The effect of palytoxin on neuromuscular junctions in the anococcygeus muscle of the rat.

Palytoxin, a highly toxic natural product isolated from zoanthids of the genus Palythoa, is accumulated by a wide range of fishes and marine invertebrates used as food in the Indo-Pacific. It is responsible for many incidents of human morbidity and mortality. The toxin is a potent smooth muscle spasmogen. The cause of the contraction of smooth muscle is unclear, but recent work strongly suggests that it is primarily initiated by the release of neurotransmitters from the motor innervation of the smooth muscle. We show here that palytoxin caused the swelling of the muscle cells and some internal organelles of the anococcygeus muscle of the rat, but no substantial structural damage to the tissue. Axons and Schwann cells were also swollen but the most dramatic feature was the depletion of synaptic vesicles from putative release sites in the axons. Some axons were physically damaged following exposure to the toxin, but this was relatively uncommon (< 10% of all axons studied). In the majority of axons there was no damage to nerve terminal membranes, but there was damage to mitochondria. The depletion of vesicles involved all types-clear, dense-cored, large and small. Our observations and pharmacological data gathered elsewhere, provide a neuropathological basis for the spasmogenic activity of palytoxin.

Nitric oxide and cyclic GMP formation following relaxant nerve stimulation in isolated human corpus cavernosum.

OBJECTIVE: To investigate further the role of the nitric oxide (NO)-cyclic GMP pathway as the mediator of relaxant neurotransmission in human corpus cavernosum and to establish whether impaired activity of this pathway contributes to the pathophysiology of impotence. PATIENTS AND METHODS: Samples of cavernosal tissue were obtained from 59 men undergoing penile operations. The controls comprised four men with penile carcinoma and 17 with Peyronie's disease. Of the impotent men, 35 had clinical evidence of penile vascular disease on pre-operative investigation, whilst three had non-vascular impotence. Each biopsy was divided into two strips which were then suspended under tension in organ bath chambers. The relaxant innervation of one strip of each pair was stimulated electrically whilst the other strip was left unstimulated. The formation of NO and cyclic GMP was calculated by comparing their respective tissue content in the stimulated and unstimulated strips. RESULTS: Overall, stimulation of the relaxant innervation produced significant increases in the tissue content of both NO and cyclic GMP. Incubation with an inhibitor of NO biosynthesis abolished the mechanical relaxant response and the formation of both NO and cyclic GMP. The magnitude of relaxant response and the formation of NO was diminished in tissue from men with vascular impotence compared to controls. The increase in cyclic GMP content was similar in both these groups. Relaxant response, NO formation and cyclic GMP formation in tissue from men with non-vascular impotence was similar to controls. CONCLUSIONS: This study provides further evidence that the NO-cyclic GMP pathway acts as the mediator of nerve-evoked smooth muscle relaxation in human corpus cavernosum. Diminished NO formation following relaxant nerve stimulation may account for impaired relaxant responses found in tissue from men with vascular impotence and may contribute to the cause of their erectile dysfunction.

Presence of neuropeptide Y in the rat seminal vesicle and its effects on noradrenaline- and nerve-induced contractions.

1. Immunohistochemical and functional studies have been performed to localize and determine the effects of neuropeptide Y (NPY) in the rat seminal vesicle. 2. An abundant presence of NPY-immunoreactive nerves, mainly concentrated in the smooth muscle layer of the seminal vesicle was found. Chronic 6-hydroxydopamine treatment (four doses of 50 mg kg-1 i.p. on days 1, 2, 4 and 6; rats killed one week after the last injection) led to a large reduction but not abolition of the NPY-immunoreactivity. 3. NPY (1-250 nM) did not affect the resting tone of the seminal vesicle. 4. The seminal vesicle was contracted by electrical field stimulation (EFS) and by exposure to 5 microM noradrenaline (NA). These contractions were abolished by phentolamine (1 microM). Tetrodotoxin (0.5 microM) abolished EFS-evoked contractions but did not affect NA-evoked contractions. 5. Seminal vesicles, from animals chronically-treated with reserpine (5 mg kg-1 i.p. on days 1 and 2; rats killed on day 3), were contracted by NA but not by EFS. 6. NPY (0.25-250 nM), concentration-dependently, inhibited EFS-evoked contractions by up to 70% maximum inhibition. Contractions evoked by EFS with short trains of pulses were inhibited by NPY to a greater degree than those with longer trains. 7. NPY had no significant effect on NA-evoked contractions. 8. These data provide strong evidence that the motor transmission in rat seminal vesicle is predominantly if not exclusively, adrenergic. It is further concluded that a rich NPY-containing innervation is present in the smooth muscle layer of rat seminal vesicle. The primary effect of NPY is modulation of adrenergic motor transmission by a prejunctional inhibition of NA release.

Corpus cavernosal relaxation in impotent men.

OBJECTIVE: To investigate smooth muscle responsiveness in isolated preparations of corpus cavernosum from men with vasculogenic impotence. PATIENTS AND METHODS: Biopsies of corpus cavernosum were obtained from 63 men undergoing penile surgery. These included a control group of eight men (mean age 59 years, range 38-82) undergoing penile amputation for carcinoma, 47 men with vasculogenic impotence (mean age 58, range 36-72) who were further sub-divided into arterial (n = 9), venous (n = 24) or mixed arterial/venous (n = 14) impotence according to the results of pre-operative haemodynamic investigation, and eight men with non-vasculogenic impotence (mean age 49 years, range 34-66). Smooth muscle contractile responses to alpha-adrenoceptor activation and relaxant responses to stimulation of intrinsic nerves and exposure to papaverine and sodium nitroprusside were recorded in tissue strips prepared from the biopsies. Morphology was assessed histologically using haematoxylin and eosin staining of tissue sections together with immunocytochemical labelling of intrinsic nerves. RESULTS: Nerve-evoked relaxation was markedly impaired in tissue from men with venous or mixed arterial/venous impotence. A lesser degree of impairment was found in tissue from men with arterial impotence alone. Tissue from men with all types of vasculogenic impotence also showed a decreased contractile response to alpha-adrenoceptor stimulation. The magnitude of relaxant responses to papaverine and sodium nitroprusside in the vasculogenic group was similar to that of the control. There were no differences in smooth muscle content or nerve density between the vasculogenic group and the control. In the non-vasculogenic group responses to relaxant nerve stimulation, alpha-adrenoceptor activation and relaxant drugs were similar to those of the control. Nerve density in this group was similar to the control but smooth muscle content was reduced. CONCLUSIONS: The results of this study demonstrate a functional impairment of smooth muscle contractility and neurogenic relaxation in corpus cavernosum from impotent men with abnormal penile haemodynamics. Altered smooth muscle responsiveness is likely to be a factor in the aetiology of impotence in such men and may contribute to the relatively poor results of vascular surgery for impotence.

Neuropeptide Y in rat detrusor and its effect on nerve-mediated and acetylcholine-evoked contractions.

1. Immunohistochemical and isolated organ bath techniques were used to detect the presence of neuropeptide Y (NPY) in the rat urinary bladder and to determine its effect on tone, spontaneous activity and contractile responses of the detrusor muscle to electrical field stimulation, acetylcholine and alpha,beta-methylene ATP (alpha,beta-MeATP). 2. A very rich presence of NPY-immunoreactive nerve fibres was found mainly within the bundles of detrusor muscle cells. Chronic treatment with 6-hydroxydopamine did not affect the density of NPY-positive nerve fibres. 3. NPY (> 1 nM) enhanced the force and frequency of spontaneous contractions and generated a rise in the resting tone of the detrusor. These effects of NPY on the tone and the spontaneous activity remained unaffected by atropine (3 microM), indomethacin (10 microM) and aspirin (100 microM) but were abolished by Ca(2+)-withdrawal from the bathing medium. 4. The enhancing effects of NPY on the spontaneous contractions and the resting tone were not prevented by the induction of purinoceptor desensitization. 5. NPY (1-250 nM) potentiated electrical field stimulation (EFS, 1-64 Hz, 0.1 ms pulses duration, 10s train duration)-evoked, tetrodotoxin (0.5 microM)-sensitive contractions. The atropine (3 microM)-resistant component of EFS-evoked contractions was also potentiated by NPY. By contrast, the nifedipine (1 microM)-resistant but atropine-sensitive component of EFS-evoked contraction was inhibited by NPY. 6. NPY (250 nM) did not affect acetylcholine-evoked contractions, but potentiated alpha,beta-MeATP-evoked contractions. 7. It is concluded that NPY-innervation of rat urinary bladder is largely confined to the detrusor muscle and is abundant and mainly non-adrenergic. It is further concluded that the enhancing effect of NPY on detrusor spontaneous activity and tone is caused by Ca2+ influx through nifedipine-sensitive Ca2+ channels and is not mediated through acetylcholine or cyclo-oxygenase-sensitive eicosanoids or ATP.8. The results are consistent with the hypothesis that intrinsic NPY in the rat detrusor innervation contributes to the motor transmission in two ways: by promoting non-cholinergic motor transmission and by inhibiting prejunctionally the cholinergic transmission.

Palytoxin-induced transmitter release in the autonomic nervous system of the rat.

A combination of pharmacological, histochemical, immunocytochemical, neurochemical and microscopic techniques has been used to test the hypothesis that smooth muscle contractions caused by palytoxin are primarily a response to toxin-induced release of transmitter from motor nerve terminals. In the anococcygeus, palytoxin caused a dose-dependent contraction; the dose-response curve was particularly steep in the dose range 0.3-100 nM. This part of the response was abolished by phentolamine and absent in the muscles of reserpinized animals. A single, large dose of palytoxin (100 nM) caused a biphasic contraction. Phentolamine blocked the first phase by 50% and the second by > 80%. Transmitter overflow studies showed that the toxin enhanced the release of 3H after loading with 3H-NA, and immunofluorescence showed the loss of adrenergic transmitters from the innervation. In the vas deferens, palytoxin caused a biphasic contraction of the muscle. Phentolamine blocked first phase by > 80% and the second by 47%. Immunofluorescence studies showed that stores of adrenergic transmitter were depleted but stores of NPY were not greatly affected. Indirect evidence suggested that palytoxin did not cause the release of purinergic transmitters. A direct component to the contraction was apparently present in most preparations, but though variable in extent it was usually slight. It is concluded that the contractions of smooth muscle caused by palytoxin are primarily the result of toxin-induced transmitter release.

Differential effects of nifedipine on nerve-mediated and noradrenaline-evoked contractions of rat anococcygeus muscle.

In rat anococcygeus muscle the inhibitory effect of nifedipine (0.01, 0.1, 1.0 and 10 microM) was determined on adrenergic twitches in response to electrical field stimulation (trains of 4 pulses, 0.1 ms pulse duration, 10 Hz) and on twitch-matching contractions evoked by noradrenaline. Nifedipine concentration-dependently reduced the neurogenic twitch with an IC50 of 0.083 microM. Nifedipine reduced the noradrenaline-evoked contraction to a markedly lesser degree (IC50 > 10 microM). The difference in the magnitude of inhibition of electrically evoked twitch and twitch-matching noradrenaline-evoked contraction was statistically significant at every concentration of nifedipine. It is concluded that inhibition of the twitch by nifedipine involves some other mechanism(s) in addition to its Ca2+ channel blocking property in smooth muscle.

Evidence against vasoactive intestinal polypeptide as the relaxant neurotransmitter in human cavernosal smooth muscle.

1. The putative role of vasoactive intestinal polypeptide (VIP) as the relaxant neurotransmitter in human cavernosal smooth muscle has been studied in isolated tissue preparations. 2. Consistent neurogenic relaxations were evoked by electrical field stimulation (EFS; 2-64 pulses/train, 0.8 ms pulse duration, 10 Hz). VIP (0.1-3 microM) relaxed cavernosal smooth muscle in a dose-dependent fashion. Relaxant responses to both EFS and VIP were reduced in tissue from impotent men. 3. Neurogenic relaxant responses were not diminished in the presence of the VIP-inactivating peptidase, alpha-chymotrypsin (alpha-CT, 2 units ml-1). In contrast VIP-induced relaxations were completely abolished. 4. Inhibition of nitric oxide synthase by NG-nitro-L-arginine (30 microM), and of guanylate cyclase by methylene blue (50 microM) caused highly significant reductions of neurogenic relaxant responses whereas VIP-evoked relaxations were unaffected. 5. It is concluded that VIP-evoked relaxations are not mediated by the NO-guanosine 3':5'-cyclic monophosphate (cyclic GMP) pathway and that VIP release is not essential for neurogenic relaxation of human cavernosal smooth muscle. VIP does not therefore act as the major relaxant neurotransmitter in this tissue.

Effect of streptozotocin diabetes on motor and inhibitory transmission in rat anococcygeus.

The effect of streptozotocin diabetes of 4-week duration on the adrenergic motor transmission and on the nonadrenergic, noncholinergic, inhibitory transmission in the rat anococcygeus was investigated by recording contractile and relaxant activity of isolated muscle preparations taken from diabetic and age-matched control animals. The neurogenic contractile responses to electrical field stimulation were significantly reduced in the preparations from diabetic rats. The inhibitory transmission remained unaffected in the diabetic rats. Concentration--response curves showed no change in sensitivity of the diabetic anococcygei to noradrenaline. The maximum tension generated was also similar in preparations from diabetic and nondiabetic animals. The contractile responses to electrical field stimulation were significantly greater in preparations from diabetic rats treated for 4 weeks with either sorbinil (20 mg.kg-1.day-1 orally) or myo-inositol (667 mg.kg-1.day-1 orally) when compared with the untreated diabetic controls; the sensitivity to noradrenaline was identical in all three groups. It is concluded that streptozotocin diabetes causes a significant reduction of adrenergic contractile responses of the anococcygeus to electrical field stimulation by a prejunctional mechanism, and the reduction can be prevented by treating the animals with the aldose reductase inhibitor sorbinil or with myo-inositol.

The action of palytoxin on the isolated detrusor muscle of the rat.

1. The effects of a coelenterate toxin, palytoxin (PTX) have been studied in the isolated detrusor muscle. of the rat. 2. PTX (1-100 nM) initiated concentration-dependent contractions of the detrusor; the contraction led to an irreversible tachyphylaxis. Muscle desensitized to PTX continued to respond to acetylcholine (ACh) and excess K+ but the contractions were reduced compared to pre-PTX contractions. 3. Contractions evoked by PTX were not affected by the presence of atropine (10 microM), indomethacin (10 microM) or tetrodotoxin (0.5 microM) but were greatly reduced by nifedipine (3 microM) and by the absence of K+. PTX could not evoke contractions in the absence of Ca2+ or in tissues depolarized by exposure to excess K+. 4. PTX abolished the neurogenic contractile responses to electrical field stimulation (EFS). 5. Combined treatment with atropine (10 microM) plus nifedipine (3 microM) abolished contractile responses to EFS and greatly reduced the contractile response to PTX. 6. The contractile response to PTX (100 nM) was reduced following exposure of the muscle to alpha, beta-methylene ATP. 7. Exposure to PTX (100 nM) for 1-3 h reduced both the ACh content of the detrusor (by more than 80%), and the immunoreactivity of neuropeptide Y-containing nerve fibres compared to control. 8. It is concluded that the primary effect of PTX is to promote the release of endogenous motor transmitters, leading to their eventual depletion.

The effect of inhibitors of nitric oxide biosynthesis and cyclic GMP formation on nerve-evoked relaxation of human cavernosal smooth muscle.

1. The inhibitory transmission in isolated preparations of cavernosal smooth muscle from human penis has been studied. 2. Electrical field stimulation (EFS; 2-64 pulses/train, 0.8 ms pulse duration, 10 Hz) evoked relaxation of preparations treated with guanethidine (50 microM). The EFS-evoked relaxations were atropine-resistant and tetrodotoxin-sensitive indicating their origin to be non-adrenergic, non-cholinergic (NANC) nerve stimulation. 3. EFS-evoked relaxation was attenuated dose-dependently by the nitric oxide (NO)-synthase inhibitor, L-NG-nitro arginine (L-NOARG; 0.3-100 microM) but not by D-NG-nitro arginine. The inhibitory effect of L-NOARG on transmission was antagonized by L-arginine (100 microM), a NO precursor, but not by D-arginine. 4. Incubation with methylene blue (10-50 microM), a known inhibitor of guanylate cyclase activation by NO, caused a concentration-related inhibition of EFS-evoked relaxation. 5. It is concluded that NANC nerve-evoked relaxation of human cavernosal smooth muscle is mediated by NO or a NO-like substance.

The effect of streptozotocin-induced diabetes on cholinergic motor transmission in the rat urinary bladder.

1. The effect of streptozotocin (STZ)-induced diabetes on cholinergic motor transmission in the rat urinary bladder was investigated by recording contractile activity of detrusor strips in vitro. 2. The Ca(2+)-channel antagonist, nifedipine, was found to be more effective in blocking the noncholinergic motor transmission than P2-purinoceptor desensitization by alpha,beta-methylene ATP. 3. The neurogenic contractile responses to electrical field stimulation in the presence of nifedipine (cholinergic) were larger in the diabetic detrusor than in the non-diabetic controls. The potentiation of the cholinergic transmission was more evident at higher frequencies. 4. Concentration-response curves for acetylcholine were identical in detrusors from diabetic and non-diabetic animals, thus excluding a postsynaptic supersensitivity to acetylcholine being responsible for the potentiation of cholinergic motor transmission. 5. It is concluded that the potentiation of cholinergic motor transmission is due to enhanced release of acetylcholine in diabetic detrusor. Possible reasons for this enhancement are discussed in relation to diabetes.

Inhibitory effect of streptozotocin-induced diabetes on non-cholinergic motor transmission in rat detrusor and its prevention by sorbinil.

1. Non-cholinergic motor transmission in the urinary bladder of streptozotocin (STZ)-diabetic rats was studied by recording contractile activity of strips of detrusor in vitro. 2. The neurogenic contractile responses to electrical field stimulation (EFS) of atropine-treated detrusor strips were decreased in 4, 8 and 12 week STZ-diabetic rats. The decrease was most marked in 12 week diabetic rats and least in 4 week ones. 3. Concentration-response curves showed no change in sensitivity of the detrusor to acetylcholine (ACh) in diabetic rats. The maximum tension generated by ACh was similar in diabetic and non-diabetic animals. 4. The contractile responses to EFS at frequencies greater than or equal to 1 Hz were not maintained during stimulation. The 'fade' was significantly greater in detrusor strips of diabetic rats. 5. The contractile response of detrusor to EFS was significantly greater in 12 week diabetic rats treated with the aldose reductase inhibitor sorbinil, than in untreated 12 week diabetic rats. The sensitivity to ACh was similar in the two groups. 6. It is concluded that the reduction of the neurogenic non-cholinergic responses of detrusor to EFS in STZ-diabetic rats is probably caused by a reduction in the release of the non-cholinergic motor transmitter. The results are discussed in relation to bladder dysfunction in human diabetes mellitus.

Effect of nifedipine on the contractile responses of the isolated rat bladder.

The effect of the calcium channel blocker nifedipine on the motor transmission in isolated preparations of rat detrusor smooth muscle has been studied. Nifedipine blocked the major part (75 to 80%) of the contractile response to electrical field stimulation, while atropine only blocked 20 to 25%. In preparations pretreated with atropine, the response to electrical field stimulation was completely abolished by nifedipine. The converse was also true; in preparations pretreated with nifedipine the response was fully blocked by atropine. The nifedipine-resistant response was greatly potentiated by the anticholinesterase eserine. The blocking action of nifedipine on motor transmission was partially antagonised by raising Ca2(+)-concentration. Acetylcholine concentration-response curve was shifted to the right by nifedipine. It is concluded that the non-cholinergic motor neurotransmitter evokes contraction of the rat detrusor smooth muscle by activating external Ca2(+)-transport channels whereas the cholinergic contraction is mediated partly or wholly by alternative mechanisms.

Presence of non-cholinergic motor transmission in human isolated bladder.

Atropine-sensitivity of the motor transmission in the isolated detrusor preparation from human bladder has been examined. The preparations were contracted by electrical field stimulation consisting either of short trains of pulses or of long trains of pulses. Part of the stable response to short-train stimuli (28%) was resistant to atropine, was not potentiated by physostigmine and was blocked by tetrodotoxin. The stable responses to long-train stimuli were fully blocked by atropine. It is concluded that the detrusor of the bladder in man, in common with other mammalian species, contains a noncholinergic component in its motor transmission, and that prolonged stimulation with long-train stimuli causes an extinction of the non-cholinergic motor transmission, probably through depletion of transmitter stores in the nerve-terminals.

Failure of non-adrenergic transmission in the rat vas deferens.

Repetitive field stimulation of the vas deferens of the rat with long trains of pulses (90 pulses, 10 Hz, 1 msec) at intervals of 30 or 60 sec produced a decline in the heights of contractile responses. This decline in responses was much more pronounced in phentolamine (5 microM)-treated than in untreated preparations and in the prostatic rather than in the epididymal end of the vas deferens. The decline in the responses was dependent on the number of pulses/train, the frequency of pulses in the train and the interval between successive trains. Potassium chloride (KCl)-induced and adenosine triphosphate (ATP)-induced contractions were largely unaltered after 30 min of stimulation. The decline in the responses was significantly reduced in low but not in high extracellular calcium. 4-Aminopyridine (4-AP) caused an initial potentiation of contractile responses, followed by a faster decline of responses. The decline in responses was more intense in vas deferens from reserpine-treated animals. The results support the view that electrical stimulation-induced decay of motor transmission in the vas deferens of the rat is caused by a specific failure of the non-adrenergic transmission.

Neuropeptide Y and platelet aggregation by adrenaline.

The effect of neuropeptide Y on platelet-rich citrated human plasma has been studied both before and after addition of adrenaline. The peptide has no aggregatory properties of its own, but in the concentrations tested it does slow and inhibit the aggregatory responses of platelets to adrenaline. This effect is dose-dependent. The implications of this finding are discussed.

Inhibition of non-adrenergic transmission in the rat vas deferens by prazosin.

Isolated preparations of rat vas deferens were induced to contract by neurogenic and non-neurogenic stimuli, and the effect of prazosin on these contractions was investigated. Prazosin (24 microM) exerted a profound inhibitory effect on neurogenic contractions and had no significant effect on non-neurogenic contractions. These experiments indicate that prazosin is capable of interacting with the neurotransmission processes in the vas deferens to produce inhibition of transmission.