The selectivity and structural determinants of peptide antagonists at the CGRP receptor of rat, L6 myocytes

1. Potency orders were determined for a series of agonists and antagonists on the calcitonin gene-related peptide (CGRP) receptor of rat L6 myocytes. The agents tested were all shown to have been active against CGRP, amylin or adrenomedullin receptors. 2. AC187 had a PIC50 Of 6.8 ± 0.10, making it 14 fold less potent as an antagonist than CGRP8-37 (PIC50, 7.95 ± 0.14). Amyline8-37 was equipotent to AC187 (PIC50, 6.6 ± 0.16) and CGRP19-37 was a fold less potent than either (pIC50 6.1 ± 0.24). 3. [Ala11]-CGRP8-37 was 6 fold less potent than CGRP8-37, (pIC50 7.13 ± 0.14), whereas [Ala18] CGRP8-37 was approximately equipotent to CGRP8-37 (pIC50, 7.52 ± 0.15). However, [Ala11,Ala18]- CGRP8-37 was over 300 fold less potent than CGRP8-37 (pIC50, 5.30 ± 0.04). 4. [Tyr0]-CGRP28-37, amylin19-37 and adrenomedullin22-52 were inactive as antagonists at concentrations of up to 1 μM. 5. Biotinyl-human α-CGRP was 150 fold less potent than human α-CGRP itself (EC50 values of 48 ± 17 nM and 0.31 ± 0.13 nM, respectively). At 1 μM, [Cys(acetomethoxy)(2'7)]-CGRP was inactive as an agonist. 6. These results confirm a role for Arg11 in maintaining the high affinity binding of CGRP8-37. Arg18 is of less direct significance for high affinity binding, but it may be important in maintaining the amphipathic nature of CGRP and its analogues.

A comparative study of the effects of hypoxia upon isolated arterial muscle from the rat

Contractile response of rat aorta, mesenteric artery and femoral artery to noradrenaline and potassium chloride were studied under standard and hypoxic conditions and the effect of hypoxia was dependent upon both the vessel and the stimulant. Hypoxia had less effect upon contractions to potassium chloride than those to noradrenaline. The effects of hypoxia on potassium chloride induced responses in different vessels were relatively similar although responses to noradrenaline were vessel dependent. Noradrenaline induced contractions of the femoral artery were most affected by hypoxia whilst those of the mesenteric artery were least affected. Hypoxia changed the well maintained response of the femoral artery to noradrenaline to a transient form; this effect of hypoxia was not evident in the aorta or the mesenteric artery. The aorta and mesenteric artery contracted in calcium free EGTA PSS suggesting that these vessels displayed a release component. Hypoxia reduced the magnitude of this component. The effects of verapamil on noradrenaline and potassium chloride induced responses were investigated and were found to be different to those of hypoxia. Verapamil exerted a greater effect on contractions to potassium chloride than on those to noradrenaline. The effects of hypoxia on 45calcium flux were also vessel dependent. In the mesenteric and femoral arteries hypoxia increased basal 45calcium accumulation. However, the magnitude of noradrenaline stimulated 45calcium accumulation was reduced in the femoral artery and aorta but was unchanged in the mesenteric artery. The effects of hypoxia on 45calcium accumulation were similar to verapamil only in the aorta. The results provide evidence that the effects of hypoxia may arise from alterations in calcium mobilisation processes and that differences between vessels in these processes accounts for the heterogeneity between vessels in their response to hypoxia.

Simultaneous estimation of global background synaptic inhibition and excitation from membrane potential fluctuations in layer III neurons of the rat entorhinal cortex in vitro

It is becoming clear that the detection and integration of synaptic input and its conversion into an output signal in cortical neurons are strongly influenced by background synaptic activity or "noise." The majority of this noise results from the spontaneous release of synaptic transmitters, interacting with ligand-gated ion channels in the postsynaptic neuron [Berretta N, Jones RSG (1996); A comparison of spontaneous synaptic EPSCs in layer V and layer II neurones in the rat entorhinal cortex in vitro. J Neurophysiol 76:1089-1110; Jones RSG, Woodhall GL (2005) Background synaptic activity in rat entorhinal cortical neurons: differential control of transmitter release by presynaptic receptors. J Physiol 562:107-120; LoTurco JJ, Mody I, Kriegstein AR (1990) Differential activation of glutamate receptors by spontaneously released transmitter in slices of neocortex. Neurosci Lett 114:265-271; Otis TS, Staley KJ, Mody I (1991) Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release. Brain Res 545:142-150; Ropert N, Miles R, Korn H (1990) Characteristics of miniature inhibitory postsynaptic currents in CA1 pyramidal neurones of rat hippocampus. J Physiol 428:707-722; Salin PA, Prince DA (1996) Spontaneous GABAA receptor-mediated inhibitory currents in adult rat somatosensory cortex. J Neurophysiol 75:1573-1588; Staley KJ (1999) Quantal GABA release: noise or not? Nat Neurosci 2:494-495; Woodhall GL, Bailey SJ, Thompson SE, Evans DIP, Stacey AE, Jones RSG (2005) Fundamental differences in spontaneous synaptic inhibition between deep and superficial layers of the rat entorhinal cortex. Hippocampus 15:232-245]. The function of synaptic noise has been the subject of debate for some years, but there is increasing evidence that it modifies or controls neuronal excitability and, thus, the integrative properties of cortical neurons. In the present study we have investigated a novel approach [Rudolph M, Piwkowska Z, Badoual M, Bal T, Destexhe A (2004) A method to estimate synaptic conductances from membrane potential fluctuations. J Neurophysiol 91:2884-2896] to simultaneously quantify synaptic inhibitory and excitatory synaptic noise, together with postsynaptic excitability, in rat entorhinal cortical neurons in vitro. The results suggest that this is a viable and useful approach to the study of the function of synaptic noise in cortical networks. © 2007 IBRO.

Oral uptake and distribution of microspheres

There is a growing body of experimental evidence suggesting that the gastrointestinal tract (GIT) may be penetrated by sub-micron sized polymeric particles which have the capacity to deliver therapeutic compounds. We investigated this, initially with Fluoresbrite™ carboxylate latex microspheres (0.87 m diameter) which were administered orally to rats. Microsphere numbers within blood samples were then quantified using fluorescence microscopy or FACS technology. These studies were prone to quantitative error, but indicated that increased microsphere translocation occurred if particles were administered in conjunction with large volumes of hypotonic liquid, and that uptake was very rapid. Test particles were detected in blood, only a few minutes after dosing. To improve quantification, GPC technology was adopted. 0.22 m latex particles were found to accumulate in greatest numbers within the Mononuclear phagocyte system tissues after gavage. Again translocation was rapid. The ability of test particles to leave the intestinal lumen and access systemic compartments was found to be highly dependent on their size and hydrophobicity, determined by hydrophobic interaction chromatography. Considerably lower numbers of 0.97 m diameter latex microspheres were detectable within extra-intestinal tissue locations after gavage. Histological studies showed that Fluoresbrite™ microspheres accumulate within the liver, spleen, Mesenteric lymph node and vasculature of rats after oral administration. Fluorescent particles were observed in both the Peyer's patches (PPs), and non lymphoid regions of rat intestinal mucosa after gavage, conductive to the acceptance that more than one mechanism of particle absorption may operate.

The role of insulin and the insulin-like growth factors in the proliferation of the rat thymic lymphocyte

Concanavalin A, provoked a 35-fold increase in the rate of proliferation of rat thymocytes. Insulin (10-6M), and insulin-like growth factor I (10-10M) approximately doubled the rate of DNA synthesis. Both of these structurally related molecules acted through the type I insulin-like growth factor receptor. The sequential addition of Concanavalin A and insulin, promoted a much greater proliferative response than to either of the two agonists alone. Insulin also increased the uptake of glucose and amino acids by the cells. Glucose uptake was enhanced at insulin concentrations of 10-6M and 10-10M. Amino acid uptake was more strongly affected at the higher concentration. Insulin-like growth factor I (10-11M) also enhanced amino acid uptake. The effects of insulin on metabolism were mediated by both insulin and type I insulin-like growth factor receptors. These effects were greatly enhanced after a pre-treatment with Concanavalin A. Concanavalin A provided a primary mitogenic signal to the cells. Amongst the responses was an increased expression of insulin and/or type I insulin-like growth factor receptors. The consequent enhanced cellular sensitivity to these agonists, enabled them to facilitate the passage of the cells through the cell cycle by: i) providing a secondary mitogenic signal, and ii) promoting the uptake of raw materials and energy substrates. The initiation of DNA synthesis and passage through the cell cycle was thus punctuated by the sequential expression of various cell surface receptors. This regulated cellular sensitivity, enabling them to react in a precisely orchestrated fashion to hormones and other molecules in their environment. The intracellular mechanism of insulin action remains an enigma. Although the presence of extracellular calcium was essential for insulin stimulation of amino acid uptake and DNA synthesis, the cation did not subserve a direct mediator function. Insulin promoted an increase in intracellular pH, which was mediated by the Na+/H+ antiport. Other mechanisms were probably also involved in mediating the full cellular response to insulin.

Effect of nitric oxide on apoptotic activity in the rat gastrointestinal tract

The effect of nitric oxide (NO) on apoptosis in the gastrointestinal mucosa was investigated. Experiments involved long-term exposure of rat gastric mucosal cells in vitro to exogenous NO delivered from the NO, donor S-nitroso-N-acetyl-penicillamine, and the effect of intravenous administration of lipopolysaccharide in vivo, in the presence and absence of the selective inhibitor of inducible NO synthase N-(3-(aminomethyl)benzyl) acetamidine (1400 W). S-nitroso-N-acetyl-penicillamine produced a dose-related inhibition of caspase 3-like activity and DNA fragmentation in isolated gastric mucosal cells. Caspase 3-like activity and DNA fragmentation in gastric, ileal and colonic mucosa were increased both 5 and 24 h after injection of lipopolysaccharide (3 mg/kg, i.v.) to rats in vivo. Administration of 1400 W (5 mg/kg, i.v.) immediately after lipopolysaccharide enhanced caspase 3-like activity and DNA fragmentation above that found with lipopolysaccharide alone. In conclusion, data obtained both in vitro and in vivo suggest that NO exerts an anti-apoptotic effect on rat gastrointestinal mucosal cells. © 2001 Elsevier Science B.V.

Separation of n-paraffins by selective adsorption

A study has been undertaken of the vapor-phase adsorptive separation of n-alkanes from Kuwait kerosene (Kuwait National Petroleum Company, heavy kerosene) using zeolite molecular sieves. Due to the shortage of information on the adsorption of multicomponent systems in the open literature, the present investigation was initiated to study the effect of feed flowrate, temperature, and zeolite particle size on the height of mass transfer zone (MTZ) and the dynamic capacity of the adsorbent for multicomponent n-alkanes adsorption on a fixed-bed of zeolite type-5A. The optimum operating conditions for separation of the n-alkanes has been identified so that the effluent would also be of marketable quality. The effect of multicycle adsorption-desorption stages on the dynamic behaviour of zeolite using steam as a desorbing agent has been studied and compared with n-pentane and n-hexane as desorbing agents. The separation process comprised one cycle of adsorption using a fixed-bed of zeolite type-5A. The bed was fed with vaporized kerosene until saturation had been achieved whereby the n-alkanes were adsorbed and the denormalized material eluted. The process of adsorption-desorption was carried out isobarically at one atmosphere. A mathematical model has been developed to predict the breakthrough time using the method of characteristics. The results were in a reasonable agreement with the experimental values. This model has also been utilized to develop the equilibrium isotherm. Optimum operating conditions were achieved at a feed flowrate of 33.33 x 10-9 m3/s, a temperature of 643 K, and a particle size of (1.0 - 2.0) x 10-3 m. This yielded an HMTZ value and a dynamic capacity of 0.206 m and 9.6S3 x 10-2 kg n-alkanes/kg of zeolite respectively. These data will serve as a basis for design of a commercial plant. The purity of liquid-paraffin product desorbed using steam was 83.24 wt%. The dynamic capacity was noticed to decrease sharply with the cycle number, without intermediate reactivation of zeolite, while it was kept unchanged by intermediate reactivation. Normal hexane was found to be the best desorbing agent, the efficiency of which was mounted to 88.2%.

Analysis of β-amyloid (Aβ) deposition in the temporal lobe in Alzheimer's disease using Fourier (spectral) analysis

To determine the spatial pattern of ß-amyloid (Aß) deposition throughout the temporal lobe in Alzheimer's disease (AD). Methods: Sections of the complete temporal lobe from six cases of sporadic AD were immunolabelled with antibody against Aß. Fourier (spectral) analysis was used to identify sinusoidal patterns in the fluctuation of Aß deposition in a direction parallel to the pia mater or alveus. Results: Significant sinusoidal fluctuations in density were evident in 81/99 (82%) analyses. In 64% of analyses, two frequency components were present with density peaks of Aß deposits repeating every 500–1000 µm and at distances greater than 1000 µm. In 25% of analyses, three or more frequency components were present. The estimated period or wavelength (number of sample units to complete one full cycle) of the first and second frequency components did not vary significantly between gyri of the temporal lobe, but there was evidence that the fluctuations of the classic deposits had longer periods than the diffuse and primitive deposits. Conclusions: (i) Aß deposits exhibit complex sinusoidal fluctuations in density in the temporal lobe in AD; (ii) fluctuations in Aß deposition may reflect the formation of Aß deposits in relation to the modular and vascular structure of the cortex; and (iii) Fourier analysis may be a useful statistical method for studying the patterns of Aß deposition both in AD and in transgenic models of disease.

Gasification of biomass in a downdraft reactor

The objective of this study was to design, construct, commission and operate a laboratory scale gasifier system that could be used to investigate the parameters that influence the gasification process. The gasifier is of the open-core variety and is fabricated from 7.5 cm bore quartz glass tubing. Gas cleaning is by a centrifugal contacting scrubber, with the product gas being flared. The system employs an on-line dedicated gas analysis system, monitoring the levels of H2, CO, CO2 and CH4 in the product gas. The gas composition data, as well as the gas flowrate, temperatures throughout the system and pressure data is recorded using a BBC microcomputer based data-logging system. Ten runs have been performed using the system of which six were predominantly commissioning runs. The main emphasis in the commissioning runs was placed on the gas clean-up, the product gas cleaning and the reactor bed temperature measurement. The reaction was observed to occur in a narrow band, of about 3 to 5 particle diameters thick. Initially the fuel was pyrolysed, with the volatiles produced being combusted and providing the energy to drive the process, and then the char product was gasified by reaction with the pyrolysis gases. Normally, the gasifier is operated with reaction zone supported on a bed of char, although it has been operated for short periods without a char bed. At steady state the depth of char remains constant, but by adjusting the air inlet rate it has been shown that the depth of char can be increased or decreased. It has been shown that increasing the depth of the char bed effects some improvement in the product gas quality.

Molecular biology and biochemistry of brain tumours

ßElucidating some molecular mechanisms and biochemistry of brain tumours is an important step towards the development of adjuvant medical therapies. The present study concentrates on cholecystokinin (CCK), a gut-brain peptide that has been described to be able to induce mitosis of rat gliomas as well as hormone secretion by the anterior pituitary, via the CCK-B receptor. The significance of a polymorphism in the growth hormone releasing hormone (GHRH) receptor (GHRH-R) gene was also determined. Finally, defects in the ß-catenin gene, an important component of the developmental pathway, in a sub-set of craniopharyngiomas were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), restriction digestion analysis and direct sequencing demonstrated expression of CCK peptide itself and its A and B receptors by human gliomas, meningiomas and pituitary tumours. CCK peptides stimulated growth of cultured gliomas and meningiomas as well as in vitro hormone secretion [growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH)] by human pituitary tumours. These biological effects were reduced or abolished by CCK antagonists. In addition, an antibody to CCK reduced mitosis by gliomas and meningiomas, and the same antibody inhibited hormone secretion by cultured human pituitary tumours. CCK peptides stimulated phosphatidylinositol (PI) hydrolysis, indicating coupling of the CCK receptors to phopsholipase C. Cyclic AMP was unaffected. In addition, caspase-3 activity was significantly and markedly increased, whilst proteasome activity was decreased. Taken together, these results may indicate an autocrine/paracrine role of CCK in the control of growth and/or functioning of gliomas, meningiomas and pituitary tumours. Primer induced restriction analysis (PIRA) of a rarer and alternative polymorphism in the GHRH-R receptor, in which Thr replaces Ala at codon 57, in human GH-secreting pituitary tumours was investigated. Whilst the rarer form correlated with an increased response of the pituitary cells to GHRH in vitro, allele distribution studies revealed that it is unlikely that the polymorphism contributes to increased risk of developing GH-secreting tumours and therefore acromegaly. Further findings of this study, using PCR and direct sequencing, were the demonstration of an association between b-catenin gene alterations and craniopharyngiomas of the adamantinomatous type. Since this gene product is involved with development, these results suggest that p-catenin mutations may contribute to the initiation and subsequent growth of congenital adamantinomatous craniopharyngiomas.

The mode of action of bradykinin

The action of bradykinin on transepithelial transfer of sodium and water in isolated rat jejunum and on smooth muscle contraction of rat terminal ileum has been investigated. (1) Bradykinin was shown to stimulate transfer at low control transfer, inhibit transfer at high control transfer and have no effect at intermediate transfer in rat jejunal sacs. Stimulation of transfer occurred only when bradykinin was in the serosal solutiun while inhibition of transfer occurred whether bradykinin was in the aerosal or mucosal solution. Bradykinin-induced stimulation of transfer was not affected by adrenalectomy, nephrectomy, combined adrenalectomy-nephrectomy,  nor maintenance on 1% saline drinking solution or low sodium diet pretreatment. Meclofenamic acid abolished the bradykinin-induced inhibition of water transfer while prostaglandins A1, E1 aud F2α all potentiated this action. Theophylline inhibited water transfer and potentiated the bradykinin-induced inhibition of water transfer. Cyclic AMP and dibutyryl cyclic AMP both inhibited water transfer and the bradykinin-induced inhibition of water transfer was potentiated by the latter. ( 2 ) Bradykinin-induced contractions of rat terminal ileum were little affected by hyoscine while those of acetylcholine were abolished. Anoxia reduced markedly responses tv bradykinin while those of acetylcholine were little affected . Theophylline reduced the responses of rat terminal ileum to bradykinin significantly more than those to acetylcholine. Aspirin and indomethacin reduced markedly the responses to bradykinin while not affecting those to acetylcholine and PGT2. Meslofenamic acid at a concentration of 3.4 µM blocked bradykinin-induced contractions but had no effect on those to acctylcholine, PGE2 or PGF2 and at a concentration of 17. 0 µM drastically reduced bradykinin responses but also reduced those to acetylcholine, PGE2 and PGF2α• Flufenamic acid drastically reduced responses to bradykinin while not affecting those to acetylcholine and PGE2 and slightly affecting those to PGF2α. Polyphloretin phosphate reduced responses to bradykinin, PGF2α and PGE2 but not acetylcholine . Diphloretin phosphate reduced responses to bradykinin, PGF2 and PGE2 in a dose dependent manner but not those to acetylcholine. SC 19220 , in a dose dependent manner, inhibited responses to bradykinin and PGE2 but not to acetylcholine and PGF2. 7 oxa - 13 -prostynoic acid non specifically reduced responses to acetylcholine, bradykinin and PGE2. Bradykinin, in the presence of SQ 20881 , increased the release of prostaglandin-like activity from rat terminal ileum and this was reduced or abolished in the presence of indomethacin, aspirin, meclofenamic acid or flufenamio acid. The extract of PG-like activity did not appear as PGE, PGA or PGFon TLC, but included a substance with similar mobility as 15-Keto-prosta-glandin E2.

Adrenoceptors and intestinal fluid and electrolyte transport in the rat

Noradrenaline was found to significantly stimulate fluid and Na absorption across everted sacs of rat jejunum. Of a number of a1, and 2-adrenoceptor antagonists tested only prazosin significantly inhibited the stimulant effect of noradrenaline and further experiments revealed an antiabsorptive effect of prazosin alone. Theophylline reduced jejunal fluid and Na absorption and this effect was not reversed by 2-adrenoceptor stimulation in contrast to previous findings in vivo. Evidence suggests the everted sac preparation is not appropriate to the study of intestinal fluid and electrolyte transport. The investigation of Jejunal ion transport in vitro was continued using an Ussing chamber preparation. Selective 2-adrenoceptor stimulation was found to depress electrogenic anion secretion, as neurotoxin tetrodotoxin indicated that this was a direct epithelial effect. 2-adrenoceptor agonists have considerable therapeutic value as antisecretory agents and the model of rat jejunum in vitro represents a convenient experimental model for research in this area. The selective 2-adrenoceptor antagonist ICI 118551 decreased basal SCC and inhibited increases in SCC in response to isoprenaline or salbutamol indicating the presence of a 2-adrenoceptor mechanism mediating both secretory tone and increases in secretory processes. Many intestinal secretagogues elicit electrolyte secretion via the stimulation of intramural secretory nervous pathways. If these pathways involve the activation of 2-adrenoceptorsthe 2-adrenoceptor antagonists may be useful in the treatment of diarrhoeal diseases. A single pass lumen perfusion technique was used to investigate possible sympathetic tone over colonic fluid and electrolyte absorption in the rat colon in vivo. The technique employed appeared to lack the necessary resolution for this study and alternative approaches are discussed

Adrenoceptor influences on fluid and electrolyte transport in the rat intestine

An investigation of rat jejunal and distal colonic electrolyte transport in-vitro was undertaken using an Ussing chamber prepartion. Selective α2-adrenoceptor stimualtion in the jejunum was found to depress theo-phylline elevated anion secretion, as evidenced by decreases in short- circuit current (SCC). or α1 -Adrenoceptor stimulation, after α2 -adrenoceptor antagonism in the jejunum, evoked transient increases in basal anion secretion, as reflected by transient increases in basal SCC. The use of the neurotoxin tetrodotoxin indicated that this was a direct epithelial secretory effect. 5-hydroxytryptamine (5-HT) on the jejunum elicited transient increases in basal anion secretion, as demonstrated by transient increases in basal SCC. The use of tetrodotoxin, reserpine and α1 -adrenoceptor antagonists, indicated that a major component of this epithelial secretory effect by 5-HT, was associated with activation of intramural nervous pathways of the sympathetic nervous system, ultimately stimulating α1-adrenoceptors. This might represent an important secretory mechanism by 5-HT in the jejunum. β2-Adrenoceptor stimulation in the distal colon was found to decrease basal SCC, as evidenced by the metoprolol resistant effect of the selective β2- adrenoceptor agonist salbutamol, and lack of effect of the selective β1-adrenoceptor agonist prenalterol. An investigation of rat distal colonic fluid and electrolyte transport in-vivo was undertaken using an colonic loop technique. Although a basal colonic absorption of Na+ and Cl-, and a secretion of K+ were observed, these processes were not under tonic α-adrenergic regulation, as evidenced by the lack of effect of selective α-adrenoceptor antagonism. The secretory effects of prostaglandin-E2 were inhibited by α-adrenoceptor activation, whereas such stimulation did not evoke pro-absorptive responses upon basal transport, unlike noradrenaline.

Studies on the anti-inflammatory effect of S-Adenosyl-L-methionine

1. S-adenosyl-L-methionine (SAMe) had no effect on cytochrome C reduction by superoxide generated from xanthine oxidase except at high concentrations. This was due to direct inhibition of the enzyme. 2. SAMe inhibited the neutrophil respiratory burst , measured by luminol enhanced chemiluminescence, to FMLP and zymosan A but not to PMA. 3. Adenosine and methylthioadenosine (MTA) inhibited the respiratory burst elicited by FMLP. 4. SAMe inhibited the phagocytosis of latex particles by neutrophils at high concentrations but methionine and S-adenosyl L-homocysteine had no effect. 5. Treatment with SAMe had no effect on cell infiltration or PGE2 production in 6-day air pouches. 6. Treatment with SAMe at the optimum dose of 50mg/kg inhibited the early phases of carrageenan induced rat hind paw inflammation but had a lesser effect on the secondary response. The antiinflammatory effect was sustained after inhibiton of polyamine synthesis. 7. SAMe increased liver putrescine levels in the presence and absence of inflammation Spermidine levels were increased in the presence of inflammation but spermine levels were unaffected by any of the treatments. 8. MT A and adenosine increased liver putrescine and spermidine levels 9. Treatment with SAMe had no effect on the polyamine status of blood. lO.Treatment with SAMe had no effect on the levels of glutathione in liver or blood. 11.SAMe and MTA inhibited histamine and platelet-activating factor (PAF) induced hind paw inflammation but had no effect on inflammation induced by dextran, zymosan, compound 48/80, 5-hydroxytryptamine, arachidonic acid or glucose oxidase. MTA was more effective than SAMe. 12. PAP-induced rat hind paw inflammation was inhibited by isoprenaline and verapamil. Combinations of these drugs with SAMe or MT A had no further enhancement of effect. 13. Incubation of rat PMNLs with [14c ] SAMe increased the intracellular levels of S-adenosyl-L-homocysteine in a dose dependent manner, but had no effect on the intracellular levels of SAMe, adenosine or MT A. 14. Pharmacokinetic studies of plasma SAMe following a single dose of the drug (50mg/kg) i.p. demonstrated that SAMe is rapidly absorbed and metabolised

Investigations of the site and mechanisms of drug-induced reductions in the reabsorption of divalent cations by the kidney

Disturbances in electrolyte homeostasis are a frequent adverse side-effect of the administration of aminoglycoside antibiotics such as gentamicin, and the antineoplastic agent cis-platinum. The aims of this work were to further elucidate the site(s) and mechanism(s) by which these drugs may produce disturbances in the renal reabsorption of calcium and magnesium. These investigations were undertaken using a range of in vivo and in vitro techniques and models. Initially, a series of in vivo studies was conducted to delineate aspects of the acute and chronic effects of both drugs on renal electrolyte handling and to select and evaluate an appropriate animal model: subsequent investigations were focused on gentamicin. In a study of the acute and chronic effects of cis-platinum administration, there were pronounced acute changes in a variety of indices of nephrotoxic injury, including electrolyte excretion. Most effects resolved but there were chronic increases in the urinary excretion of calcium and magnesium. The renal response of three strains of rat (Fischer 344, Sprague-Dawley (SD), and Wistar) to a ranges of doses of gentamicin was also investigated. Drug administration produced substantially different responses between strains, in particular marked differences in calcium and magnesium excretion. The results suggested that the SD rat was an appropriately sensitive strain for use in further investigations. Acute infusion of gentamicin in the anaesthetised SD rat produced rapid, substantial increases in the fractional excretion of calcium and magnesium, while sodium and potassium output were unaffected, confirming previous results of similar experiments using F344 rats. Studies using lithium clearance measurements in the anaesthetised SD rat were undertaken to investigate the effects of gentamicin on proximal tubular calcium reabsorption. Lithium clearance was unaffected by acute gentamicin infusion, suggesting that the site of acute gentamicin-induced hypercalciuria may not be located in the proximal tubule. Inhibition of Ca2+ ATPase activity was investigated as a potential mechanism by which calcium reabsorption could be affected after aminoglycoside administration. In vitro, both Ca2+ ATPase and Na+/K+ ATPase activity could be similarly inhibited by the presence of aminoglycosides, in a dose-related manner. Whilst inhibition of Na+/K+ ATPase could be demonstrated biochemically after in vivo administration of gentamicin, there were no concurrent effects on Ca2+ ATPase activity, suggesting that inhibition of Ca2+ ATPase activity is unlikely to be a primary mechanism of aminoglycoside-induced reductions of calcium reabsorption. Histochemical studies could not discern inhibition of either Na+/K+ ATPase or Ca2+ ATPase activity after in vivo administration of gentamicin. Selection of renal cell lines for further investigative in vitro studies on the mechanisms of altered cation reabsorption was considered using MTT (3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Neutral Red cytotoxicity assays. The ability of LLC-PK1 and LLC-RK1 cell lines to correctly rank a series of nephrotoxic compounds with their known nephrotoxic potency in vivo was studied. Using these cell lines grown on semi-permeable inserts, alterations in the paracellular transport of 45Ca was investigated as a possible mechanism by which gentamicin could alter calcium reabsorption in vivo. Short term exposure (I h) of LLC-RK1 cells to gentamicin, via both cell surfaces, resulted in a reduction in paracellular permeability to both transepithelial 3H-mannitol and 45Ca fluxes. When LLC-RK1 cells were exposed via the apical surface only, similar dose-related reductions were seen to those observed when cells were exposed to the drug from both sides. Short-term basal exposure to gentamicin appeared to contribute less to the observed reductions in 3H-mannitol and 45Ca fluxes. Experiments investigating transepithelial movement of 45Ca and 3H-mannitol on LLC-PK1 cells after acute gentamicin exposure were inconclusive. Longer exposure (48 h) to gentamicin caused an increase in the permeability of the monolayer and a consequent increase in transepithelial 45Ca flux in the LLC-RK1 cell line; increases in permeability of LLC-PK1 cells to 45Ca and 3H-mannitol were not apparent under the same conditions. The site and mechanism at which gentamicin, in particular, alters calcium reabsorption cannot be definitively described from these studies. However, indirect evidence from lithium clearance studies suggests that the site of the lesion is unlikely to be located in the proximal tubule. The mechanism by which gentamicin exposure alters calcium reabsorption may be by reducing paracellular permeability to calcium rather than by altering active calcium transport processes.