In vivo alpha 2-adrenoceptor-mediated inhibition of isoproterenol-stimulated free fatty acid output in dog bone marrow adipose tissue

Clomdine, an α₂ agonist, inhibited the isoproterenol-induced free fatty acid outflow from perfused bone marrow adipose tissue of dog tibia. This effect was suppressed by the α₂-antagonist, yohimbine. These in vivo experiments clearly demonstrate an α₂-adrenoceptor-mediated inhibition of isoproterenol-induced lipid mobilization and argue for a physiological interplay between β- and α₂-site stimulation in the regulation of lipolysis.     

Regulation of plasma insulin level by alpha 2-adrenergic receptors

Phentolamine, yohimbine or dihydroergotamine markedly increased plasma immunoreactive insulin (IRI) and inhibited epinephrine-induced hyperglycemia in fasted mice. On the other hand, phenoxybenzamine or prazosin only slightly increased plasma IRI and enhanced epinephrine-induced hyperglycemia. These results indicate that there is a distinct difference in the effects of alpha-adrenergic blockers on the plasma IRI and glucose levels, and that alpha-adrenergic receptors responsible for the plasma IRI level resemble alpha 2-adrenergic receptors more closely.     

Non-adrenergic inhibition at prejunctional sites by agmatine of purinergic vasoconstriction in rabbit saphenous artery

We investigated the effects of agmatine, clonidine, xylazine and moxonidine on the purinergic vasoconstriction induced by electrical stimulation in the rabbit isolated saphenous artery without endothelium. Transmural electrical stimulations induced reproducible responses in the arterial preparations, which were abolished by tetrodotoxin at 0.1 microM or pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium salt (PPADS, 30 microM), but were not affected by 1 microM prazosin. Clonidine, xylazine and moxonidine induced transient and concentration-independent vasoconstriction, with threshold concentrations of 1, 3 and 30 microM, respectively. Agmatine, in contrast, did not produce any vascular response even at 1 mM. Lower concentrations of clonidine, xylazine and moxonidine (0.01-0.3 microM) concentration-dependently decreased vasoconstrictor responses to electrical stimulation, whereas agmatine (0.1-1 mM) induced an inhibitory followed by a facilitatory effect on electrically evoked responses. Agmatine, clonidine and moxonidine but not xylazine significantly enhanced the vasoconstriction elicited by 1 mM ATP. The concentration-response curve for NA was shifted to the left slightly by 1 mM agmatine, but not affected by 0.3 microM of other three agonists. Phenoxybenzamine did not affect the vasoconstrictive responses to 1mM ATP and to electrical stimulations, but abolished those to NA. Agmatine at 1mM evoked only an inhibitory effect on electrical stimulation-induced vasoconstriction in the preparation pretreated with phenoxybenzamine, and the inhibitory action was enhanced to 38.6% from the control value (without treatment with phenoxybenzamine) of 22.5%. The non-imidazoline compound xylazine at 0.3 microM lost its inhibitory effect on the neurogenic vasoconstriction in the presence of phenoxybenzamine. In conclusion, agmatine produces a biphasic effect on the purinergic vasoconstriction induced by sympathetic nerve stimulation in the rabbit isolated saphenous artery. The monophasic inhibition of agmatine in the artery treated with phenoxybenzamine is due to an alpha-adrenoceptor-independent mechanism at prejunctional sites, and the potentiation effect of agmatine is mainly dependent on its enhancement of vasoconstriction at postjunctional sites.     

Evidence for the alpha 2 nature of the alpha-adrenergic receptor inhibiting lipolysis in human fat cells

Theophylline-stimulated human subcutaneous adipocytes were incubatged in vitro in the presence of selected alpha-adrenergic agents in order to characterize the alpha-adrenoceptor of human fat cells. Inhibition of theophylline-induced lipolysis occurred with the agents tested; clonidine was the most potent agonist while methoxamine had no effect. The relative order of potency of the various agonists was: clonidine > adrenaline > phenylephrine > methoxamine; this order is consistent with the classification of agonists described for the presynaptic alpha 2-receptor. Moreover, selected antagonists were used in order to antagonise clonidine inhibition of theophylline-induced lipolysis. The order of potency of the alpha-antagonists for the human alpha-adrenoceptor of adipocytes was: yohimbine > piperoxane > phenoxybenzamine > prazosin. This order is consistent with an alpha 2 type receptor. In conclusion, the results demonstrate that the alpha-adrenergic receptor which inhibits lypolysis in human fat cells is of the alpha 2 type. it is noteworthy that although localized postsynaptically this alpha-receptor can be classified as alpha 2 like the commonly known presynaptic alpha-adrenergic receptor which inhibits noradrenaline release from sympathetic neurons.     

In vitro adrenaline and collagen-induced mobilization of platelet calcium and its inhibition by naftopidil, doxazosin and nifedipine

Aims The aim of the study was to obtain further information regarding the modes of action of doxazosin, naftopidil and nifedipine on platelet function.Methods We conducted an in vitro study of drug influences on adrenaline and collagen-induced mobilization of platelet calcium.‘fn2\Results In the presence of fibrinogen (300 μg ml⁻¹ ) both collagen (5 μg ml⁻¹ ) and adrenaline (16 μm ) stimulated the aggregation of washed platelets. Collagen induced a transient rise (+4.97±0.63 μm ) in platelet Ca²⁺ concentration, [Ca²⁺]_i, as measured using the photoprotein aequorin, which coincided with the onset of aggregation. Adrenaline induced a smaller rise (+3.6±0.96 μm ) which, however, occurred after the onset of aggregation. Naftopidil, an α₁-adrenoreceptor antagonist produced a concentration-dependent inhibition of collagen-induced Ca²⁺ mobilization, maximum inhibition (22.9±4%, P<0.05) occurring with 40 μm naftopidil. The inhibition of Ca²⁺ mobilization was not reflected by a concentration-dependent inhibition of platelet aggregation, although 40 μm naftopidil produced statistically significant inhibition (23.3±11.7%, P<0.05). The adrenaline-induced rise in [Ca²⁺]_i was inhibited dose dependently by naftopidil (e.g. 40 μm naftopidil, 100±0%, P<0.05), as was aggregation (40 μm naftopidil, 100±0%, P<0.05). Doxazosin, another α₁-adrenoreceptor blocker, inhibited Ca²⁺ mobilization induced by collagen to similar extents as for naftopidil (30 μm doxazosin, 17.4±2.5%, P<0.05), but did not inhibit platelet aggregation. It also inhibited the adrenaline-induced rise in [Ca²⁺]_i in a concentration-dependent manner (30 μm doxazosin, 37.6±13.7%, P<0.05), significant inhibitions of platelet aggregation also being produced (30 μm, 49.6±17.2%, P<0.05). As expected, the calcium channel blocker nifedipine produced concentration-dependent inhibitions of both collagen-induced Ca²⁺ mobilization (e.g. 28 μm nifedipine, 47.8±2.7%, P<0.05) and aggregation (28 μm, 55.1±9.2%, P<0.05).Conclusions These data indicate that the α₁-adrenoreceptor blockers, naftopidil and doxazosin, inhibit Ca²⁺ mobilization, this mechanism being possibly the means whereby these drugs inhibit platelet aggregation.     

Antinociception mediated by alpha(2)-adrenergic activation involves increasing tumor necrosis factor alpha (TNFalpha) expression and restoring TNFalpha and alpha(2)-adrenergic inhibition of norepinephrine release

The central component that establishes chronic pain from peripheral nerve injury is associated with increased tumor necrosis factor-alpha (TNFalpha) production in the brain. This study examined TNFalpha and its reciprocally permissive role with alpha(2)-adrenergic activation during peak and progressive decline of thermal hyperalgesia in sciatic nerve chronic constriction injury (CCI). Accumulation of TNFalpha mRNA (in situ hybridization) increases in the hippocampus and locus coeruleus during the onset of neuropathic pain and persists as hyperalgesia abates. Activation of alpha(2)-adrenergic receptors in control rats decreases TNFalpha mRNA accumulation in these brain regions. In contrast, during hyperalgesia, alpha(2)-adrenergic activation enhances TNFalpha mRNA accumulation. Whether this enhanced TNFalpha production is associated with changes in the regulation of norepinephrine (NE) release was tested. Hippocampal slices were electrically depolarized to evaluate alpha(2)-adrenergic and TNFalpha regulation of NE release. While inhibition of NE release by TNFalpha is maximal during peak hyperalgesia, it subsequently transforms to facilitate NE release. In addition, alpha(2)-adrenergic receptor activation with clonidine (0.2mg/kg, i.p.) in CCI rats experiencing hyperalgesia restores TNFalpha and alpha(2)-adrenergic inhibition of NE release. While TNFalpha directs the development of hyperalgesia, it also directs its resolution. Transformed sensitivity to alpha(2)-adrenergic agonists during hyperalgesia demonstrates a mechanism for therapy.     

Further evidence for central histamine H2-receptor involvement in the hypotensive effect of clonidine in the rat

In urethane-anaesthetised rats, the administration of the specific histamine H₂-receptor antagonist metiamide intracerebroventricularly (i.c.v.) raised the blood pressure and increased the heart rate. Metiamide (i.c.v.) antagonised the hypotensive effect of clonidine (i.c.v.) in an apparently competitive manner. 4-Methylhistamine i.c.v. did not significantly change the blood pressure. The results are consistent with the concept that the hypotensive effect of clonidine is at least partly due to a stimulation of cerebral H₂-receptors. The existence of cerebral H₂-receptors mediating hypotensive effects is supported by the hypertensive effect of metiamide but not by the lack of hypotensive effects of 4-methylhistamine.     

Fluoride-induced changes in the tooth glycosaminoglycans: an in vivo study in the rabbit

Effect of high fluoride ingestion on the tooth matrix glycosaminoglycans was studied in rabbits administered 10 mg NaF/kg body weight orally at 24-h intervals for a period of 9 months. Fluoride-treated tooth showed a significant reduction in glycosaminoglycan content as compared to the normal tooth. Sephadex G-75 chromatography and DEAE-cellulose ion exchange chromatography revealed the presence of small molecular weight glycosaminoglycan molecules and an increase in the charge density heterogeneity in the sulphated glycosaminoglycans of the fluoride-treated rabbit tooth as compared to the controls. These changes may be related to the dedifferentiated tooth matrix and an increase in the dermatan sulphate content in the fluoride-treated tooth matrix reported earlier.     

Involvement of Ca+ mobilization in the amplifying effect of serotonin on responses of rabbit isolated ear artery to exogenous noradrenaline

Summary The present study examined the involvement of Ca⁺ mobilization in the amplifying effect of serotonin on steady-state responses of rabbit isolated perfused ear artery to exogenous noradrenaline (NA; 0.001–3 ol/l). In contrast to its marked amplifying effect on responses to NA, serotonin in the subconstrictor concentration of 100 nmol/l had no effect on responses to KCl. The Ca⁺-entry blocking drug diltiazem (10 gmol/1) decreased responses to NA; in addition, the amplifying effect of serotonin on responses to NA was reduced by diltiazem. Lowering the concentration of Ca⁺ in the Krebs-Henseleit perfusion solution from 2.5 to 0.25 mmol/l also reduced both responses to NA and the amplifying effect of serotonin. Using the method of Manzini et al. (1982), separate intra- and extracellular Ca⁺-dependent responses to NA were obtained. Serotonin had no effect on intracellular Ca⁺-dependent responses to NA but enhanced extracellular Ca²⁺-dependent NA responses. These results suggest that the amplifying effect of serotonin on responses of rabbit ear artery to exogenous NA involves a selective enhancement of the component of the NA response which is dependent on extracellular Ca⁺; serotonin may increase NA-evoked entry of Ca²⁺ into the vascular smooth muscle cells through receptor-operated Ca⁺ channels. Send offprint requests to A. G. Meehan at the above address     

In vivo inhibition of prostaglandin synthesis in rabbit kidney by non-steroidal anti-inflammatory drugs

To define dose- and time-response properties for in vivo inhibition of renal prostaglandin (PG) synthesis, aspirin, diclofenac sodium, indomethacin and d-naproxen were injected intravenously in different doses to unanaesthetized rabbits. After 30 min. the animals were killed and the post mortem accumulation of PGE2 and PGF2alpha in the renal medulla was determined by mass fragmentography. In control animals, the accumulated levels of PGE2 and PGF2alpha in the medulla were 9.2+/-2.2 (S.D.) and 1.5+/-0.6 microgram/g, respectively. Dose-dependent inhibition was demonstrated with all the drugs. The ED95 was for aspirin 15 mg/kg, for diclofenac sodium 1.5 mg/kg, for indomethacin 1.5 mg/kg and for d-naproxen 5 mg/kg. The duration of inhibition was studied by radioimmunoassay in anaesthetized rabbits by following the urinary excretion of PGF2alpha and PGE2 after an intravenous injection of solvent or test drug in doses twice the ED95. For three hours following aspirin, diclofenac sodium, indomethacin and d-naproxen, the decreases in urinary excretion of PGF2alpha ranged from 64 to 88, 87 to 95, 64 to 90 and from 40 to 77 per cent of the control, respectively, and the decreases in PGE2 excretion were of similar magnitude. Together these results indicate that diclofenac sodium might be used as a long-lasting and potent alternative to indomethacin and aspirin in experimental studies on the renal PG system in vivo.     

Effect of serotonin depletion on 5-HT2A-mediated learning in the rabbit: evidence for constitutive activity of the 5-HT2A receptor in vivo

Rationale Associative learning during Pavlovian eyeblink conditioning has been shown to be regulated by 5-HT_2A receptors. The existence of inverse agonists that retard learning through an action at the 5-HT_2A receptor suggests the existence of constitutive activity at that receptor and that depletion of serotonin should have minimal effects on learning. Objectives We examined whether depletion of serotonin would impair trace eyeblink conditioning or the enhancement of conditioning produced by the agonist lysergic acid diethylamide (LSD) and the retardation of conditioning produced by the inverse agonist MDL11,939. Methods Animals received bilateral intraventricular injections of 5,7-dihydroxytryptamine (5,7-DHT) at doses of 760 or 1,140 μg/side (1.88 or 2.82 μmol/side) and were later exposed to eight daily conditioning sessions. Results Serotonin depletion produced by the lower dose of 5,7-DHT was 71 and 72% in cortex and hippocampus, respectively, with no change in 5-HT_2A receptor density, no effect on learning, and no effect on the ability of LSD to enhance and MDL11,939 to retard learning. The higher dose of 5,7-DHT produced serotonin decreases of 85 and 90% in cortex and hippocampus, respectively, accompanied by a 96% decrease in the density of the serotonin transporter, but no significant effect on learning. Conclusions Pavlovian trace eyeblink conditioning is regulated predominantly by the constitutive activity of the 5-HT_2A receptor rather than by serotonin release onto the receptor during learning. It was suggested that the 5-HT_2A receptor regulates learning by modulating the release of dopamine, acetylcholine, and glutamate, transmitters known to affect eyeblink conditioning.     

Norepinephrine, clonidine, and tricyclic antidepressants selectively stimulate carbohydrate ingestion through noradrenergic system of the paraventricular nucleus

Using a self-selection feeding procedure, the present experiments examined the impact of central and peripheral injection of the alpha-adrenergic agonist clonidine (CLON) and the tricyclic antidepressant drugs amitriptyline (AMIT) and chlorimipramine (CIMIP) on nutrient selection in the adult male rat. In tests with mixed diets or with separate sources of the 3 macronutrients (carbohydrate, protein, and fat) simultaneously available, the following results were obtained: Peripheral and paraventricular nucleus (PVN) injection of CLON stimulated total food intake and preferentially increased ingestion of carbohydrate. Little or no change in protein or fat intake was observed. This pattern of response is similar to that observed with norepinephrine. PVN injection of AMIT and peripheral injection of CIMIP also selectively enhanced carbohydrate intake. These drug effects on carbohydrate selection occurred under a variety of conditions, including with mixed diets and pure dietary nutrients; under ad lib and restricted feeding conditions; in short (1 hr) as well as long (6 hr) test intervals; and in the absence or presence of a change in total calorie intake. Based on this and other evidence, it is proposed that noradrenergic neurons innervating the PVN in the rat play a role in regulating carbohydrate selection, and that this neurochemical system mediates the stimulating action of CLON and antidepressants on carbohydrate ingestion.     

Effects of adrenergic blockers on the inhibition of muricide by desipramine and noradrenaline injected into the amygdala in olfactory bulbectomized rats

Muricide in olfactory bulbectomized rats (OB rats) is readily inhibited by systemic administration of desipramine (DMI) or microinjection of DMI and noradrenaline (NA) into the medial amygdaloid nucleus. The present experiment investigated whether the muricide inhibition produced by these forms of drug treatment was mediated by alpha- or beta-noradrenergic receptors in the central nervous system. Muricide inhibition produced by systemic administration of DMI was antagonized by an alpha-blocker phenoxybenzamine but unaffected by a beta-blocker sotalol, although administration of these adrenergic blockers alone had no effect on muricide. Muricide inhibition induced by the microinjection of DMI and NA into the medial amygdaloid nucleus was similarly antagonized by pretreatment of phenoxybenzamine injected into the same site, but sotalol had no effect. Injection of phenoxybenzamine or sotalol alone into the medial amygdaloid nucleus did not elicit any changes in muricide. These findings suggest that mechanisms mediated by brain noradrenergic alpha-receptor play an important role in muricide inhibition by tricyclic antidepressants in rats and that the medial amygdaloid nucleus is an important site of action of these drugs.     

Characterization of postsynaptic alpha1-adrenoceptors in the rabbit iris dilator smooth muscle

Summary Interactions of several alpha-adrenoceptor agonists and antagonists with their receptors were studied in rabbit and guinea pig iris dilator smooth muscle and rabbit aortic strips using pharmacological procedures.In rabbit iris dilators and aortic strips, noradrenaline acted as a full agonist, while oxymetazoline, clonidine and tizanidine acted as partial agonists. The dissociation constants of full and partial agonists in the dilators, calculated after irreversible blockade of a proportion of the active receptors with phenoxybenzamine, were similar to those in the aortic strips. Furthermore, the relative intrinsic efficacies of partial agonists were practically equal in the two tissues, suggesting that these drugs act on the same alpha-adrenoceptors. Since the alpha₂-agonists clonidine and tizanidine had low affinity in the rabbit dilators, the alpha-adrenoceptors in this tissue appear to be of alpha₁-type. These results were further supported by the fact that the pA₂-value of prazosin, an alpha₁-antagonist, was approximately 2 log units higher than that of yohimbine, an alpha₂-antagonist.However, pA₂-values of four quinazolines (prazosin, bunazosin, SM911 and SM2470) and two yohimbine alkaloids (yohimbine and corynanthine) were significantly lower in the rabbit dilator muscle than in rabbit aortic strips. Two imidazoline antagonists (phentolamine and tolazoline) and a phenethanolamine (labetalol) acted on the alpha₁-adrenoceptors in the two tissues nonselectively. These results suggest that alpha₁-adrenoceptors in the rabbit dilator muscle and aortic strips may not be identical and that both selective and nonselective antagonists which act on these receptor sites exist. The pA₂-values of prazosin, SM911 and SM2470 against noradrenaline in guinea pig iris dilator smooth muscle were also significantly lower than those in the rabbit aortic strips, but practically equal to those in the rabbit iris dilators, suggesting that the alpha₁-adrenoceptors in the dilators are the same in these two species.     

Potentiation by yohimbine of alpha-adrenoceptor-mediated vasoconstriction in response to clonidine in the rabbit ear vein

We investigated the pharmacological profile of the vasoconstrictive response to clonidine in the isolated rabbit ear vein, and compared the characteristics of clonidine with those of noradrenaline and moxonidine. The maximal vasoconstrictive responses to clonidine and moxonidine in the rabbit ear vein were 35.94+/-11.18% and 88.78+/-11.54% of the maximum response to noradrenaline, respectively. Prazosin 0.1 microM inhibited the vasoconstriction induced by lower concentrations of noradrenaline, and the concentration-dependent response curve for noradrenaline was significantly shifted to the right by 1 microM prazosin. Yohimbine (0.1 and 0.5 microM) only decreased the vasoconstrictive response to lower concentrations of noradrenaline, but did not affect the response to higher concentrations. Vasoconstrictive responses to lower but not higher concentrations of clonidine and moxonidine were inhibited by 0.1 microM yohimbine. In contrast, the same concentration of yohimbine significantly potentiated the maximal response to a high concentration of clonidine by 24.06%. In isolated rabbit ear vein pretreated with 0.1 microM yohimbine, prazosin competitively inhibited the concentration-response curve for clonidine with a pA(2) value of 8.05+/-0.06. We conclude that clonidine acts mainly on alpha(2)-adrenoceptors to produce vasoconstriction in the rabbit ear vein; however, in the preparation pretreated with yohimbine, the clonidine-induced vasoconstriction is mediated via alpha(1)-adrenoceptors and its maximal vasoconstriction is significantly potentiated.     

In vivo tonic modulation of the noradrenaline release in the rat cortex by locus coeruleus somatodendritic alpha(2)-adrenoceptors

The regulation of noradrenaline release in the rat cingulate cortex by somatodendritic alpha(2)-adrenoceptors placed in the locus coeruleus was evaluated by dual-probe microdialysis. The alpha(2)-adrenoceptor antagonists BRL44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole), RS79948 ((8,12,13)-decahydro-3methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]-naphthyridine) and RX821002 (2-methoxyidazoxan) administered by reverse dialysis into the locus coeruleus increased concentration-dependently (0.01-100 microM) noradrenaline release in the cortex (maximal effects 170+/-30%, 543+/-17%, 195+/-26%, respectively). Administration of the alpha(2)-adrenoceptor antagonist idazoxan increased at lower (0.1-10 microM) but decreased at the highest dose (100 microM) noradrenaline in the cortex. These data demonstrate that somatodendritic alpha(2)-adrenoceptors in the locus coeruleus exert an inhibitory tonic modulation on noradrenaline release in noradrenergic terminal areas.     

Pharmacological evidence that alpha2A- and alpha2C-adrenoceptors mediate the inhibition of cardioaccelerator sympathetic outflow in pithed rats

It has been suggested that the alpha(2)-adrenoceptors mediating cardiac sympatho-inhibition in pithed rats closely resemble the pharmacological profile of the alpha(2A)-adrenoceptor subtype. However, several lines of evidence suggest that more than one subtype may be involved. Thus, the present study has pharmacologically re-evaluated the receptor subtype(s) involved in the inhibitory effect of the alpha(2)-adrenoceptor agonist, B-HT 933, on the tachycardic responses elicited by selective cardiac sympathetic stimulation (0.03, 0.1, 0.3, 1 and 3 Hz) in desipramine-pretreated pithed rats. I.v. continuous infusions of B-HT 933 (30 microg/kg min), which failed to modify the tachycardic responses to exogenous noradrenaline, inhibited those induced by preganglionic (C(7)-T(1)) stimulation of the cardiac sympathetic outflow at all frequencies of stimulation (0.03-3 Hz). This cardiac sympatho-inhibitory response to B-HT 933 was: (1) unaltered by saline (1 ml/kg) or the antagonists BRL44408 (100 microg/kg; alpha(2A)) or imiloxan (3000 and 10,000 microg/kg; alpha(2B)); (2) partially antagonized by BRL44408 (300 microg/kg) or MK912 (10 microg/kg; alpha(2C)) given separately; and (3) completely antagonized by rauwolscine (300 microg/kg; alpha(2)), MK912 (30 microg/kg) or the combination of BRL44408 (300 microg/kg) plus MK912 (10 microg/kg). Moreover, the above doses of antagonists, which are high enough to block their respective receptors, failed to block per se the tachycardic responses to sympathetic stimulation. These results suggest that the cardiac sympatho-inhibition induced by B-HT 933 in pithed rats is mainly mediated by stimulation of alpha(2A)- and alpha(2C)-adrenoceptors.     

Stereospecific inhibition by ozolinone of stimulated chloride secretion in rabbit colon descendens

Summary The effects of the diuretic drug ozolinone on electrogenic Cl^– secretion by rabbit colonic mucosa were investigated in vitro. Electrical properties and unidirectional Cl^– fluxes were measured in stripped preparations mounted in Ussing-type chambers. After abolition of electrogenic Na⁺ absorption by amiloride (10^–4 mol/l) on the mucosal side electrogenic Cl^– secretion was induced by addition of PGE₁ (10^–6mol/l, serosal side) and theophylline (10^–2mol/l, both sides). Under these conditions, the monitored short-circuit current (I_sc) equals the amount of Cl^– secreted as evidenced by determination of unidirectional Cl^– fluxes. After establishing a stable Cl^– secretion its sensitivity to the enantiomers of the diuretic was studied. Only levorotatory (-)-ozolinone, but not the dextrorotatory (+)form, inhibited Cl^– secretion on serosal application. This effect was fully accounted for by a reduction in the serosal-to-mucosal Cl^– fluxes (J _sm ^Cl ). It was readily reversible and concentration-dependent with a K _i value of 6×10^–4mol/l, but absent when the drug was added to the mucosal side. The results are in agreement with the hypothesis that loop diuretics inhibit a coupled NaCl entry mechanism across the baso-lateral membrane into colonic epithelial cells. This mechanism is though to account for Cl^– influx into the cells.     

In vivo diminution by chelators of snake venom-provoked hemorrhage and in vitro inhibition of proteolytic activity

Topical application of alcohol-soluble metal-complexing agents, particularly α-mercapto-β-(2-furyl)acrylic acid (MFA)1, diminished the severity of hemorrhages caused by s.c. injection to mice of thirteen hemorrhagic pit viper venoms and two viper venoms. Metal dependency of the alkaline protease activity ranged from 0 to 100% for these venoms, plus one non-hemorrhagic pit viper venom; MFA completely inhibited this activity in six pit viper and two viper venoms. The presence of zinc-dependent proteases in venoms of Crotalus atrox and Bitis arietans was indicated. Less compelling evidence suggested the presence of zinc metalloproteases in at least 11 of 14 other venoms. The potencies of hemorrhagic and alkaline protease activities were not related in the venoms and we did not investigate if metalloproteases were the targets of chelator antagonism of hemorrhage development. Topically active chelators may be useful as adjunctive therapy in some snake envenomations.