Characterization of a putative nitric oxide synthase in the neuromuscular system of the parasitic nematode, Ascaris suum.

In this paper we report on the biochemical presence of nitric oxide synthase (NOS)-like activity in Ascaris suum tissue and examine the pharmacological effect of NO donors on A. suum muscle strip preparation. NOS activity was determined by monitoring the formation of [3H]citrulline from [3H]L-arginine and NO formation via the oxyhaemoglobin assay. Neuromuscular tissue from A. suum which stained positively for NADPH diaphorase, contained NOS activity. Neither NOS activity nor NADPH diaphorase staining was detected in intestinal tissue. The absence of Ca2+, NADPH and other co-factors normally required for mammalian neuronal NOS activity only partially reduced the formation of both citrulline and NO by A. suum neuromuscular homogenate. The results of the biochemical assays indicate the presence of an enzyme capable of producing NO and citrulline, but with a different profile from that of rat neuronal NOS. We also present preliminary evidence for the action of NO (NO donors) in the neuromuscular system of A. suum.

Immunocytochemical localization of a putative inhibitory amino acid receptor subunit in the parasitic nematodes Haemonchus contortus and Ascaris suum.

A rabbit antiserum was raised against a synthetic peptide corresponding to a region near the N-terminus of the Haemonchus contortus inhibitory amino acid receptor subunit, HG1. The antiserum recognized a recombinant form of the N-terminal domain of the subunit on Western blots and reacted with the ventral nerve cord of H. contortus in immunofluorescence experiments. Immunofluorescence was also detected in specific head neurons of H. contortus: these were tentatively identified as ring motor- and inter-neurons, plus a possible sensory neuron equivalent to the AQR cell of Caenorhabditis elegans. In the roundworm Ascaris suum, immunoreactivity was limited to the muscle arms, the post-synaptic component of the neuromuscular junction. The possible ligand of receptors containing the HG1 subunit is discussed in the light of this expression pattern.

NADPH diaphorase activity in peptidergic neurones of the parasitic nematode, Ascaris suum.

The histochemical marker for nitric oxide synthase, NADPH diaphorase, is known to co-localize in mammalian neurones with various classical neurotransmitters and neuropeptides. The nervous system of the parasitic nematode Ascaris suum has previously been shown to contain both NADPH diaphorase activity and neuropeptide immunoreactivity. This study examined the possibility that NADPH diaphorase and neuropeptide immunoreactivity may co-exist in the same neurones. Two antisera were used, one raised to KYSALMFamide, a C-terminal synthetic analogue of SALMFamide 1 (GFNSALMFamide), and another that recognizes calcitonin-gene-related peptide (CGRP). We provide evidence that in a distinct subset of neurones in the ventral, dorsal and lateral ganglia NADPH diaphorase staining and SALMFamide-like immunoreactivity are co-localized, suggesting a possible role for nitric oxide in modulating neuropeptide activity in these regions. CGRP-like immunoreactivity was less widely distributed, and was not consistently co-localized with NADPH diaphorase.

Histochemical mapping of NADPH diaphorase in the nervous system of the parasitic nematode Ascaris suum.

NADPH diaphorase has recently been discovered to be responsible for neuronal nitric oxide (NO) synthase activity in mammals. It thus serves as a histochemical marker for the localization of NO synthase in the nervous system. The histochemical technique was used to map out potential NO-producing neurones in the nervous system of the parasitic nematode, Ascaris suum. Positive staining for NADPH diaphorase was present in various parts of the central nervous system, in particular within selective cell bodies and fibres in the ventral ganglion, the retrovesicular ganglion, ventral and dorsal cords and sublateral lines. Intense staining was also present in the motorneurone commissures, indicating a potential role for NO as a neurotransmitter at the neuromuscular junction. NADPH disphorase-positive neurones were not confined to the central nervous system. Selective staining was also present in the enteric nervous system, in particular the pharynx and in the peripheral nervous system innervating the sensory organs.

Quantitative studies on the effect of prostacyclin on freshly isolated rat osteoclasts in culture.

Prostaglandins exert marked but transient inhibitory effects on bone resorption. The present study examines the effects of prostacyclin (0.15 to 25 microM) on the morphology of freshly disaggregated rat osteoclasts. An area descriptor, rho, represented changes in total cell spread area, and a motility descriptor, mu, represented overall changes in cell motility. The application of prostacyclin intercepted the trend of an increasing cell spread area with time and produced a transient reduction of rho, an R effect. Its magnitude depended upon concentration and was marked at 25 microM prostacyclin. The subsequent recovery (+0.8/min) of rho at this concentration resembled the persistent spreading seen in the absence of the agonist. There was also a sustained decrease in mu to approximately 60% of its pretreatment value (a Q effect) following the application of 25 microM prostacyclin. The extracellular application of 20 mM [Ca2+] produced a similarly transient cell retraction preceded by a rise of cytosolic [Ca2+], but without a corresponding decrease in mu. In contrast, prostacyclin did not elevate cytosolic [Ca2+], suggesting the triggering of an alternative transduction pathway. A fully reversible retraction together with incomplete quiescence may explain the transience characteristic of the antiresorptive action of prostacyclin.

Estimation of intracellular calcium activity in confluent monolayers of primary cultures of quail medullary bone osteoclasts.

The present study was concerned with an investigation of the relative utilities of the fluorescent indicators fura-2 and fluo-3 for spectrofluorimetric estimation of the intracellular calcium concentration ([Ca2+]i) in confluent osteoclast monolayers that had been isolated from medullary bone of quail hens and maintained in tissue culture for 6-8 days. Additionally, we have determined the effects of raised extracellular calcium ([Ca2+]o) and chicken calcitonin (CT) on [Ca2+]i in this preparation. Relative to fura-2, fluo-3 was poorly incorporated into the osteoclasts and had a high apparent equilibrium binding constant (Kd) for Ca2+ binding (809 nM). The osteoclasts were only weakly sensitive to the calcium ionophore, ionomycin. It is concluded that fura-2 is of greater utility than furo-3 in this preparation. In contrast to its lack of effect in freshly isolated cells, elevated [Ca2+]o up to 20 mM stimulated a concentration-dependent increase in [Ca2+]i in cultured osteoclasts, but CT was without effect. These findings further support the idea that quail osteoclasts are able to acquire a Ca2+ sensor or 'receptor' and thus to respond to [Ca2+]o in a similar manner to mammalian osteoclasts when they are removed from the bone microenvironment, but retain a refractoriness to CT under these conditions.

Effect of raised extracellular calcium on cell spread area in quail medullary bone osteoclasts.

The present study reports on the effects of extracellular calcium ([Ca2+]o) elevation and ionomycin on cell spread area of medullary bone osteoclasts freshly isolated from egg-laying Japanese quail. The responses were compared with those demonstrated in osteoclasts cultured for periods of 5-8 days and also to those previously demonstrated in neonatal rat osteoclasts. Freshly isolated medullary bone osteoclasts, unlike rat osteoclasts, were refractory to 20 mM [Ca2+]o, in that they showed no change in cell spread area. They did, however, show a modest (15%) reduction in cell spread area to ionomycin (7-50 microM), applied for 15-30 min. When medullary bone osteoclasts were precultured for 5-8 days, they exhibited a well-developed response to 20 mM [Ca2+]o with a 46% reduction in cell spread area. They also showed a similar reduction in cell spread area in response to ionomycin (4 microM). It is concluded that, unlike freshly isolated neonatal rat osteoclasts, those obtained from quail medullary bone appear refractory to inhibitory factors such as [Ca2+]o. However, when the avian cells are cultured for a few days they appear to recover their ability to respond to [Ca2+]o.

Osteoclasts from medullary bone of egg-laying Japanese quail do not express the putative calcium 'receptor'.

The present study reports the contrasting effects of extracellular calcium ([Ca2+]e) elevation on cytosolic free calcium levels ([Ca2+]i) of osteoclasts, freshly isolated either from medullary bone of the egg-laying Japanese quail or from rat cortical bone. [Ca2+]i was measured in single osteoclasts using the Ca(2+)-sensitive fluorochrome, Indo-1. We found that elevation of [Ca2+]e failed to induce a rise of [Ca2+]i in quail osteoclasts, whilst causing an elevation of [Ca2+]i in rat osteoclasts. The calcium ionophore, ionomycin, led to a sustained elevation of [Ca2+]i in both cell types. These findings suggest that osteoclasts isolated from egg-laying quail do not possess the calcium sensor or 'receptor' that appears to be vital for the survival and function of rat osteoclasts.

Calcitonin gene-related peptide promotes transient radiocalcium uptake into chick bone in vivo.

We have examined the in vivo effects in chicks of intravenously injected chicken (c-) and rat (r-) calcitonin gene-related peptides (CGRP) on uptake into bone of a simultaneously administered 45Ca label. Both peptides caused transient (10 min) increases in 45Ca uptake into a variety of bone types. In dose-response experiments at 10 min, CGRP doses of 0.26-1.04 nmol/100 g body wt were found to give maximal responses. These were well developed in chicks fasted for 22 h but absent in those which were continuously fed. This contrasts with the hypercalcaemic effect of CGRP which is apparent in fed rather than fasted chicks.

Abnormal redox status without increased lipid peroxidation in sugar cataract.

In conflict with a previous report, we find that phenolic inhibitors of lipid peroxidation (butylated hydroxytoluene [BHT] and butylated hydroxyanisole [BHA]) do not have significant inhibitory effect on galactosemic cataract formation. This is consistent with the lack of enhancement of stable products of lipid peroxidation (measured by the thiobarbituric acid assay) in the lenses of galactosemic rats. This does not imply that oxidative stress plays no role in galactosemic cataract formation (indeed, we find that galactosemic lens homogenates contain increased amounts of an Fe2+ oxidant, possibly a peroxide), but rather that BHT- and BHA-inhibitable lipid peroxidation specifically has no role to play. In instances where drugs appear to inhibit galactosemic cataract formation, other effects caused by the drugs, e.g., inhibition of feeding or induction of general detoxification pathways, must be considered.

Effects of rat and chicken calcitonin gene-related peptides (CGRP) upon calcium metabolism in chicks.

In vivo effects of intravenously injected chicken(c-) and rat(r-) calcitonin gene related peptide (CGRP) upon plasma total (Cat), ionized (Cai) calcium, inorganic phosphate (Pi) and clearance of an acutely administered 45Ca label have been examined in chicks. Both peptides were hypercalcaemic in fasted chicks, unlike previously reported hypocalcaemic response in mammals. r-CGRP was hypercalcaemic at doses of both 0.26 and 1.31 nmol/100 g body wt, the lower dose produced a significant elevation of Cat one hour after injection into 12-h-fasted chicks, the upper dose had a similar effect at 20 min. Cai was also non-significantly elevated by r-CGRP. Pi was slightly increased by r-CGRP at both doses, 20 and 60 min after injection. c-CGRP produced a dose (0.26-4.17 nmol/100 g body wt) dependent elevation of Cat and Cai in 22-h-fasted chicks. A greater response was however seen in fed animals. Peak responses were observed 45 min after injection. c-CGRP (1.04 nmol/100 g body wt) caused a significant decline in plasma Pi (P less than 0.05) in fasted chicks. Pi was elevated in control fed animals compared with fasted controls. c-CGRP (1.04 nmol/100 g) did not effect plasma Pi in fed chicks. Whilst both peptides elevated plasma Ca, clearance of an acutely administered 45Ca label from plasma was greater in both r-CGRP treated 12-h-fasted chicks and c-CGRP treated 22-h-fasted chicks. In contrast, the rate of 45Ca clearance in fed chicks was not affected by c-CGRP treatment. The differential effects of these peptides upon plasma 45Ca clearance and other plasma parameters of Ca metabolism, suggest a complex mode of action of the peptide upon avian Ca homeostasis, possibly involving direct actions upon kidney and bone.

Antioxidant characteristics of some potential anticataract agents. Studies of aspirin, paracetamol, and bendazac provide support for an oxidative component of cataract.

It is conceivable that drugs with anticataract potential possess antioxidant activity. Using defined chelating agents and reducing agents in a number of assays for antioxidant activity, we have demonstrated that paracetamol, aspirin, bendazac, and its metabolite 5-hydroxybendazac possess substantial reducing and/or metal-chelating activity. We found that paracetamol and 5-hydroxybendazac reduced the diphenylpicrylhydrazyl free radical; inhibited butyl peroxide-induced erythrocyte lysis as well as the haematin-catalyzed oxidative coupling of 4-aminoantipyrine and phenol by butyl peroxide. Furthermore, paracetamol, bendazac, and aspirin competed with 3-(2-pyridyl)-5,6-Diphenyl-1,2,4-triazine-4', 4"-disulphonic acid (ferrozine) for ferrous ion.

The calcitonin gene peptides: biology and clinical relevance.

The calcitonin/CGRP multigene complex encodes a family of peptides: calcitonin, its C-terminal flanking peptide, katacalcin, and a third novel peptide, calcitonin gene-related peptide (CGRP). The 32-amino acid peptide calcitonin inhibits the osteoclast, thereby conserving skeletal mass during periods of potential calcium lack, such as pregnancy, growth, and lactation. This hormonal role is emphasized by observations that lower circulating calcitonin levels are associated with bone loss and that calcitonin replacement prevents further bone loss. Structurally, CGRP resembles calcitonin and has been implicated in neuromodulation and in the physiological regulation of blood flow. Here we review the molecular genetics, structure, and function of the calcitonin-gene peptides as analyzed in the laboratory and focus on more recent clinical studies relating to disorders and therapeutics.

"Autoxidative glycosylation": free radicals and glycation theory.

Studies have shown that glycation in vitro is complicated by the ability of glucose to oxidise, in the presence of trace amounts of transition metal, generating protein-reactive ketoaldehydes, hydrogen peroxide and diverse free radicals. Protein exposed to glucose undergoes fragmentational and conformational alterations, and these, as well as thiol oxidation, appear to be caused by hydroxyl radicals. Glycofluorophore formation is dependent upon ketoaldehyde formation. It is suggested that glucose autoxidation contributes to oxidative stress in pathophysiology associated with diabetes and ageing via this newly described process of "autoxidative glycosylation".