Hydrostatic pressure mimics gravitational pressure in characean cells

Summary Hydrostatic pressure applied to one end of a horizontalChara cell induces a polarity of cytoplasmic streaming, thus mimicking the effect of gravity. A positive hydrostatic pressure induces a more rapid streaming away from the applied pressure and a slower streaming toward the applied pressure. In contrast, a negative pressure induces a more rapid streaming toward and a slower streaming away from the applied pressure. Both the hydrostatic pressure-induced and gravity-induced polarity of cytoplasmic streaming respond identically to cell ligation, UV microbeam irradiation, external Ca²⁺ concentrations, osmotic pressure, neutral red, TEA Cl^–, and the Ca²⁺ channel blockers nifedipine and LaCl₃. In addition, hydrostatic pressure applied to the bottom of a vertically-oriented cell can abolish and even reverse the gravity-induced polarity of cytoplasmic streaming. These data indicate that both gravity and hydrostatic pressure act at the same point of the signal transduction chain leading to the induction of a polarity of cytoplasmic streaming and support the hypothesis that characean cells respond to gravity by sensing a gravity-induced pressure differential between the cell ends.     

Cellular mechanisms of paired electrical stimulation in ferret ventricular myocardium: relationship between myocardial force and stimulus interval change

Summary The subcellular mechanisms of twitch-force potentiation with paired electrical stimulation was studied in ferret ventricular myocardium using the bioluminescent calcium indicator aequorin. It is demonstrated for the first time that interpolation of an extrasystole in a train of conditioned twitches results in a beat-to-beat change in [Ca²⁺]_i and force. Steady-state twitch force and Ca _i ²⁺ were increased with paired stimulation. Increased [Ca²⁺]₀ in the setting of paired stimulation resulted in an increase in the amplitude of the postextrasystole and associated Ca²⁺ transient. Verapamil, a Ca²⁺ channel antagonist, had the opposite effect of increased [Ca²⁺]₀. Postextrasystole potentiation was still present, but diminished in amplitude. These results indicate that postextrasystole potentiation is in part due to a verapamil-depletable store (Ca²⁺). Postextrasystole potentiation is therefore predominantly dependent on sarcoplasmic reticulum (SR) Ca²⁺ loading. Ryanodine, an alkaloid which induces Ca²⁺ leakage from the SR, abolished postextrasystole potentiation; however, in the presence of ryanodine the extrasystole was potentiated. Caffeine, a phosphodiesterase inhibitor which induces SR Ca²⁺ release and impairs uptake, also abolished postextrasystole potentiation. As with ryanodine there was resultant potentiation of the extrasystole. In the case of caffeine the calcium transient consisted of a second slow component associated with extrasystole twitch potentiation. The results are consistent with sarcolemmal Ca²⁺ influx playing a role in potentiation of the extrasystole in the presence of an impaired SR. These data indicate that transsarcolemmal Ca²⁺ influx in the presence of impaired intracellular Ca²⁺ buffering can directly activate the myofilaments in agreement with reports on human myocardium.Abbreviations C conditioned stimulus - ESI extrasystolic interval - L_max active tension - PES postextrasystole - PESI postextrasystolic interval - SR sarcoplasmic reticulum - T test stimulus     

Activation of two types of fibres in ferret,Mustela putorius furo,cremaster muscle

Summary Some contractile, histochemical, morphological and electrophysiological properties of ferret, Mustela putorius furo, cremaster muscle have been estimated. Histochemical fibre typing revealed the presence of two types of fibres (type I 66.2%, type II 33.8%). Morphometry performed on ATPase-stained transverse sections showed that type I was composed of a large amount (40%) of small(<400 m²) cells. In mammalian Ringer two groups of fibres could be recognized on the basis of the values of resting potential (-69.7 mV and-59.1 mV) and intracellular sodium activity (8.3 mmol·l^-1 and 14.1 mmol·l^-1, respectively). In experiments on fibre bundles, the elevation of extracellular potassium concentration to 15–200 mmol·l^-1 produced contractures that consisted of a well-defined transient or phasic tension followed by a sustained or tonic tension. Properties of activation and inactivation of the tension analysed in small bundles of cut fibres (lengths 0.5–1.0 cm) were of fast- and slow-twitch type for phasic and tonic phase, respectively. In contrast to the phasic component of K contractures, the tonic phase was abolished by Ca²⁺ withdrawal and inhibited by Ni²⁺, Cd²⁺, Co²⁺, Gd³⁺ and gallopamil (D600). In Ca²⁺-free medium the sustained tension was restored by adding Sr²⁺. It is concluded that in ferret cremaster muscle the presence of slow-twitch fibres would give rise to the tonic component of the K contracture in which an extracellular source of activator Ca²⁺ is involved. The ability of these fibres to contract with a maintained tension for prolonged periods of time might participate in the temperature regulation of the testes.Abbreviations a _i ^Na intracellular sodium activity - ATPase myosin adenosine triphosphatase - D600 gallopamil - E _m membrane potential - E _r resting potential - EDL muscle, extensor digitorum longus muscle - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - e.c. excitation-contraction - SDHase succinate dehydrogenase - NADHase nicotinamide adenine, dinucleotide hydrogen-diaphorase - SOL muscle, soleus muscle - T time constant of relaxation - TEACI tetraethylammonium chloride - [Ca]_o, [K]_o, [Na]_o extracellular calcium, potassium, sodium concentration     

Functional properties of caltrin proteins from seminal vesicle of the guinea pig.

Caltrin proteins from seminal vesicle content of the guinea pig bind with great specificity to different regions of the spermatozoa. Indirect immunofluorescence studies with polyclonal antibodies showed that caltrin I binds to the head, on the acrosomal cup, while caltrin II binds on the principal tail and the neck. No fluorescence was detected either in the midpiece or in the post-acrosomal area of the head when sperm were exposed to either of the caltrins. Calcium-induced hyaluronidase release, which occurs during the acrosomal reaction, was dramatically inhibited by caltrin I (approximately 85% inhibition). Caltrin II was less effective in preventing the enzyme release (approximately 50% inhibition). Chemical modification of the structure modified the biological activity of the two caltrins. Reduction and carboxymethylation of the cysteine residues diminished the inhibitory activity on 45Ca2+ uptake and reduced the ability of the proteins to react with their antibodies. Removal of the carbohydrate portion by chemical deglycosylation transformed the inhibitor proteins into enhancers of calcium uptake into the spermatozoa. Caltrin proteins from the guinea pig appear to play the same physiological role as bovine caltrin, regulating specifically calcium transport across the spermatozoal membranes related with the acrosome reaction and hyperactivation process. The dual behavior of caltrins to inhibit or enhance Ca2+ uptake enables them to fulfill this function. Nevertheless, molecular mechanisms different from those described for bovine caltrin seem to be involved in the control of the functional activity of the guinea pig caltrins.     

Dependence of the photophobic response of the blue-green alga,Phormidium uncinatum,on cations

It is suggested that photophobic responses caused by a sudden step-down in light intensity require the presence of cations in the blue-green alga, Phormidium uncinatum.Drastic removal of cations abolishes the phobic response, which recovers after addition of Ca²⁺ ions. Calcium can be substituted for partially by other cations with an effectivity following the sequence Ca>Mg>Na>Ba>Co=0. During the photophobic response there is a 25% increase in ⁴⁵Ca binding by the cells related to a step-down in light intensity. Three seconds after a light-dark transition there is a sharp increase in the binding of labelled calcium, followed by a subsequent release.Flushing the filaments with high cation concentrations, esp. calcium causes a reversal of movement in the absence of a light stimulus similar to a photophobic reversal. This stimulus could trigger the same sequence of events in the transduction chain bypassing the primary photoresponse.Abbreviations EDTA Ethylene diaminetetraacetic acid - EGTA ethylene glycol-bis (2-aminoethylether) N,N tetraacetic acid     

Stimulus-Secretion Coupling in Isolated Adrenal Chromaffin Cells: Calcium Channel Activation and Possible Role of Cytoskeletal Elements

Abstract: The catecholamine secretory function of a preparation of isolated bovine adrenal chromaffin cells has been further characterized under conditions designed to elucidate the mechanism of calcium channel activation and the possible role of cytoskeletal elements in stimulus-secretion coupling. Three related sets of data were obtained: (1) Differences in kinetics, Ca dependence, strength, and additivity of the secretory response to acetylcholine (ACh) versus excess K; (2) the effects on secretion of the Ca channel-blocking agents, Ni, Mg, and verapamil; and (3) the Ca dependence of vinblastine action on ACh- and K-evoked secretion. The results suggest that a major portion of the Ca influx required for catecholamine release enters the cell via voltage-dependent Ca channels with some additional Ca influx via the ACh receptor channel. Comparison of the present secretion data with corresponding known electrophysiological properties of isolated chromaffin cells provides added evidence for a role of chromaffin cell action potentials in regulation of Ca influx and the secretory response. Elevated Ca concentrations enhanced K-evoked secretion to levels comparable to that of ACh but did not induce a vinblastine block of K-evoked release. This provides further evidence against a role of microtubules in the common exocytosis event per se. However, a role of cytoskeletal elements in directing the movement of secretory granules, or an action of vinblastine at cholinergic receptors, remain distinct possibilities.     

Influence of Calcium on Dopamine Synthesis and Tyrosine Hydroxylase Activity in Rat Striatum

Abstract: We have investigated three aspects of the relationship between calcium and tyrosine hydroxylase activity in rat striatum. In the first series of experiments, we examined the hypothesis that the rise in dopamine synthesis during increased impulse flow results from a calcium-induced activation of tyrosine hydroxylase. Calcium (12.5–200 μ M ) had no effect when added to crude enzyme or enzyme partially purified by gel filtration. Moreover, incubation of synaptosomes with excess calcium (up to 3.5 m M ) had little or no effect on dopamine synthesis. Incubation with the depolarizing alkaloid veratridine (75 μ M ) did increase dopamine synthesis, but did not alter the activity of tyrosine hydroxylase subsequently prepared from the synaptosomes, despite the presumed rise in intracellular calcium. In the second series we examined the hypothesis that increased dopamine synthesis after axotomy results from activation of tyrosine hydroxylase owing to a decrease in intracellular calcium. Addition of the calcium chelator EGTA (100 μ M ) to crude or partially purified enzyme was without effect, whereas incubation of synaptosomes with EGTA (500 μM ) decreased cell-free enzyme activity. In the third experimental series we examined the relationship between calcium and activation of tyrosine hydroxylase by dibutyryl cyclic AMP. EGTA failed to alter the increase in the activity of tyrosine hydroxylase prepared from synaptosomes incubated with dibutyryl cyclic AMP. However, it blocked the increase in synaptosomal dopamine synthesis and dopamine content normally produced by the cyclic AMP analogue. Thus, tyrosine hydroxylase does not appear to be activated by either increases or decreases in calcium availability. However, calcium may be important for the maintenance of basal tyrosine hydroxylase activity, and may play an indirect role in the expression of tyrosine hydroxylase activation produced by other means.     

Effects of Pentobarbitone on 45Ca2+ Transport by Rat Brain Mitochondria

The effects of pentobarbitone on the transport of 45Ca2+ by rat brain mitochondria were studied, using the Ruthenium Red-EGTA quench technique. In the presence of succinate and inorganic phosphate, mitochondria rapidly accumulate 45Ca2+. Pentobarbitone (0.1-1.0 mM) stimulates the initial rate of Ca2+ transport. In contrast, pentobarbitone (1 mM) did not affect the NaCl (50 mM)-induced efflux of 45Ca2+ from mitochondria. Dibucaine (60 micro M), a clinically used local anaesthetic, inhibits both 45Ca2+ uptake an efflux. The results suggest that barbiturate stimulation of mitochondrial Ca2+ uptake may, in combination with effects on other Ca2+ sequestering processes, contribute to the inhibitor of transmitter release observed at a number of synapses.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Calcium Binding Sites of Synaptosomes from Insect Nervous System as Probed by Trivalent Terbium Ions

Terbium binding to synaptosomes from the central nervous system of the locust was studied by fluorescence spectroscopy and electron microscopy. The protein-sensitized fluorescence of terbium was used to characterize the calcium binding sites of synaptosomes. As judged by electron microscopy and x-ray analysis, terbium ions produced electron-dense patches in regular arrays on the outer surface of synaptosomal membranes and induced marked aggregation of synaptic vesicles in isolated terminals.