Biophysical model for organic acid excretion in Ascaris suum.

To develop a model for the mechanisms of organic acid excretion in nematodes, we measured the concentrations of volatile fatty acids (VFAs), pH and electrical potentials across hypodermal and muscle membranes and across the composite body wall (consisting of hypodermis, muscle and cuticle) of Ascaris suum using standard chromatographic and microelectrode recording techniques. In incubates containing one parasite in 20 ml modified Ascaris Ringer's solution, the level of combined VFAs excreted into the medium increased linearly for about 18 h, then plateaued at a concentration of 4.2 mM; the medium acidified rapidly to a plateau at about pH 5.0 within 4-6 h. Following 24 h incubations, the concentrations of VFAs in the hypodermis, muscle, and pseudocoelomic compartments were 62.4 +/- 8.1, 62.3 +/- 7.8 and 74.4 +/- 3.2 mM, respectively. The pseudocoelomic fluid was more acidic (pH 6.52 +/- 0.06) than the hypodermis (pH 6.78 +/- 0.03) or muscle (pH 6.77 +/- 0.03). These data and the electrical potentials across hypodermal (-57.9 +/- 6.3 mV) and muscle (-30.3 +/- 0.8 mV) membranes were used to determine the equilibrium concentrations for protonated (HVFA) and anionic (VFA-) forms of the acids across these membranes and across the cuticle. Under these conditions, little transmembrane or transmural excretion of HVFAs is expected to occur in A. suum. However, a 16-27 mV driving force for VFA- excretion exists across hypodermal and muscle membranes, and a larger driving force is predicted to exist for these anions across the cuticle. This driving force could provide potential energy for VFA- excretion through anion channels which exist in muscle and hypodermal membranes of this parasite, or for facilitated transport systems.

Characterization and function of Ca(2+)-activated K+ channels in arteriolar muscle cells.

We examined the functional role of large-conductance Ca(2+)-activated K+ (KCa) channels in the hamster cremasteric microcirculation by intravital videomicroscopy and characterized the single-channel properties of these channels in inside-out patches of membrane from enzymatically isolated cremasteric arteriolar muscle cells. In second-order (39 +/- 1 microns, n = 8) and third-order (19 +/- 2 microns, n = 8) cremasteric arterioles with substantial resting tone, superfusion with the KCa channel antagonists tetraethylammonium (TEA, 1 mM) or iberiotoxin (IBTX, 100 nM) had no significant effect on resting diameters (P > 0.05). However, TEA potentiated O2-induced arteriolar constriction in vivo, and IBTX enhanced norepinephrine-induced contraction of cremasteric arteriolar muscle cells in vitro. Patch-clamp studies revealed unitary K(+)-selective and IBTX-sensitive currents with a single-channel conductance of 240 +/- 2 pS between -60 and 60 mV (n = 7 patches) in a symmetrical 140 mM K+ gradient. The free Ca2+ concentration ([Ca2+]) for half-maximal channel activation was 44 +/- 3, 20 +/- 1, 6 +/- 0.4, and 3 +/- 0.5 microM at membrane potentials of -60, -30, +30, and +60 mV, respectively (n = 5), with a Hill coefficient of 1.9 +/- 0.2. Channel activity increased e-fold for a 16 +/- 1 mV (n = 6) depolarization. The plot of log[Ca2+] vs. voltage for half-maximal activation (V1/2) was linear (r2 = 0.9843, n = 6); the change in V1/2 for a 10-fold change in [Ca2+] was 84 +/- 5 mV, and the [Ca2+] for half-maximal activation at 0 mV (Ca0; the Ca2+ set point) was 9 microM. Thus, in vivo, KCa channels are silent in cremasteric arterioles at rest but can be recruited during vasoconstriction. We propose that the high Ca0 is responsible for the apparent lack of activity of these channels in resting cremasteric arterioles, and we suggest that this may result from expression of unique KCa channels in the microcirculation.

Isolation, localization, and bioactivity of the FMRFamide-related neuropeptides GYIRFamide and YIRFamide from the marine turbellarian Bdelloura candida.

Two FMRFamide-related neuropeptides, GYIRFamide and YIRFamide, were isolated from the marine turbellarian Bdelloura candida. The peptides elicited a dose-dependent contraction of isolated turbellarian muscle fibers, and both were more potent than FMRFamide. Structure-activity studies, using a range of analogues of the tetrapeptide amide, indicated that the structure of the endogenous peptides was optimal for peak activity. Immunocytochemistry, using an autologous antiserum, revealed a widespread distribution of peptide immunoreactivity within central and peripheral neurons and their processes. This study indicates an important role for GYIRFamide and YIRFamide in the control of neuromuscular function in turbellarians.

Inhibitory effects of nematode FMRFamide-related peptides (FaRPs) on muscle strips from Ascaris suum.

A large number of FMRFamide-related peptides (FaRPs) are found in nematodes, and some of these are known to influence tension and contractility of neuromuscular strips isolated from Ascaris suum body wall. Relaxation of these strips has been noted with five nematode FaRPs. The inhibitory actions of SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2) appear to be mediated by nitric oxide, as previously demonstrated with inhibitors of nitric oxide synthase (NOS). This present study showed that the effects of PF1 were also depended on external Ca++ and were reduced by the Ca(++)-channel blocker verapamil, observations consistent with the finding that nematode NOS is Ca(++)-dependent. KSAYMRFamide (PF3), KNIRFamide (PF4) and KNAFIRFamide (an alanine substituted analog of KNEFIRFamide, AF1, termed A3AF1) also relaxed A. suum muscle strips, but these responses were not affected by NOS inhibitors. PF3 inhibited the activity of strips prepared from the dorsal side of the worm, but contracted ventral strips. Both effects were dependent on the presence of ventral/dorsal nerve cords (unlike PF1/PF2) and were attenuated in medium which contained high K+ or low Ca++. PF4-induced muscle relaxation and hyperpolarization were independent of nerve cords, but were reversed in Cl-free medium, unlike PF1 or PF3. The PF4 effect physiologically desensitized muscle strips to subsequent treatment with PF4 and/or GABA. However, PF4 and GABA were not synergistic in this preparation. The effects of GABA, but not PF4, were reduced in muscle strips treated with the GABA antagonist, NCS 281-93. Following PF4 (or GABA) relaxation, subsequent treatment with higher doses of PF4 caused muscle strip contraction. A3AF1 was found to relax muscle strips and hyperpolarize muscle cells independently of the ventral and dorsal nerve cords, K+, Ca++, and Cl-, and mimicked the inhibitory phase associated with the exposure of these strips to AF1. On the basis of anatomical and ionic dependence, these data have delineated at least four distinct inhibitory activities attributable to nematode FaRPs. Clearly, a remarkably complex set of inhibitory mechanisms operate in the nematode neuromuscular system.

Nitric oxide mediates the inhibitory effects of SDPNFLRFamide, a nematode FMRFamide-related neuropeptide, in Ascaris suum.

1. The physiological effects of two Phe-Met-Arg-Phe-NH2 (FMRFamide)-related neuropeptides isolated from the free-living nematode Panagrellus redivivus, SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2), were examined using neuromuscular preparations from the parasitic nematode Ascaris suum. 2. PF1 and PF2 hyperpolarized muscle membrane and induced sustained flaccid paralysis, independent of external Cl-, in both innervated and denervated preparations. 3. PF1 reversed spastic contractions induced by the cholinomimetic levamisole, elevated K+, or the excitatory nematode FMRFamide-related neuropeptides KNEFIRFamide or KHEYLRFamide. 4. PF1 reversal of levamisole contraction was blocked by pretreatment with agents that interfere with nitric oxide (NO) synthesis (e.g., N-nitro-L-arginine), whereas sodium nitroprusside, which releases NO in solution, mimicked PF1 and PF2. 5. NO synthase activity, monitored by the conversion of [3H]arginine to [3H]citrulline, was twice as abundant in A. suum hypodermis as in muscle, but was not present in reproductive tissue. The relative abundance of NO synthase activity in these tissues was similar to the observed specific binding of [3H]PF1. 6. These results suggest that the inhibitory effects of PF1 and PF2 on nematode somatic muscle are mediated by NO, and that the hypodermis may serve a role in this process analogous to that of the endothelium in vertebrate vasculature.

Comparison of anticoagulation regimens after Carpentier-Edwards aortic or mitral valve replacement.

BACKGROUND: To identify the optimal use of anticoagulants after Carpentier-Edwards valve replacement, a retrospective study of all patients undergoing Carpentier-Edwards aortic (N = 378) or mitral (N = 370) valve replacement was done. METHODS AND RESULTS: At the time of hospital discharge, 103 patients were managed with warfarin, 509 with aspirin alone, and 136 with no anticoagulation or antiplatelet therapy. Over the first 90 days after aortic or mitral valve replacement, the linearized rate of hemorrhage was greater for warfarin than for aspirin or no therapy (16.7 +/- 7.6%, 3.4 +/- 1.7%, and 3.1 +/- 3.1% per patient-year, respectively; P = .03). After aortic valve replacement, aspirin provided a low rate of thromboembolism (0.8 +/- 0.2% per patient-year), not significantly different from warfarin or no treatment (2.9 +/- 1.6% and 1.5 +/- 0.6% per patient-year) (P = .07). After mitral valve replacement, no single treatment was most advantageous because the rate of hemorrhage over the first 90 days for warfarin was equivalent to the 90-day rate of thromboembolism with aspirin or no therapy. CONCLUSIONS: Anticoagulation after Carpentier-Edwards mitral valve replacement may be best guided by individual patient characteristics. Within the limits of a retrospective analysis, these data support the routine use of aspirin alone after Carpentier-Edwards aortic valve replacement, both in the first 90 days and long-term.

Schistosoma mansoni: myogenic characteristics of phorbol ester-induced muscle contraction.

The myogenic nature of phorbol ester-induced muscle contractures of Schistosoma mansoni was investigated using muscle physiology and electrophysiological techniques. The contracture is dependent of extracellular Ca2+, is blocked by Ca2+ channel blockers, and appears to be associated with an increase in the permeability of the muscle to Ca2+ but not to Na+ or H+. The musculature is not depolarized during phorbol ester-induced contracture, but surface electrical activity decreases. Threshold treatments of phorbol ester and depolarization or praziquantel produce synergistic contractures of the parasite. The contracture could not be explained by altered release of or sensitivity to putative neurotransmitters, decreased Ca2+ efflux, or an increase in the sensitivity of the contractile system to Ca2+. These results support the hypothesis that activation of protein kinase-C in the schistosome with phorbol esters leads to muscle contracture by enhancing sarcolemmal Ca2+ channel activity.

Serotonin and acetylcholine: further analysis of praziquantel-induced contraction of magnesium-paralysed Schistosoma mansoni.

The nature of stimulus-induced flaccid paralysis produced in Mg(2+)-paralysed Schistosoma mansoni was investigated. Serotonin induced a dose-dependent, heterologous flaccid paralysis with an IC50 of 600 nM. This flaccid paralysis was a function of the extracellular Mg2+:Ca2+ ratio and was reversible. Tonic contractions produced by phorbol-12,13-dibutyrate or 60 mM K+ were reversed by the application of serotonin and flaccid paralysis was induced. These actions of serotonin were mimicked by forskolin and synergized by IBMX but the potassium channel blocker, 3,4-DAP, did not produce flaccid paralysis. When Mg(2+)-paralysed parasites were stimulated with 3,4-DAP, IBMX produced a dose-dependent flaccid paralysis with an IC50 of 11 microM. Membrane permeable analogues of cAMP and cGMP did not synergize with IBMX. Cholinergic agonists, but not other inhibitory substances, prevented the serotonin- and forskolin-induced and the IBMX-synergized flaccid paralysis but not that produced by praziquantel. The possible interactions of these agents with the muscle are discussed.

Praziquantel: physiological evidence for its site(s) of action in magnesium-paralysed Schistosoma mansoni.

The mechanism whereby praziquantel produces a contraction and subsequent flaccid paralysis (a loss of sensitivity to subsequent stimuli) of Schistosoma mansoni in a medium containing an elevated Mg2+:Ca2+ ratio was investigated. In RPMI, praziquantel produced a concentration-dependent tonic contraction of the parasite with an EC50 of 200 nM. Magnesium inhibited the contraction in such a manner as to convert the tonic contraction to a phasic one without altering the peak force generated. The Mg(2+)-dependent block was non-competitive with praziquantel but was competitive with extracellular Ca2+, ratios of 7.5:1;Mg2+:Ca2+ being needed to inhibit the tonic contraction and to induce flaccid paralysis. Flaccid paralysis was associated with a reduced ability of the parasite to take up 45Ca2+ from the bath compared to parasites that had not entered into flaccid paralysis and flaccid paralysis was reversible. Recovery from flaccid paralysis was accelerated by treatments that are expected to increase Ca2+ uptake by the parasite. At a concentration of 500 nM, praziquantel produced 2 distinct phasic contractions in intact parasites incubated in an elevated [Mg2+] medium but only 1 phasic contraction in parasites lacking their surface tegumental membranes. In zero Ca2+ I-RPMI, 10 microM praziquantel produced a phasic contraction of intact parasites but did not stimulate contraction of detegumented parasites until Ca2+ was reintroduced into the bath. These results indicate that praziquantel interacts with specific Ca(2+)-permeable sites in the tegumental and sarcoplasmic membranes of the parasite and that under these conditions of elevated Mg2+:Ca2+ ratios, these sites become blocked by Mg2+, leading to flaccid paralysis of the parasite.

Studies on muscle cells isolated from Schistosoma mansoni: a Ca(2+)-dependent K+ channel.

Muscle cells from adult male Schistosoma mansoni have been isolated using a combination of papain digestions and mechanical dissociation procedures. The muscle fibres isolated in this way lacked nuclei but they did contract and relax in response to high [K+], a response which was blocked in the presence of Co2+. From this we conclude that the isolation procedure yields viable muscle fibres useful for physiological studies. Patch-clamp recordings taken from the isolated fibres show a variety of discrete ionic conductances. In inside-out patches one prominent channel was a Ca(2+)-activated K+ channel with a conductance of 195 pS and a selectivity greater than 10:1 for K+ over Na+, Cs+ or NH4+. Percentage open time was dependent on [Ca2+] at the intracellular face. With [Ca2+] at 1 microM or greater percentage open time was greater than 95%; at 0.1 microM it was less than 2%. No voltage sensitivity could be detected in the voltage range from -50 to -10 mV membrane potential. Ba2+ (10 mM), but neither tetraethylammonium nor 3,4-diaminopyridine blocked the channel from the intracellular face. This Ca(2+)-activated K+ channel in the muscle membrane of this acoelomate animal is similar in most respects to the maxi-K+ channels which have been described in a variety of cells from more highly evolved animals.

Schistosoma mansoni: evidence for protein kinase-C-like modulation of muscle activity.

The phorbol esters, phorbol-12,13-dibutyrate, phorbol-12-myristate-13-acetate, phorbol-12,13-didecanoate, and phorbol-12,13-diacetate, as well as mezerin at concentrations as low as 10 nM produce a spastic paralysis of the schistosome musculature. The action of these protein kinase-C activators is dependent on the sites of esterification and is stereo-specific since phorbol-13,20-diacetate, phorbol-12,13,20-triacetate, 20-oxo, 20-deoxy-beta-phorbol-12,13-dibutyrate, alpha-phorbol-12,13-didecanoate, and alpha-phorbol are inactive. A phospholipid and phorbol ester-dependent protein kinase is identified. This kinase is stimulated by all of the phorbol esters that increase muscle tone but is not stimulated by phorbol esters that do not affect muscle tone. A high affinity, stereo-specific phorbol ester receptor is identified. Dose-response curves of phorbol-12,13-dibutyrate-induced muscle tension and -stimulated kinase activity and receptor binding indicate that these responses are mediated by the same system. These results indicate that protein kinase-C-like enzyme may play an important role in modulating activity of the schistosome musculature.