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Uterine secretory cells receive a sympathetic cholinergic secremotor innervation. Nitric oxide (NO) has been suggested to be a second messenger of neurogenic modulated glandular secretion of the seminal vesicle. Thus a similar pattern for nervous induced carbohydrate secretion of the endometrium was assumed. The nitric oxide synthase (NOS) activity was estimated via formation of L-citrulline from L-arginine and histochemically with the nicotinamide-adenine dinucleotide phosphate diaphorase (NADPH-d) nitro blue technique. The carbohydrate secretion from everted uterine horns placed in organ baths was estimated. A calcium dependent formation of citrulline was found in the uterine horn suggesting an NOS activity. Strong NADPH staining cells were found in the glandular ducts of the endometrium and in the epithelial linings of the oviduct. Carbachol induced carbohydrate secretion of the endometrium while N-nitro L-arginin (L-NNA) and N-nitro L-arginin methyl ester (L-NAME) inhibited the carbachol induced secretion. The isomer D-NAME had no effect on carbachol induced secretion. When L-arginine was administered together with L-NNA no inhibitory effect on carbachol induced secretion was seen. L-arginine only had no effect on carbohydrate secretion. The NO donor glyceryl tritrate increased carbohydrate secretion but no synergistic effect was seen in combination with carbachol. The results suggest that glandular NO production is a prerequisite for muscarinic carbohydrate secretion of the endometrium.

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The presence of NADPH-diaphorase activity and acetylcholinesterase in the testis, epididymis, vas deferens, seminal vesicle, pelvic plexus, prostate and urethra of man and guinea-pig was investigated with the nitro blue NADPH technique and the thiocholine method, respectively. In human material NADPH-diaphorase activity was found in the Leydig cells, Sertoli cells and the epithelial linings of the rete testis, the excretory ducts, seminal vesicle, prostate and urethra. The guinea-pig material showed staining of the Leydig cells and spermatozoa and similar epithelial staining of the tract as man. Nerves beneath the epithelium and in the muscle layers of cauda epididymis, vas deferens, seminal vesicle, prostate and urethra were also stained. NADPH-diaphorase-positive nerve cells were seen in the pelvic plexus. Some cells also displayed acetylcholinesterase activity but others showed activity for only one of the enzymes or no activity for either enzyme. In the cauda epididymis, vas deferens, seminal vesicle, prostate and urethra acetylcholinesterase-positive nerve fibres formed a plexus beneath the secretory cells. It is concluded that NADPH-diaphorase, generally accepted as a nitric oxide synthase, is present in glandular cells of the male genital tract. The enzyme is also present in nerves, where it is partly co-localized with acetylcholinesterase.

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The significance of nitric oxide (NO) formation in seminal secretion was studied in guinea-pig seminal vesicles. The nitric oxide synthase (NOS) activity was estimated and reduced nicotinamide-adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry was performed. Furthermore, cyclic guanosine 3,5-monophosphate (cGMP) concentration as well as fructose secretion from isolated vesicles was estimated. High Ca2+-dependent NOS activity as well as prominent glandular NADPH-diaphorase staining was found in the secretory epithelium. The NOS inhibitors N(G)-nitro L-arginine methyl ester (L-NAME) and N(G)-nitro L-arginine (L-NNA) inhibited carbachol-induced fructose secretion but the D-isomer to L-NAME had no effect. When L-arginine was administered together with L-NAME, no inhibitory effect on the carbachol-induced fructose secretion could be seen. Nerve-induced fructose secretion was also inhibited by L-NAME. The NO donor glyceryl trinitrate (GTN) increased the fructose secretion. Carbachol or GTN did not increase cGMP levels, nor was fructose secretion inhibited by a guanylate cyclase inhibitor (ODQ). Our results suggests that glandular NO production is a prerequisite for muscarinic fructose secretion in the seminal vesicle via a cGMP-independent pathway.

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While the crucial role of neurally produced nitric oxide in mediating penile erection is well established, the understanding of the peripheral neuroanatomy of the nitric oxide-ergic pathways is still incomplete. This study was designed to elucidate further the distribution of nitric oxide synthase, and its relation to the distribution of neuropeptides and tyrosine hydroxylase in all penis-projecting neural pathways. A triple-labelling technique was employed, with the retrograde tracer Fluoro Gold combined with neuropeptide immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, a marker of nitric oxide synthase. The presence within the penis of scattered nerve cell bodies exhibiting NADPH-diaphorase activity was revealed. Most (76%) of the penis-projecting neurons in the major pelvic ganglion exhibited NADPH-diaphorase activity and immunoreactivity to vasoactive intestinal peptide, while none of them contained tyrosine hydroxylase. Sympathetic paravertebral postganglionic neurons, in turn, contained tyrosine hydroxylase, but did not exhibit NADPH-diaphorase activity. In the afferent, sensory neurons projecting to the penis from the dorsal root ganglia, NADPH-diaphorase activity coexisted with immunoreactivity to both substance P (8%) and calcitonin gene-related peptide (26%). Preganglionic neurons originating in the spinal cord intermediolateral column at the thoracolumbar level T11-L3 terminated, not only in the major pelvic ganglion, but also within the penis. The majority (81%) of the penis-projecting neurons exhibited NADPH-diaphorase activity. The results indicate that the rat penis receives several different nitric oxide-ergic neural projections. It is therefore possible that nitric oxide affects penile erection at several neuronal levels.

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Fructose secretion of everted guinea-pig seminal vesicles was studied in vitro. Carbachol produced dose dependent increase in fructose secretion. The effect was blocked by scopolamine but not by hexamethonium, mecamylamine, tetrodotoxin or previous denervation. High concentrations of acetylcholine also increased fructose secretion. This response was not augmented by physostigmine. Methoxamine reduced secretion. Methoxamine, terbutaline, clonidine and vasoactive intestinal peptide counteracted carbachol. Field stimulation produced increased secretion that was not blocked by autonomic drugs, tetrodotoxin or previous denervation. Stimulation of the hypogastric nerve produced frequency dependent increase in fructose secretion. The effect was blocked by tetrodotoxin and scopolamine but not enhanced by physostigmine. If the hypogastric nerve was stimulated close to the seminal vesicle the response was unaffected by hexamethonium but proximal stimulation was blocked. After chronic proximal denervation of the hypogastric nerve, stimulation close to the seminal vesicle produced enhanced response. Destruction of the peripheral ganglia at the base of the seminal vesicle abolished the response. Sections showed that most secretory nerves enter the organ at its base. Phentolamine or yohimbine but not prazosine or propranolol or guanethidine enhanced the secretory response to distal hypogastric nerve stimulation. Tyramine counteracted the response but after reserpinization it was enhanced by tyramine. It is concluded that the secretory cells of the guinea-pig seminal vesicle have a sympathetic secretomotor innervation by short cholinergic neurones with a preganglionic supply via the hypogastric nerve. Inhibitory alpha 1 and beta 2-adrenoreceptors are present on the cells but neurogenic adrenergic inhibition of the secretion is essentially prejunctional and due to activation of inhibitory alpha 2-receptors on the secretomotor nerves.

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Inhibition of relaxation of strips of the bovine retractor penis muscle induced by a standard dose of nicotine (30 microM) was used for quantitative assessment of the ganglion-blocking activity of eight neuromuscular blocking agents that are in clinical use. The order of potency of the drugs studied was (+)-tubocurarine >> alcuronium > vecuronium > metocurine >> pancuronium > atracurium >>> suxamethonium > gallamine. The results have been compared to those obtained with other methods. On the basis of the present results, it is concluded that inhibition of the nicotine-induced relaxation of the bovine retractor penis muscle can be used as an alternative, sensitive in vitro method for the assessment of the ganglion-blocking activity of a neuromuscular blocking agent relative to that of, for example, (+)-tubocurarine. Earlier results have showed that this method is useful also for the assessment of the ganglion-blocking activity of other drugs, because it has yielded comparable and reproducible results at the quantitation of this property of actual ganglion-blocking and various antimuscarinic agents. In addition, this method may be useful for rapid screening of ganglion-blocking activity.

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The nervous control of the retractor penis muscle (rp) was investigated in the anaesthetized goat. Also, isolated field stimulated strips of the muscle were studied. The noradrenaline (NA) and acetylcholine (ACh) content of the rp was determined, and histochemistry for adrenergic and acetylcholinesterase (AChE) positive nerves was performed. The muscle exhibited spontaneous activity that persisted after section of all nerves. There was, however, also a tendency of the activity to follow the general vasomotor tone, which disappeared after section of the sympathetic chains. The excitatory adrenergic nerves which innervate the muscle come from the sympathetic chains and run along the pudendal, the hypogastric and the pelvic nerves. The rp has a dense network of adrenergic fibres and is very sensitive to excitatory adrenergic stimulation. It has a fairly large NA content, which is higher in old goats (5.95 +/- 0.42 micrograms g-1) than in young goats (2.87 +/- 0.78 micrograms g-1). Inhibitory non-adrenergic non-cholinergic (NANC) innervation reaches it via the pelvic and the hypogastric nerves. The maximum inhibitory response is reached at low frequencies (2-4 Hz). Cholinergic prejunctional inhibition of the excitatory response to sympathetic chain stimulation was effected by simultaneous stimulation of the hypogastric nerves. In vitro experiments confirmed the presence of endogenous cholinergic muscarinic suppression of the excitatory adrenergic neurotransmission. Significant amounts of ACh (0.81 +/- 0.18 micrograms g-1) are present in the muscle, and it contains strongly AChE positive nerve fibres and nerve cell bodies. It is concluded that the goat rp is innervated by sympathetic adrenergic excitatory nerves and parasympathetic NANC inhibitory nerves. It further has a direct sympathetic inhibitory NANC innervation, and an indirect inhibitory cholinergic innervation which at least in part is sympathetic.

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The relative potency in inhibiting nicotine-induced relaxation of the bovine retractor penis muscle (BRP) was estimated for the racemates of seven beta-adrenoceptor antagonists, both of the optical isomers of propranolol, and lidocaine. The order of potency of the drugs studied was (+)-propranolol greater than (-)-propranolol greater than propranolol greater than alprenolol greater than metoprolol greater than lidocaine greater than acebutolol greater than pindolol greater than sotalol greater than atenolol. It is concluded that the inhibition of the relaxation was not due to blockade of beta-adrenoceptors but to the nonspecific effects of the beta-adrenoceptor antagonists. It is also concluded that the neurotransmitter(s) which was (were) released from the non-adrenergic non-cholinergic inhibitory nerves in the BRP did not relax the muscle by activating the beta-adrenoceptors. It is suggested that the beta-adrenoceptor antagonists inhibited the release of the inhibitory neurotransmitter(s) by a mechanism which is significantly correlated to their lipophilicity.

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The influence of albumin, 4.5 and 45 g/l, on the effects of Hg2+, 10(-9)-10(-3) M, on the neuromuscular transmission of the isolated guinea-pig ileum and vas deferens was investigated. Hg2+, 10(-9)-10(-6) M, transiently increased the basal tone of the ileum in Tyrode solution without albumin. Albumin, 4.5 g/l, reversed this stimulant effect but enhanced the contractile response to direct muscle stimulation. This contractile response also increased in the vas deferens. Albumin, 45 g/l, obliterated the stimulant effects of Hg2+ on the smooth muscle of the ileum but not of the vas deferens. The effects caused by higher concentrations of Hg2+, 10(-5)-10(-4) M, were only partly inhibited when albumin was present. When neurogenic contractions were elicited in the presence of albumin (45 g/l), Hg2+, 10(-9)-10(-4) M, reduced the contractions in both organs. Consequently, Hg2+ in concentrations presently considered acceptable in blood plasma (10(-9)-10(-8) M) suppressed both cholinergic and adrenergic neuromuscular transmission even in the presence of albumin.

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The influence of albumin, 4.5 and 45 g/l, on the effects of Cu2+, 10(-9)-10(-3) M, on the neuromuscular transmission of the isolated guinea-pig ileum and vas deferens was investigated. Low concentrations of Cu2+, 10(-9)-10(-6) M, caused a slight and transient increase of the contractile response to direct muscle stimulation of the vas deferens. Albumin at 4.5 g/l inhibited this effect. The basal tone of the ileum increased transiently when Cu2+ was added in the presence of albumin, 4.5 g/l. A stimulant action of Cu2+, 10(-9)-10(-6) M, was seen on both organs, when contractions were induced by nerve stimulation. All these effects were inhibited by albumin, 45 g/l. Higher concentrations of Cu2+, 10(-5)-10(-4) M, increased the basal tone of the ileum and the vas deferens. In the presence of albumin, 45 g/l, this stimulating effect of Cu2+ appeared in higher concentrations. The results suggest that Cu2+ is more likely to influence the function of neuronal tissues when the concentration of protein is low.

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1. The relative potency in blocking the nicotine-induced relaxation of the bovine retractor penis muscle (BRP) was estimated for 12 drugs known to have ganglion-blocking properties. 2. The order of potency of the drugs studied was mecamylamine greater than chlorisondamine greater than pentolinium greater than propantheline greater than (+)-tubocurarine greater than hexamethonium greater than emepronium greater than tetraethylammonium greater than glycopyrrolate greater than decamethonium greater than butylscopolamine greater than scopolamine. 3. The results conform well to those obtained with other pharmacological methods used for the estimation of ganglion-blocking activity. 4. It is concluded that blockade of the nicotinic relaxation of the BRP can be used as an alternative method for quantitative assessment of ganglion-blocking activity. 5. Advantages of this technique are that it discriminates well between antinicotinic and antimuscarinic activity and that it satisfies most or all ethical and economical demands. 6. It is also possible that this method has certain value in predicting whether a drug has enough ganglion-blocking activity to be likely to cause impotence.

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The presence of single nerve cell bodies and small ganglia in the retractor penis muscle and the penile artery of the bull was demonstrated by using antisera to neurofilament protein and neuron specific enolase. In the retractor penis muscle the findings were confirmed by staining for acetylcholinesterase. It was also shown that relaxation of strips of the retractor penis muscle induced by 70 microM acetylcholine was totally blocked by a 2.0 microM concentration of the ganglionic blocking drug chlorisondamine. The hypothesis is presented that the relaxation of the bovine retractor penis muscle and the bovine penile artery induced by nicotinic ganglionic stimulating drugs is at least in part mediated via receptors located on the nerve cell bodies described in this study.

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Testicular and germ cell line morphology in rats were studied 2 weeks, 10 months and 14 months after cessation of a 61-day inhalation exposure to 1000 ppm n-hexane. Androgen biosynthetic capacity of testis, testosterone blood concentration, vas deferens morphology and noradrenaline (NA) concentration, epididymal sperm morphology, and fertility were also studied. Severe testicular atrophy involving the seminiferous tubules with loss of the nerve growth factor (NGF) immunoreactive germ cell line was found. Total loss of the germ cell line was found in a fraction of animals up to 14 months post-exposure, indicating permanent testicular damage. No impairment of androgen synthesis or androgen dependent accessory organs was observed. Simultaneous administration of 1000 ppm n-hexane and 1000 ppm toluene, or 1000 ppm n-hexane and 1000 ppm xylene, did not cause germ cell line alterations or testicular atrophy. Toluene and xylene were thus found to protect from n-hexane induced testicular atrophy.

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Relaxations of the isolated bovine retractor penis muscle elicited by nicotine and the three other nicotinic agonists acetylcholine, carbachol, and dimethylphenylpiperazinium were studied. Nicotine (10-45 microM) induced dose-dependent relaxations that closely resembled those evoked by transmural stimulation of inhibitory nerves. The relaxations induced by this dose level of nicotine were abruptly abolished by hypoxia and totally blocked by 1.2 microM mecamylamine, 45 microM lidocaine, and 13 microM methylene blue. They were reduced 50-90% by 0.16 microM tetrodotoxin, but they were unaffected by 17 microM scopolamine. Provided that sufficient concentrations of scopolamine and eserine were present, the relaxations caused by acetylcholine (30-140 microM) were exactly like those evoked by nicotine, and they were identically affected by hypoxia and the blocking drugs. Also carbachol and dimethylphenylpiperazinium induced relaxations qualitatively identical to those effected by the above-mentioned doses of nicotine. Relaxations induced by larger doses of nicotine were less susceptible to hypoxia and the blocking drugs. The results suggest that nicotine concentrations ranging from about 10-45 microM relax the bovine retractor penis muscle by a rather selective activation of inhibitory nerves, whereas higher concentrations may additionally activate some other less specific inhibitory mechanism. They further strongly suggest the presence of nerve cell bodies in this muscle. It is suggested that one physiological role of acetylcholine in the development of penile erection is nicotinic activation of inhibitory nerves. Moreover, nicotinic activation of these nerves of the bovine retractor penis muscle can be used as a model for further characterization of mammalian erectile nerves.

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Isolated strips from isthmic and ampullary muscle layers of the human fallopian tube were investigated with the sucrose-gap technique and in organ baths. Generally good correlation existed between electrical and mechanical activity, but dissociation was also noted. The isthmic external longitudinal layer (IEL) was characterized by slow waves of depolarization and broad 'spikes'. Tonic contraction followed depolarization, and phasic waves followed the spikes. In the other layers, regular spikes and phasic contractions dominated. Late in the menstrual cycle, more irregular patterns and slow waves, which could synchronize spike activity, were found. Automaticity tended to be highest in the late proliferative phase. Active response to stretch was best demonstrated in IEL. Excitatory alpha-adrenergic responses (hypopolarization, firing and contraction) to noradrenaline and nerve stimulation dominated in the IEL. In the other layers, beta-adrenergic inhibition prevailed. Non-adrenergic, non-cholinergic neurogenic inhibition was demonstrated. Decrease in spike frequency and hyperpolarization seemed to be as prominent as during beta-adrenergic inhibition. Muscarinic stimulation by acetylcholine was recorded as depolarization, spikes and contraction. However, a role for cholinergic nerves could not be defined. Thus, the human oviductal smooth muscle belongs to the single unit category; its activity can be modified during the menstrual cycle, by stretch and by excitatory and inhibitory nerves.

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The possible biological significance of low concentrations of Cu2+ on the guinea-pig ileum has been investigated. Responses to nerve stimulation by single electrical shocks, acetylcholine (ACh), and histamine and the response to ACh of depolarized muscle were examined. In summary, Cu2+ has diverse and dose-dependent effects on the ileum. A stimulant effect dominates in intestine accommodated in physiological saline solution. The excitatory effect of the ion, in the concentration range 10 nM-1 microM, is probably due to stimulation of a depolarization-coupled initiation of the contraction. The inhibitory effect of Cu2+ (10 nm-1 microM) is presumably due to a decreased Ca2+ availability for the contractile process. In a higher concentration range (10 microM-100 microM) the stimulant action could also, in part, be related to a copper-induced release of Ca2+ from a storage site. No effect that certainly could be ascribed to a neuronal site of action could be observed. The possible effect of Cu2+, released during corrosion of dental alloys, on oral excitable tissue such as taste and pain receptors is discussed.

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The resistance to atropine or scopolamine of the neurogenic contractile response to field stimulation of intramural nerves of the rat detrusor was studied during the early phase of Wallerian degeneration in vitro and in vivo. The neurogenic contractile response to low frequency stimulation of control bladders was completely resistant to atropine or scopolamine in vitro. As a matter of fact the response was enchanced by the drugs. In vivo a transient shortening of the response could be seen. 26-34 h after bilateral extirpation of the pelvic ganglia there was prominent degeneration activity in the bladders in vitro as well as in vivo. Atropine or scopolamine inhibited this activity in vivo but enhanced it in vitro. The neurogenic contractile response to field stimulation of the bladder nerves was blocked in vivo but not in vitro by the antimuscarinic drugs 26-34 h after neurectomy. The results support the concept that the excitatory neurotransmission in the detrusor is essentially cholinergic. An explanation of the difference between the in vivo and the in vitro results based on differences in degree of blockade of pre- and post-junctional muscarinic receptors and diffusion of the antimuscarinics in the tissue is proposed.

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The effect of phentolamine on the neurogenic contractile response in vasa deferentia from rat and guinea pig was studied during Wallerian degeneration. This response was also investigated after partial denervation (surgery or chemical sympathectomy by guanethidine treatment) in vasa deferentia from guinea pig. During Wallerian degeneration the response showed a gradual increase in sensitivity to phentolamine and was abolished in the late stages. The neurogenic contractile response of the partially denervated vas deferens was blocked by low concentrations of phentolamine. It is concluded that decreased intimacy of the neuroeffector junctions leads to increased susceptibility to alpha-adrenoceptor blockade of the contractile response to nerve stimulation. It is further concluded that the motor transmission in the vas deferens is essentially adrenergic. The resistance to alpha-adrenoceptor blockade of the initial phase of the contractile response to nerve stimulation of intact vasa deferentia from guinea pig and rat might well be explained by the "proximity theory" of Dale & Gaddum although participation of supplementary mediators cannot be excluded.

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