Effects of chronic bretylium treatment on the sympathetic neuron and the smooth musculature of the rat

The effects of chronic i.p. injection of high doses of bretylium on sympathetic nerves on the smooth musculature of the vas deferens of adult and newborn rats were examined using fluorescence histochemistry, light and electron microscopy and organ bath physiological techniques. Bretylium treatment caused mitochondrial swelling, loss of cristae and the formation of electron-dense inclusions in the mitochondria of sympathetic neurons. However, neuron degeneration was not observed and fluorescent histochemical appearance of adrenergic neurons was normal. A small transient supersensitivity of the isolated vas deferens of bretylium-treated rats to noradrenaline, but not to acetylcholine, occurred. There was, however, considerable increase in the maximal contractile response to both noradrenaline and acetylcholine. In high calcium concentrations acetylcholine-induced contractions of vasa deferentia from bretylium-treated rats were significantly greater than control; there was no difference in magnitude of noradrenaline-induced contractions.     

Buthus martensi karsch venom: prejunctional adrenergic activity in the rat isolated anococcygeus muscle

The effect of Buthus martensi Karsch venom (MKV) on adrenergic responses was investigated using the rat isolated anococcygeus muscle (Acm), since several scorpion venoms can cause peripheral sympathetic nerve stimulation with enhanced adrenergic responses. The effects of phentolamine (5 microM), guanethidine (5 microM), tetrodotoxin (2 microM), desipramine (1.5 microM) and reserpine pretreatment in vivo (5 mg/kg s.c. x 24 hr and 5 mg/kg i.p. x 3 hr) on contractile responses of the rat Acm to field stimulation, noradrenaline (3 microM), tyramine (10 microM), crude MKV (2 micrograms/ml), carbachol (3 microM) and potassium chloride (50 mM) were compared. Phentolamine, guanethidine, tetrodotoxin and reserpine pretreatment completely blocked the contractile responses of the Acm to MKV and to field stimulation but desipramine potentiated the responses. The responses to NA were completely blocked by phentolamine, but were potentiated by guanethidine, desipramine and reserpine pretreatment. The contractile responses to tyramine were completely blocked by phentolamine, desipramine and reserpine pretreatment. The low doses (0.1 microgram/ml x 3) of MKV, which did not produce any observable increase in tone of the anococcygeus muscle, potentiated the contractile responses to field stimulation, but not the responses to exogenous NA. Thus, the adrenergic agonist action of MKV in the rat isolated anococcygeus muscle is mediated by some prejunctional mechanism(s) of action, presumably stimulating the release of the neurotransmitter noradrenaline.     

The effects of denervation, cocaine, 6-hydroxydopamine and reserpine on the characteristics of drug-induced contractions of the depolarized smooth muscle of the rat and guinea-pig vas deferens

Previous studies have suggested that postjunctional supersensitivity of the vas deferens is due in part to altered electrophysiological properties, the sensitivity of the muscle being increased to any agonist which initiates contraction by means of depolarizing the cell membrane. Results of the present study indicate that altered electrical properties are not the only postjunctional changes which can account for the enhanced response. Dose-response curves for stimulant agonists were obtained in isolated vasa deferentia which were depolarized by a K-rich, Na-free solution. Chronic denervation resulted in a 2- to 3-fold displacement of the dose-response curve for norepinephrine to the left of control. Cocaine (10-(5)M) did not potentiate the response to norepinephrine of the innervated, depolarized smooth muscle. Supporting the contention that the supersensitivity of the depolarized tissue is postjunctional in nature was the finding that the denervated vas deferens was supersensitive to methoxamine, an agent which is not taken up by the neuronal amine transport system. Pretreatment of rats with reserpine (1.0 mg/kg/day for 5-7 days) also produced supersensitivity of the depolarized vas deferens. The increased maximal response to drugs of the denervated rat vas deferens which is observed in normally polarized tissues is absent in depolarized tissues suggesting that the phenomenon of increased maximum requires the existence of a membrane potential in order to be manifest. The denervated vas deferens, but not the vas deferens from reserpine-pretreated animals, exhibits an increase in the duration of drug-induced contractions. This effect occurs in both normal and depolarizing salt solutions suggesting that the change which leads to this phenomenon differs from those alterations which lead to postjunctional supersensitivity and to the enhanced maximal response.     

Poly(ethylene glycol) on the liposome surface: on the mechanism of polymer-coated liposome longevity

The mode of action of muramyl dipeptide (MDP), a compound with immunopharmacological properties, was studied in isolated nerve smooth muscle preparations with different receptor systems. The amplitudes of contractions evoked directly by stimulants as well as neurogenic twitches or relaxations were registered. In the rat stomach strip EC50 of acetylcholine, serotonin (5-HT) and KCl was estimated. MDP (50 nmol/l) but not levamisole potentiated selectively the contractions evoked by 5-HT and significantly (p < 0.01) lowered the respective EC50. In the rat vas deferens MDP selectively potentiated the twitches enhanced by 5-HT but not those enhanced by noradrenaline. Such potentiation was blocked by 5-HT3 antagonists tropisetron and MDL 72,222 (1 alpha H,3 alpha,5 alpha-H-tropan-3-yl 3,5-dichlorobenzoate) but not by the 5-HT2 antagonist ketanserin. The antagonist methiothepin nonselectively abolished the potentiation by MDP as well as the enhancement of twitches by 5-HT and noradrenaline, whereas l-propranolol and isamoltan influenced neither the enhancement of twitches by 5-HT nor the potentiation by MDP. In the isolated longitudinal muscle of guinea pig proximal colon, 5-HT caused a biphasic response in the presence of atropine; the initial neurogenic relaxation was potentiated in the presence of MDP and was suppressed in the presence of tropisetron. Thus, the potentiating effect of MDP in the isolated organs studied was selective with respect to the serotoninergic system and might be mediated by 5-HT3 receptors.     

An analysis of the postjunctional component of denervation supersensitivity in the isolated vas deferens of the guinea-pig

An attempt was made to devise evidences for a role of calcium ions in the postjunctional component of denervation supersensitivity. The evidences obtained are: chronic postganglionic denervation increases sensitivity and maximum response of the vas deferens to cumulative concentrations of calcium and alters the pattern of response to low-calcium, potassium-rich Krebs solution; denervation supersensitivity could not be demonstrated after depolarization, and in KC1-Ringer or in Ca2+/--free Krebs solution the rate of loss of responsiveness to acetylcholine was delayed after denervation whereas the rate of loss of responsiveness to noradrenaline was unaffected. It is suggested that the postjunctional component of denervation supersensitivity in the isolated vas deferens of the guinea-pig is due, at least partially, to an increased availability of a membrane-bound calcium store(s) associated to an enhanced cell membrane permeability to the ion.     

Homogeneous or heterogeneous distribution of systemically administered adrenaline: organ dependence

In incubation experiments it was shown that exogenous adrenaline or noradrenaline does not distribute homogeneously into the adrenergic varicosities of the rat vas deferens (wall with thick and compact muscle layer) but does distribute homogeneously in the rat spleen capsule (thin and loose muscle layer, containing more extracellular space than the vas deferens). To circumvent any hypothetical role of the muscular layer in the distribution of the amine, 100 micrograms.kg-1.h-1 adrenaline was administered to rats in vivo either i.v. (during 90 min) or i.p. (under pentobarbital anaesthesia, an Alzet minipump was implanted which delivered that dose during 6 days). The rats also received 100 mg.kg-1 pargyline (to inhibit MAO) and 100 mg.kg-1 tropolone (to inhibit COMT). At the end of adrenaline administration, vasa deferentia and spleen capsule were removed, washed and then exposed to 100 mumol.l-1 tyramine for 20 min. At the end of this exposure, the ratio noradrenaline/adrenaline in the tissue and in the medium was compared. In the vas deferens both after i.v. and i.p. administration of adrenaline, the ratio noradrenaline/adrenaline was about 3 times higher in the medium than in the tissue, while in the spleen capsule the ratio noradrenaline/adrenaline was not significantly different in the medium and in the tissue. We conclude that, even when the amine reaches the storage sites from the blood, it distributes homogeneously in the spleen capsule and heterogeneously in the vas deferens, perhaps because there are more than one kind of storage vesicles in the vas deferens.     

Defining the precapillary sphincter

Influence of denervation on phospholipid metabolism in the vas deferens and effect of phospholipase C treatment on sensitivity of the vas deferens for noradrenaline were studied. The incorporation of 32P-orthophosphate into phosphatidylethanolamine and phosphatidylcholine was markedly increased by denervation. Incorporation of 32P-orthophosphate into proteolipid was accelerated more than that of 3H-leucine. Sensitivity of the denervated vas deferens for noradrenaline was strongly reduced by phospholipase C treatment. These data suggest that the supersensitivity of the denervated vas deferens for noradrenaline was mainly due to the increase in phospholipids.     

Prevention by calcium administration of reserpine action on rat brain noradrenaline stores: a reappraisal

The conditions under which pretreatment with a calcium salt may prevent the action of reserpine on brain noradrenaline stores in the rat were investigated. The results show that only after subcutaneous administration of reserpine in the same site as a previous CaCl2 injection, was the action of reserpine prevented and reduced levels of this drug were found in the brain. Conversely, the depletion of encephalic noradrenaline following reserpine, as well as the reserpine brain concentration, were not affected by subcutaneously administered calcium chloride, when reserpine was administered either intravenously, or subcutaneously in a site different from that selected for pretreatment with the calcium salt. In essence calcium chloride, a well known irritant, acts accordingly at the site of subcutaneous administration, thus limiting by a non specific mechanism the absorption of reserpine. Under similar conditions, in fact, the absorption of a different drug, i.e. harmaline, was likewise altered. In view of these findings the significance of some studies on calcium-reserpine interaction appearing in the literature requires a reappraisal.     

KCl contractions in the rat intact and bisected vas deferens: contribution of endogenous noradrenaline release

1. KCl produced a biphasic contraction in the intact rat vas deferens. Both components were larger and the initial rapid phasic component was faster in the prostatic portion than the epididymal portion. In some experiments the epididymal phasic response was a single slow contraction, while in others it had a mixture of fast and slow responses. 2. Phentolamine reduced the phasic response but not the tonic response of the intact vas deferens. This effect was not observed after denervation produced by chronic guanethidine treatment. 3. Both phases of the response to KCl 160 mmol/l were substantially reduced by phentolamine in the epididymal portion. In the prostatic portion phentolamine produced only slight inhibition of the phasic component and had no effect on the tonic component. 4. Isoprenaline had no effect on the response to KCl 160 mmol/l but reduced both phases of the response to KCl 50 mmol/l. This effect was antagonized by propranolol. 5. It is concluded that part of the phasic component of the response to KCl in the rat vas deferens is due to the release of noradrenaline from intramural nerves.     

Activation of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase in rat vas deferens and distal colon is not accompanied by inhibition of contraction

There is good evidence that in vascular smooth muscle, the relaxant effects of sodium nitroprusside (SNP) are mediated by increases in cGMP levels and activation of cGMP-dependent protein kinase (PKG). However, in rat vas deferens and rat distal colon, cGMP-elevating agents such as SNP and atrial natriuretic factor (ANF) have been shown to elevate cGMP without inducing relaxation. The lack of relaxation might be explained by either lack of activation of PKG by these agents or low levels of PKG in these tissues. The object of the present study was to investigate these possibilities by simultaneously monitoring cGMP levels, PKG activity and contractility in isolated strips of rat vas deferens, rat proximal colon and distal colon exposed to high concentrations of SNP or ANF. Verification of the specificity of the assay for PKG was obtained using MonoQ chromatography to resolve soluble smooth muscle extracts, followed by immunoblotting with a PKG-specific antibody to identify the kinase. In rat vas deferens, 5 mM SNP increased cGMP levels (14-fold) and PKG activity ratios (3.4-fold) but did not inhibit phenylephrine-induced contractions. In both rat proximal and rat distal colon, 100 nM ANF significantly elevated cGMP levels and PKG activity ratios, but only in the proximal colon was inhibition of spontaneous contractions observed. Total PKG activity was much lower (approximately 16 pmol PO4/min/mg protein) in rat vas deferens, which was not relaxed by SNP, than in rabbit aorta (approximately 148 pmol PO4/min/mg), which was relaxed. However, in the rat proximal colon, despite low PKG levels (approximately 11 pmole/min/mg), ANF did inhibit contractions. Thus the inability of the cGMP-elevating agents SNP and ANF to inhibit contractions in rat vas deferens and rat distal colon cannot be explained by either of the possibilities suggested above.     

Prejunctionally mediated inhibition of neurotransmission by isoprenaline in rat vas deferens

Effects of isoprenaline on monophasic contractions evoked by electric field stimulation were studied in rat isolated prostatic vas deferens. Isoprenaline reduced electrically evoked contractions (EC50: 0.27 +/- 0.05 microM), and propranolol concentration-dependently antagonized the effect of isoprenaline. In contrast, isoprenaline (0.3-3 microM) did not affect the contractile response induced by exogenous noradrenaline or ATP, while forskolin (100 nM) attenuated agonist-induced contraction. In some tissues, adrenergic and purinergic components of the electrically evoked contraction were isolated by exposure to alpha,beta-methylene ATP (3 microM) and prazosin (3 microM), respectively. Isoprenaline induced a greater inhibition of purinergic than adrenergic component of the electrically evoked contraction. Iberiotoxin (50 nM), glibenclamide (3 microM), 4-aminopyridine (0.3 mM) and tetraethylammonium ions (1 mM) attenuated the effect of isoprenaline. These results indicate that isoprenaline-induced inhibition of the electrically evoked (both purinergic and adrenergic) contraction was mediated primarily through activation of prejunctional beta-adrenoceptors, which probably inhibited release of contractile transmitters from sympathetic nerves supplying vas deferens. Lack of effect of isoprenaline on agonist-induced contraction does not favour a functional role of beta-adrenoceptors in vas smooth muscle.     

Evidence for participation of nitric oxide in excitatory neurotransmission in rat vas deferens

A depression of the fast, non-adrenergic, and also of the slow, adrenergic, components of muscle contraction in response to intramural nerve stimulation was induced by the blocker of nitric oxide synthase NG-nitro-L-arginine methyl ester (L-NAME), in rat vas deferens. Effects of exogenous noradrenaline or ATP were not reduced by L-NAME. However, L-arginine also caused an inhibition of electrically induced effects in most of the preparations, contrary to the expectations for a precursor of nitric oxide synthesis. In spite of these difficulties L-arginine antagonized the action of L-NAME. These results indicate that nitric oxide is involved in excitatory nerve-muscle transmission in vas deferens.     

Comparative study of the effects of clozapine and clothiapine in different preparations of guinea pig and rat isolated organs

1. A study has been made to know the effects of clozapine and clothiapine on the responses of rat isolated vas deferens to norepinephrine, dopamine and potassium, those of the rat isolated uterus to serotonin and potassium, and that of guinea pig isolated ileum to histamine. 2. Clozapine was a noncompetitive antagonist to norepinephrine, dopamine, serotonin and histamine; it inhibited potassium-induced contraction in isolated rat uterus and vas deferens. 3. Clothiapine was a competitive antagonist to serotonin and a noncompetitive antagonist to norepinephrine, dopamine and histamine; it inhibited potassium-induced contractions in isolated rat uterus and vas deferens.     

Linear-programming method for obtaining effective cluster interactions in alloys from total-energy calculations: Application to the fcc Pd-V system

Vasa deferentia taken from mice treated with delta 9-tetrahydrocannabinol (20 mg/kg i.p., once daily for 2 days) showed tolerance to the inhibitory effect of the cannabinoid, R-(+)-arachidonyl-1'-hydroxy-2'-propylamide, on electrically evoked twitches. This treatment did not induce tolerance to the inhibitory effects on the twitch response of morphine or clonidine or of selective mu-, delta- or kappa-opioid receptor agonists. Nor did it affect the contractile potencies of noradrenaline or beta,gamma-methylene-L-ATP. We suggest that cannabinoid tolerance in the vas deferens is attributable neither to downregulation of opioid receptors or alpha 2-adrenoceptors nor to an increased sensitivity of this tissue to its main contractile transmitters noradrenaline and ATP. A concentration of delta 9-tetrahydrocannabinol that inhibits electrically evoked twitches of the vas deferens (100 nM) did not alter the ability of noradrenaline or beta,gamma-methylene-L-ATP to induce contractions suggesting that delta 9-tetrahydrocannabinol inhibits the twitch response by acting prejunctionally.     

Geometry, kinetics and plasticity of release and clearance of ATP and noradrenaline as sympathetic cotransmitters: roles for the neurogenic contraction

The paper compares the microphysiology of sympathetic neuromuscular transmission in three model preparations: the guinea-pig and mouse vas deferens and rat tail artery. The first section describes the quantal release of ATP and noradrenaline from individual sites. The data are proposed to support a string model in which: (i) most sites (> or = 99%) ignore the nerve impulse and a few (< or = 1%) release a single quantum of ATP and noradrenaline; (ii) the probability of monoquantal release is extremely non-uniform; (iii) high probability varicosities form 'active' strings; and (iv) an impulse train causes repeated quantal release from these sites. Analogy with molecular mechanisms regulating transmitter exocytosis in other systems is proposed to imply that coincidence of at least two factors at the active zone, Ca2+ and specific cytosolic protein(s), may be required to remove a 'fusion clamp', form a 'fusion complex' and trigger exocytosis of a sympathetic transmitter quantum, and that the availability of these proteins may regulate the release probability. The second section shows that clearance of noradrenaline in rat tail artery is basically > or = 30-fold slower than of co-released ATP, and that saturation of local reuptake and binding to local buffering sites maintain the noradrenaline concentration at the receptors, in spite of a profound decline in per pulse release during high frequency trains. The third section describes differences in the strategies by which mouse vas deferens and rat tail artery use ATP and noradrenaline to trigger and maintain the neurogenic contraction.     

Somatostatin-induced inhibition of neurotransmission in the mouse isolated vas deferens is resistant to pertussis toxin

The potential effects of pertussis toxin pretreatment on the inhibitory effect of somatostatin (SRIF) and the selective SRIF receptor agonist, seglitide, were studied in mouse vas deferens and these were compared with its effect on the negative chronotropic action of carbachol in mouse atria. Somatostatin and seglitide caused a concentration-dependent inhibition of neurogenically mediated contractile responses in the vas deferens (EC50 values of 15 and 0.6 nM respectively). There was no difference in their potencies in preparations removed from pertussis toxin pretreated mice. In contrast, the negative chronotropic action of carbachol in mouse atria was abolished by pretreatment with pertussis toxin. We conclude that, in contrast to muscarinic receptor activation in mouse atria, the inhibitory effect of somatostatin in the vas deferens is not mediated by a pertussis toxin sensitive G-protein. The high potency of seglitide suggests that the SRIF receptor involved is of the SRIF1 type.     

Alpha 1-adrenoceptors and calcium sources in adrenergic neurogenic contractions of rat vas deferens

1. The involvement of alpha 1-adrenoceptor subtypes in adrenergic neurogenic contractions of different type was studied in epididymal and prostatic portions of the rat vas deferens. 2. The adrenergic component of neurogenic contractions was isolated by suramin (300 microM). Twitch-like and tonic contractions were elicited by appropriate pulse patterns of electrical field stimulation, and contractions relying on intracellular calcium mobilization and calcium entry were isolated by means of nifedipine (10 microM) and ryanodine (20 microM), respectively. Increasing concentrations of 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)- amino)-propyl)benzeneacetonitrile (HV 723), prazosin and 5-methylurapidil progressively, monophasically and with potency decreasing in that order reduced and finally abolished all types of contraction, with one exception: concentration-effect curves of 5-methylurapidil in epididymal segments in the presence of ryanodine levelled off at about 75% inhibition. In the presence of both nifedipine (10 microM) and ryanodine (20 microM), contractions were abolished. 3. Contractions elicited by exogenous noradrenaline were also studied in the presence of either nifedipine 10 microM (prostatic segments) or ryanodine 20 microM (epididymal segments). Increasing concentrations of tamsulosin, WB 4101, benoxathian, HV 723, prazosin, 5-methylurapidil and urapidil progressively, monophasically and with potency decreasing in that order reduced and eventually abolished both kinds of contraction, with two exceptions: in epididymal segments in the presence of ryanodine, the concentration-effect curve of 5-methylurapidil was biphasic and the curve of urapidil levelled off at only partial inhibition. 4. In slices prepared from the prostatic end and preincubated with [3H]-noradrenaline, WB 4101, HV 723, prazosin and 5-methylurapidil, at the highest concentrations tested against neurogenic contractions, increased only slightly the overflow of tritium elicited by trains of 50 pulses at 5 Hz. 5. It is concluded that two alpha l-adrenoceptor subtypes mediate adrenergic neurogenic contractions of rat vas deferens. The main one, pharmacologically alpha 1A, activates both calcium mobilization and entry. In addition there is a second receptor, not previously detected in the vas deferens and not corresponding to any named alpha l subtype, characterized by high and similar affinity for tamsulosin, WB 4101, benoxathian,HV 723 and prazosin and very low affinity for 5-methylurapidil and urapidil, and linked exclusively to calcium entry. Both subtypes and their respective transduction pathways also contribute to contractions elicited by exogenous noradrenaline. An alpha 1B-adrenoceptor-mediated contraction was not found under any experimental conditions.     

Desensitization of muscarinic receptor-coupled inositol phospholipid hydrolysis in human detrusor cultured smooth cells

PURPOSE: The aim of this study was to investigate the desensitization characteristics of muscarinic M3 receptors in primary cultures of human detrusor smooth muscle cells. MATERIALS AND METHODS: Cell cultures were prepared from cold cup pinch biopsies of the human detrusor muscles by explant culture methods. Accumulation of (3)H-inositol phosphates was measured on confluent monolayers as described previously in detail. Desensitization was achieved by preincubating the cells with carbachol or histamine for 5 to 60 minutes. RESULTS: Carbachol induced a concentration-dependent increase in phosphoinositide turnover in naive cells, the response being rapid and evident after only a 30-second exposure to the agonist. Preincubation of the cells with carbachol produced a concentration-dependent decrease in the inositol phosphate response to a second challenge with carbachol. Preexposure to carbachol for only 5 minutes prior to a rechallenge reduced the mean size of response of the second stimulation to 49% of that observed in naive cells. Preexposure of the cells to histamine did not alter the response of the cells to a subsequent challenge with carbachol and vice versa. CONCLUSIONS: The muscarinic receptors retained by human detrusor smooth muscle cells in culture are susceptible to a desensitization of the carbachol-induced increase phosphoinositide turnover observed in these cells. This desensitization is rapid, and the results indicate that it is homologous and does not occur via a postreceptor mechanism but at the level of the receptor itself.