component observed in mammalian cells. The block of hSkM1 Na⁺ currents by the extracellular toxins tetrodotoxin (TTX) and -conotoxin (CTX) was measured. The IC₅₀ values were 25 nM (TTX) and 1.2 M (CTX) in oocytes. The potency of TTX is similar to that observed for the rat homolog rSkM1, but the potency of CTX is 22-fold lower in hSkM1, primarily due to a higher rate of toxin dissociation in hSkM1. Single-channel recordings were obtained from outside-out patches of oocytes expressing hSkM1. The single-channel conductance, 24.9 pS, is similar to that observed for rSkM1 expressed in oocytes.     

Curing of Saccharomyces cerevisiae 2-μm DNA by transformation

Summary A general procedure for the curing of 2-m in Saccharomyces cerevisiae is described. The method is based on the displacement of endogenous 2-m DNA by the recombinant plasmid pMP78-1, which carries the yeast leu2 gene and the 2 -m DNA replicon, but cannot be maintained stably in a yeast cell without endogenous 2-m DNA. After transformation with pMP78-1 cells are grown selectively to displace 2-m DNA. During the non-selective growth which follows, plasmid pMP78-1 is lost and up to 100% of the cells completely lack plasmids. In conjunction with a kanamycin resistance marker, as present in plasmid pMP81, this method should be applicable to cure any wild-type yeast strain. The stability of recombinant plasmids in cir ⁺ and cir ⁰ strains has been compared.     

Microsphere size and determination of intrarenal blood flow distribution in the rat

The influence of microsphere size upon the estimation of cardiac output (CO), renal blood flow (RBF) and its cortical distribution (ICBFD) was evaluated by simultaneous injection of 8.5±0.8 m (SD) and 12.7±1.2 m (SD) spheres in control conditions and after hemorrhagic hypotension (HH rats). The values of CO and RBF were unaffected whilst the ratio of flow to outer and inner halves of cortex (OCF/ICF) was 32% higher with 12.7 m than with 8.5 m spheres in both groups.Microscopic analysis of cleared kidney slices confirmed that large spheres were more concentrated in outermost and less concentrated in innermost glomeruli than small spheres. In addition, the ratio of sphere number per outermost to that per innermost glomerulus (f _s /f _jm ), an approximation of glomerular blood flow distribution was 1.74 and 1.76 with large spheres and 0.98 and 1.06 with small spheres in control and HH rats respectively.It is concluded that the artifact due to sphere size was not minimized in low flow conditions (HH rats) and that 8.5 m spheres may be a more realistic marker of glomerular blood flow distribution in the rat than 12.7 m spheres.Attaché de Recherches I.N.S.E.R.M.     

Length-dependent effects of osmotic compression on skinned rabbit psoas muscle fibers

The goal of this study was to characterize the interrelationship between sarcomere length and interfilament spacing in the control of Ca²⁺ sensitivity in skinned rabbit psoas muscle fibers. Measurements were made at sarcomere lengths 2.0, 2.7 and 3.4 m. At 2.7 m the fiber width was reduced by 17% relative to that at 2.0 m and the pCa₅₀ for force development was increased by 0.3 pCa units. In the presence of 5% Dextran T-500 the fiber width at sarcomere length 2.0 m was also decreased by 17% and the Ca²⁺ sensitivity was increased to the same value as at 2.7 m. In contrast, at sarcomere length 2.7 m the addition of as much as 10% Dextran T-500 had no effect on Ca²⁺ sensitivity. At sarcomere length 3.4 m there was an additional 7% compression and the Ca²⁺ sensitivity was increased slightly (0.1 pCa units) relative to that at 2.7 m. However at 3.4 m the addition of 5% Dextran T-500 caused the Ca²⁺ sensitivity to decrease to the level seen at 2.0 m. Given that the skinning process causes a swelling of the filament lattice it is evident that the relationship between sarcomere length and Ca²⁺ sensitivity observed in skinned fibers may not always be applicable to intact fibers. These data are consistent with measurements of Ca²⁺ in intact fibers which indicate that there might be a decline in Ca²⁺ sensitivity at long sarcomere lengths.     

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.     

Axon terminals of the intrinsic neurons in the nucleus lateralis of the cerebellum an electron microscope study

Summary The large projection neurons of the lateral nucleus have long axons, which leave the cell mass in the superior cerebellar peduncle. These axons emit myelinated recurrent collaterals which have synaptic varicosities en passant. The varicosities are 2–5 m in diameter and contain round, agranular synaptic vesicles ranging between 280 and 480 Å with diameters of approximately 400 Å. The vesicles lie in a moderately dark axoplasmic matrix with a mean packing density of 281/m². The varicosities synapse through Gray's type 1 junctions with dendrites and thorns of large and small neurons. They constitute 22% of the total axonal population on dendrites of large neurons and 10% on dendrites of small neurons. The recurrent collateral system may provide a means for positive feedback to the same neuron and other neurons of the neuropil.The small neuron or interneuron has a short axonal plexus. The axon is myelinated, and is distinctive with a light axoplasmic matrix and varicosities containing elliptical synaptic vesicles. The vesicles are loosely dispersed with a mean population density of 44/m². These varicosities synapse through an intermediate type of junction upon the somata of certain large and small neurons and they consitute 14% and 22% of the axosomatic synapses respectively. They also make synapses on dendrites, constituting 12% and 25% of the total population of axons synapsing with dendrites of large neurons and those of small neurons respectively. It is suggested that these are the inhibitory interneurons of the lateral nucleus.The corticonuclear input through Purkinje axons is the dominant influence on the lateral nucleus neurons. This inhibitory input is considerably larger on the large neurons than on the small ones. It is speculated that the axosomatic synapses are inhibitory. Excitatory influences, through the collaterals of mossy and climbing fibers and the recurrent collaterals of the large intrinsic neurons, impinge upon the dendrites, where the axons of both Purkinje cells and interneurons also terminate.Supported in part by U.S. Public Health Service grants NS10536, NS03659, Training grant NS05591 from the National Institute of Neurological Diseases and Stroke, and a William F. Milton Fund Award from Harvard University.     

Pertussis-toxin-sensitive inhibition by (-) baclofen of Ca signals in bovine chromaffin cells

Ca signals in bovine adrenal chromaffin cells were studied both in Fura-2/AM-loaded intact cells, and in voltage-clamped cells under whole-cell patch-clamp conditions. The effects of gamma-aminobutyric acid b subtype (GABA_b) receptor activation on K⁺-depolarization-induced signals and on voltage-activated Ca²⁺ currents were investigated. Both GABA (20 M) plus bicuculline (20 M) and (-)baclofen (20–100 M), effectively inhibited the Ca signal in intact cells. The effects caused by baclofen continued to develop during the time interval between two successive stimuli. The restoration of the Ca signal during washout of baclofen was also delayed and continued in some experiments for 10–20 min. The inhibitory effect of baclofen on the Ca signal was eliminated by pre-treatment of the cells with pertussis toxin (PTX, 1g/ml, for 4–6h at 37°C). Baclofen (50 M) inhibited Ca²⁺ current in whole-cell mode by at most 20%. The effect developed quickly and was reversible. Infusion into the cells of a non-hydrolyzable analogue of guanosine 5-triphosphate GTP S (100 M), led to complete inhibition of the Ca²⁺ conductance and of voltage-evoked intracellular Ca ([CA]_i) transients within 2 min. In paired cells intracellularly perfused with GTPS-free solution, the Ca²⁺ current amplitude decreased by only about 30% for 5–6 min. It is concluded that bovine chromaffin cells have functional GABA_b receptors the activation of which, mediated by a PTX-sensitive GTP-binding protein, inhibits the evoked increase in cytosolic free Ca²⁺. The small size of the effect on Ca²⁺ current in whole-cell mode as compared to that on the Ca signal in intact cells may be explained by washout of some regulatory element during cell dialysis, or by a relatively small contribution of the normal voltage-activated Ca²⁺ current to the Ca signal. Alternatively, it might indicate GABA_b effects on mechanisms other than Ca²⁺ channels.     

The hypocalciuric effect of thiazides: subcellular localization of the action

The acute administration of thiazides results in a decrease in the urinary Ca²⁺/Na⁺ ratio, whereas chronic administration of these diuretics decreases calciuria. In both situations, Ca²⁺ transport is enhanced in the early part of the distal tubule. The purpose of our study was to determine whether the hypocalciuric action of thiazides was due to a change in the active transport of Ca²⁺ through the basolateral membrane of the nephron or to an effect (direct or indirect) on the permeability of the distal tubule luminal membrane to calcium. In order to detect intrinsic differences between membranes of the proximal and distal tubules, the effect of the diuretic was examined in proximal and distal tubule preparations, and in basolateral and luminal membranes from the two segments separately.Preincubation of microdissected distal tubules in hypotonic solution containing 500 M hydrochlorothiazide (HCTZ) did not influence the Ca²⁺-dependent ATP hydrolysis (Ca²⁺=1 M) nor the Mg²⁺-dependent ATP hydrolysis (Mg²⁺=100 M). Similarly 100 M HCTZ did not change the Ca²⁺ ATPase activity in intact proximal and distal tubule suspensions, at Ca²⁺ concentrations ranging from 0.05 M to 1 M.ATP-dependent Ca²⁺ transport was present in basolateral membrane vesicles from proximal and distal tubule suspensions. Preincubation of the membranes with 100 M HCTZ did not influence this transport. A Na⁺/ Ca²⁺ exchanger, present in the basolateral membranes from the distal tubule, was also insensitive to HCTZ. In contrast, preincubation of luminal membranes from the distal tubules (but not proximal tubules) with 500 M HCTZ significantly increased the Ca²⁺ uptake by these membranes. This increase in Ca²⁺ uptake, in the presence of Na⁺, was dose-dependent; the minimal and the maximal effects of the diuretic were observed at concentrations of 25 M and 100 M respectively. HCTZ increased the V _maxCa²⁺ from2.5±0.3 pmol g^–1 (10 s^–1) to 3.7±0.6 pmol g^–1 (10 s^–1) (P<0.01), but did not influence the K _m (1.43±0.25 mM and 1.37±0.1 mM Ca²⁺ in experimental and control membranes, respectively). Na⁺ was necessary for this effect. Na⁺ per se decreased Ca²⁺ uptake in a concentration-dependent manner and HCTZ partially reestablished Ca²⁺ uptake to the levels observed in a Na⁺-free medium. The anion of the Na⁺ salt also modulated the effect of HCTZ on Ca²⁺ transport. While Cl^– and SCN^– permitted HCTZ to enhance Ca²⁺ uptake, the SO ₄ ^2– anion did not. It is therefore concluded that (a) the hypocalciuric effect of thiazides is primarily due to an increase in the Ca²⁺ uptake of the luminal membrane from the distal tubule, (b) Na⁺ and Ca²⁺ transports are tightly related in the distal luminal membrane, (c) HCTZ modulates this interrelationship by decreasing the inhibitory effect of Na⁺ on Ca²⁺ uptake. Whether the Ca²⁺ and Na⁺ carriers are the same molecule or different entities needs further investigation.     

Membrane specialization and axo-glial association in the rat retinal nerve fibre layer: freeze-fracture observations

Summary The ultrastrucrure of non-myelinated ganglion cell axolemma within the retinal nerve fibre layer of adult rats was examined by thin section and freeze-fracture electron microscopy. Most of the axolemma within the nerve fibre layer does not exhibit any membrane specializations; intramembranous particles are partitioned with a density of 1750 m^–2 on the P-fracture face and 225 m^–2 on the E-face of the non-specialized axolemma. The nerve fibres also exhibit specialized foci of axolemma, at which the axons are abutted by the tips of blunt, radially oriented processes from Müller cells. At such sites of axo-glial association, an electron-dense undercoating is present beneath the axon membrane. Freeze-fracture analysis revealed a substantial increase in the density of E-face particles (>500 m^–2) at sites of association between the tips of blunt glial processes and the axon. These findings demonstrate that non-myelinated axolemma of the retinal nerve fibre layer can exhibit spatial heterogeneity, with patches of node-like membrane at regions of specialized association with glial cell processes. On the basis of their morphological similarity to nodes of Ranvier, we suggest that these specialized axon regions represent foci of inward ionic current.     

Light microscopical study of dendrites and perikarya of interneurones mediating la reciprocal inhibition of cat lumbar alpha-motoneurones

Summary Interneurones which mediate disynaptic inhibition from la muscle spindle afferents of the quadriceps nerve to lumbar alpha-motoneurones were stained with intracellular injection of horseradish peroxidase. Seven best stained and most satisfactorily preserved cells were selected for analysis, and the light microscopic morphology of their cell bodies and dendrites were quantitatively investigated in parasagittal sections. The perikarya were located dorsal or dorso-medial to the motoneurones; they had mean diameters of 51 × 27 m and a mean volume of 35820 m³. The cells had 3 to 7 dendrites, which were arranged asymmetrically around the parent somata. The dendrites extended mainly in the dorso-ventral direction, in which the mean tip to tip distance for each cell was 1742 m. The dendrites had few spines and they branched almost only in bifurcations. On the average, each process divided 3.5 times and in each cell they gave rise to 14.9 branching points as well as a total combined length of more than 7000 m. Primary dendrites had a mean length of 193 m which was generally shorter than the lengths of the branches of higher order. A more detailed analysis of two cells revealed the mean width of primary dendrites to be 5.6 m while that of the 5th order processes was 1.5 m. The mean tapering of individual dendritic branches per unit length was 17%, being somewhat more pronounced for the distally located segments, while at branching points the sum of daughter processes approximately equalled the diameter of the parent process. The surface area and volume of the dendrites constituted 90% and 83% of the total surface area and 46% and 37% of the total volume of the two cells, respectively, excluding the axons. The Ia interneurones differed considerably among themselves with respect to the quantitively investigated parameters. They resembled the inhibitory Renshaw cells of the cat with regard to the number of dendrites, the poverty of spines, and the relationships between cell body diameter and width of primary dendrites.     

Agonist concentration influences the pattern and time course of intracellular Ca2+ oscillations in human arterial smooth muscle cells

Oscillations in intracellular Ca²⁺ were recorded in cultured human uterine artery vascular smooth muscle cells. In the absence of external Ca²⁺, prolonged application of 3 M histamine activated a large transient increase in Ca²⁺ followed by a burst of Ca²⁺ spikes. The time course and frequency of the spikes were approximately constant until the last two to three spikes, when the inter-spike interval progressively increased. At 30 M histamine the response was different; the amplitude of the spikes decreased rapidly to zero, the rate of rise of successive transients fell and the time between spikes increased. The cessation of oscillatory activity was not associated with the depletion of intracellular Ca²⁺ stores, since increased doses of agonist or the sulphydryl reagent thimerosal could reactivate Ca²⁺ release. The changes in the pattern of intracellular Ca²⁺ spikes seen with increasing agonist concentration may reflect the involvement of different inactivation mechanisms in the termination of Ca²⁺ transients. In the presence of external Ca²⁺, histamine (3–30 M) activated regular Ca²⁺ oscillations. The frequency, but not the amplitude, of the oscillations was dependent on agonist concentration, the highest frequency of spiking was observed at 30 M histamine. In cells depolarised with 30 mM K⁺, histamine was still able to activate Ca²⁺ oscillations, but the dependence of spike frequency upon agonist concentration was abolished. Ca²⁺ oscillations could be activated in the presence of verapamil and nifedipine (10 M). These data suggest that in human uterine artery vascular smooth muscle cells histamine-induced Ca²⁺ oscillations are generated largely by a cytosolic oscillator and are modified by the influx of Ca²⁺ across the surface membrane.     

Activity and expression of Na+/H+ exchanger isoforms in the syncytiotrophoblast of the human placenta

The purpose of this study was to compare Na⁺/H⁺ exchanger (NHE) activity in the microvillous (MVM) and basal (BM) plasma membrane of the human placental syncytiotrophoblast and to determine the relative contribution of various NHE isoforms to this activity. Uptake of ²²Na into isolated MVM vesicles in the presence of a H⁺ gradient, at initial rate, was four- to fivefold higher than that by BM vesicles (214±28 vs. 49±9 pmol/mg protein per 30 s, respectively, means±SEM, n=8, 6, P<0.001). The ²²Na uptake by MVM, but not by BM, was reduced in the absence of a H⁺ gradient and in the presence of 500 M amiloride. To determine the contribution of NHE1, NHE2 and NHE3 isoforms to NHE activity in MVM, we investigated the effect of amiloride analogues which show isoform selectivity. HOE 694, an analogue selective for NHE1 at low concentrations, inhibited ²²Na uptake with an EC₅₀ of 0.13±0.05 M (n=6), whereas S3226, an analogue selective for NHE3 at low concentrations had an EC₅₀ of 3.01±0.85 M (n=5). To investigate this further, we measured recovery of syncytiotrophoblast intracellular pH (pH_i) from an acid load using a H⁺-selective, fluorescent dye (BCECF) loaded into isolated intact placental fragments. This recovery was blocked in the absence of Na⁺ and the presence of amiloride (500 M) and concentrations of HOE 694 and S3226 were comparable to those used in vesicle experiments. Overall these data show that under the conditions used NHE activity in the term placental syncytiotrophoblast is absent from BM. NHE activity in the MVM is attributable predominantly to NHE1.     

Intimate apposition of the glomus and smooth muscle cells (g-s connection) in the carotid labyrinth of juvenile bullfrogs

Summary Intimate apposition of the glomus and smooth muscle cells (g-s connection) was found in almost all glomus cells of the carotid labyrinths in juvenile bullfrogs, Rana catesbeiana. There were three types of g-s connection: between thin processes (0.1–0.2 m in width) without dense-cored vesicles of glomus cells and smooth muscle cells; between thick processes (1.0–1.5 m in width) with dense-cored vesicles of glomus cells and smooth muscle cells; and between the tonguelike projections of smooth muscle cells and the flat surface of the glomus cell. In some cases, a single glomus cell made g-s connections with several smooth muscle cells. Exocytosis often occurs at the g-s connection. Afferent and efferent synapses were found on the glomus cells with g-s connections. Reciprocal synapses were also observed. On the basis of these findings, the second and third types of g-s connection are presumed to participate in vascular regulation.     

2,6-Diamino-N-([1-(1-oxotridecyl)-2-piperidinyl]methyl)hexanamide (NPC 15437): A selective inhibitor of protein kinase C

NPC 15437 inhibited protein kinase C (PKC) activity and [³H]phorbol 12,13-dibutyrate (PDBu) binding to the enzyme in a concentration-dependent manner (IC₅₀ values, 19±2 M and 23±4 M, respectively). No inhibition of cAMP-dependent protein kinase A (PKA) or calcium/calmodulin-dependent myosin light chain kinase (MLCK) was observed. A detailed kinetic analysis of the interaction of NPC 15437 and a homogeneous preparation of PKC-alpha revealed a competitive type of inhibition with respect to activation of the enzyme by both phorbol 12-myristate 13-acetate (PMA) (K _i =5±3 M) and phosphatidylserine (PS) (K _i =12±4 M). Mixed inhibition (predominantly of the non-competitive type), with respect to activation of the enzyme by calcium, was also observed. These studies indicate that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the molecule. NPC 15437 inhibited phorbol ester-induced ear edema in mouse (IC₅₀=175 g/ear) demonstrating the ability of NPC 15437 to inhibit PKC-mediated activity in intact cells.     

Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones

The influence of internal Ca²⁺ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca²⁺ current (I _Ca). Internal free Ca²⁺ ([Ca²⁺]_i) was varied between 0.01 and 10 M by addition of Ca²⁺-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca²⁺]_i depressed the 5-HT effect. The dose/ effect curve for the Ca²⁺ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants K _d1=0.063 M and K _d2=1 M. Addition of calmodulin (CM) antagonists (50 M trifluoperazine or 50 M chlorpromazine), phosphodiesterase (PDE) antagonists [100 M isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 M okadaic acid (OA)] in the perfusion solution caused anticalcium action and modified the Ca²⁺ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca²⁺ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with K _d1=0.04 M and K _d2=0.69 M were obtained describing the activation of the retained unblocked enzyme — PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 M), a blocker of Ca²⁺-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca²⁺-induced suppression of the cAMP-dependent upregulation of Ca²⁺ channels is due to involvement of two Ca²⁺-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca²⁺ concentration of less than or equal to 10 M.