Investigating sodium valproate as a treatment for McArdle disease in sheep.

McArdle disease is due to an absence of the enzyme muscle glycogen phosphorylase and results in significant physical impairment in humans. We hypothesised that sodium valproate, an HDAC inhibitor, might have the ability to up-regulate the enzyme. We treated McArdle sheep with sodium valproate given enterically at 20-60 mg/kg body wt. Compared with untreated control animals, there was increased expression of phosphorylase in muscle fibres. The response was dose dependent and reached a maximum 2 hours after the application and increased with repeated applications. Improvement in mobility could not be demonstrated. These findings suggest that sodium valproate is a potential therapeutic treatment for McArdle disease.

Phosphorylase re-expression, increase in the force of contraction and decreased fatigue following notexin-induced muscle damage and regeneration in the ovine model of McArdle disease.

McArdle disease is caused by a deficiency of myophosphorylase and currently a satisfactory treatment is not available. The injection of notexin into, or the layering of notexin onto, the muscles of affected sheep resulted in necrosis followed by regeneration of muscle fibres with the expression of both non-muscle isoforms of phosphorylase within the fibres and a reduction of the amount of glycogen in the muscle with an increase in the strength of contraction and a decrease in fatiguability in the muscle fibres. The sustained re-expression of both the brain and liver isoforms of phosphorylase within the muscle fibres provides further emphasis that strategies to enhance the re-expression of these isoforms should be investigated as a possible treatment for McArdle disease.

Mechanical properties and innervation of the smooth muscle layers of the urethra of greyhounds.

OBJECTIVE: To investigate the properties of the smooth muscle layers in the urethral wall of male and female greyhounds, and to consider their roles in continence and micturition. MATERIALS AND METHODS: The distribution and innervation of the smooth muscle layers of the prostate capsule and membranous urethra of male greyhounds were assessed. Strips of smooth muscle from these regions were used to determine the neuropharmacological properties by assessing the excitatory and inhibitory responses to nerve stimulation, and the effects of blocking agents. These were compared with strips from the proximal urethra and from the female urethra. RESULTS: The smooth muscle of the membranous urethra comprised 9% of the wall and received its innervation exclusively in branches from the pelvic plexus. The cholinergic innervation in the male produced 80% of the total contractile response in the longitudinal membranous urethra, 50% in the prostate capsule and 13% in the circular muscle of the proximal urethra. In the female all areas had poor contractile responses. Inhibitory fibres produced relaxation in all parts of male and female urethrae with the major effect caused by nitric oxide. Adrenergic nerves contributed to both residual excitation (alpha receptors) and inhibition (beta receptors). CONCLUSIONS: The longitudinal smooth muscle of the male membranous urethra probably shortens the urethra during micturition, through the activity of cholinergic nerves, whereas the circular smooth muscle of the proximal urethra, under adrenergic control, may be contracted during continence and ejaculation. In the female, the smooth muscle plays a minor role.

The innervation and properties of the urethral striated muscle.

The striated muscle forms an outer sleeve around the urethra and occupies about 80% of the wall. In humans more than 60% of the muscle consists of slow, fatigue resistant type I fibres whereas in male greyhounds only about 6% are type I. Most of the remainder (70% of fibres) are type IIa which are classified as fast but can contract for several hours with little decline in amplitude. Stimulation of pudendal but not pelvic nerves produces contraction of the striated muscle and a rapid increase in urethral pressure. Pelvic nerve branches enter the striated muscle sleeve but pass through it to supply the inner layer of smooth muscle. The striated muscles are well suited to maintain contraction necessary for continence.

Continence and some properties of the urethral striated muscle of male greyhounds.

OBJECTIVE: To determine the properties of the striated muscle of the greyhound (dog) urethra and to consider its role in maintaining continence. Materials and methods The thickness of the muscle layers and the muscle types were determined by examining sections stained with haematoxylin and eosin or Masson's trichrome. These factors were correlated with the mechanical and electrical responses of muscle strips to nerve stimulation, and compared with muscle from other breeds of dog and other parts of the animal. RESULTS: The striated muscle formed approximately 70% of the membranous urethra and was predominantly (68%) type IIa muscle (i.e. fast but fatigue-resistant). The mean resting membrane potential was -74 mV; nerve stimulation produced an action potential with a mean amplitude of 97 mV and contraction lasting about 200 ms. All responses were abolished by D-tubocurarine. The contractions were well maintained with continuous or intermittent stimulation. The properties were intermediate between those of the anconeus (slow) and the extensor carpi radialis (fast) muscles. CONCLUSIONS: The distribution, fibre type and contractile characteristics would enable the striated urethral muscle to maintain tension for continence at rest and provide additional continence during sprints.

Increase in calcium in smooth muscle cells of the rabbit bladder induced by acetylcholine and ATP.

Cultured smooth muscle cells from rabbit urinary bladder were loaded with fura-2. Changes in intracellular Ca concentration [Ca2+]i produced by acetylcholine (ACh) or adenosine triphosphate (ATP) were estimated by measuring the fluorescence ratio F340/F380. Western blot analysis and immunohistochemical techniques showed that the cultured cells retained alpha-smooth muscle actin. ATP produced a rapid but transient increase in [Ca2+]i and ACh produced a delayed, prolonged increase. Application of ACh after ATP in Ca-free solution failed to elevate [Ca2+]i suggesting that both ACh and ATP release Ca2+ from the same intracellular stores. Following application of ACh but not ATP in Ca-free Krebs solution, reintroduction of Ca2+ produced elevation of [Ca2+]i, indicating that ACh causes prolonged opening of channels in the membrane. The sustained increase induced by ACh was abolished by nicardipine (blocker of Ca2+ voltage dependent channel ICa(V)) or quinine (blocker of non-selective cation channels). Although the elevations to ACh or ATP were abolished by neomycin (an inhibitor of phospholipase C) the different time courses suggest that the mechanisms of release of Ca2+ from intracellular stores or the pathway for refilling the stores is different.

Innervation of the striated muscle of the membranous urethra of the male dog.

PURPOSE: To identify the functional innervation of the striated muscle layer of the post-prostatic urethra of male dogs. MATERIALS AND METHODS: Detailed anatomic dissection of the pelvic and pudendal nerves was carried out. The pressure and contractile responses to stimulation of these nerves were recorded in vivo and in vitro. RESULTS: Small branches of the pelvic nerve entered the membranous urethra but passed through the striated muscle to the inner smooth muscle layer. Stimulation of the nerve with 1 msec pulses at 10 Hz produced a slow contraction of the urethra which was unaffected by d-tubocurarine. Pudendal nerve branches entered the striated layer from the caudal end. Stimulation produced a rapid, visible contraction that was abolished by d-tubocurarine. Field stimulation of isolated strips of striated muscle resulted only in rapid, d-tubocurarine sensitive contractions. CONCLUSIONS: The striated muscle of the membranous urethra is innervated exclusively by the pudendal nerve.

Urinary continence after radical retropubic prostatectomy. Analysis and synthesis of contributing factors: a unified concept.

OBJECTIVE: To assess the effects of three types of apical dissection on urinary continence after radical retropubic prostatectomy and to evaluate possible contributing factors, e.g. preservation of the bladder neck and preprostatic sphincter, age, anastomotic strictures, previous transurethral resection and nerve-sparing surgery. PATIENTS AND METHODS: Having undergone one of three types of apical dissection, 280 patients were evaluated: in Group 1 (sphincter-damaging) 134 patients underwent the original technique of ligating and transecting the venous complex; in Group 2 (sphincter-repairing), 76 patients had the venous complex with part of striated sphincter incorporated within anastomotic suture(s); and in Group 3 (sphincter-preserving), 70 patients had the venous complex alone ligated using the 'bunching' technique of Myers. The outcome was analysed for the number becoming continent and the time to continence. RESULTS: Continence was achieved in 93% overall, with 90%, 93% and 99% achieving continence in Groups 1, 2 and 3, respectively. The mean time to continence was 68 days overall, taking 100, 52 and 30 days for the respective groups. Twenty patients (7%) did not achieve full continence; 15 had minor incontinence and five severe, with none of the latter being in Group 3. The group (preservation of external sphincter), age and freedom from development of anastomotic strictures were the most important factors both in regaining continence and decreasing the time to continence. CONCLUSIONS: Preservation of as much as possible of the normal anatomy of the sphincter mechanisms and their nerve supplies results in an excellent return to continence after radical retropubic prostatectomy.

Activity of glibenclamide-sensitive K+ channels under unstimulated conditions in smooth muscle cells of pig proximal urethra.

The membrane potential in pig proximal urethra was examined by use of the microelectrode technique. In cells 1-2 cm from the bladder neck the membrane potential was quiescent, with a value of -37.2+/-2.5 mV (n = 16). In some cells small spontaneous de- and hyperpolarizations were seen. Glibenclamide (1 microM) caused a small but significant depolarization in tissue strips (12+/-3 mV, n = 3) and also in dispersed cells using whole-cell patch electrodes (13+/-3 mV, n = 5). In the conventional whole-cell voltage-clamp configuration, glibenclamide reduced the noise level of the basal membrane current at -50 mV and inhibited the membrane current in symmetrical 140 mM K+ conditions. In cell-attached patches, brief openings of a glibenclamide-sensitive 43 pS K+ channel (K(GS)-43 pS) were seen even under unstimulated conditions and greater activation occurred in the same membrane patch on subsequent application of 100 microM levcromakalim. These results provide direct evidence that glibenclamide-sensitive K+ channels may play a role in maintaining the resting membrane potential of pig proximal urethra under unstimulated conditions.

The electrical properties and responses to nerve stimulation of the proximal urethra of the male rabbit.

OBJECTIVE: To identify the nature of neurotransmitters acting on the smooth muscle cells of the proximal urethra and the electrical activity underlying the mechanical responses to nerve stimulation. MATERIALS AND METHODS: The electrical activity of longitudinal strips of proximal urethra obtained from male rabbits was recorded with microelectrode and double sucrose-gap techniques. Intramural nerves were stimulated with 200 microseconds pulses. Drugs which selectively affected neurotransmission were added to the perfusing fluid. RESULTS: The mean resting potential of smooth muscle cells was -39 mV and they had infrequent spontaneous action potentials. The frequency was increased by noradrenaline without depolarization; ATP produced depolarization with increased action-potential frequency. Small (< or = 16 mV) spontaneous depolarizations and hyperpolarizations could be recorded from < 50% of cells; large (< or = 40 mV) hyperpolarizations also occurred. Electrical field stimulation evoked excitatory and inhibitory junction potentials (EJPs and IJPs). Most EJPs reached a maximum within 500 ms, decayed with a time-constant of 200-300 ms and were blocked by alpha, beta-methylene ATP. IJPs had a latency of > 1 s and showed much variation in amplitude and duration. IJPs could still be recorded in the presence of N-nitro-L-arginine methyl ester (L-NAME). In the double sucrose-gap, depolarization and associated contraction were reduced by alpha, beta-methylene ATP. Further reduction occurred with phentolamine and the remaining response was blocked by atropine. A large hyperpolarization could occur in response to a single stimulus but this decreased progressively with repeated stimulation. The hyperpolarization was partially blocked by L-NAME. CONCLUSIONS: The proximal urethra receives excitatory innervation involving three neurotransmitters, i.e. ATP, acetylcholine and noradrenaline, and inhibition is associated with hyperpolarization which results at least in part from action of non-nitrergic nerves.

[The control of smooth muscle tissues by nonadrenergic noncholinergic (NANC) nerve fibres in the autonomic nervous system].

Smooth muscle cells distributed in the visceral organs are under the control of the autonomic nervous system, and contraction or relaxation of the muscle cells plays an important physiological role in the control of blood pressure, motility of the digestive, respiratory and urinary tracts and secretion. Recent physiological, pharmacological and histochemical investigations indicate that neurotransmitters other than acetylcholine or noradrenaline are involved in peripheral autonomic neuro-effector transmission, and these neurotransmitters are generally termed non-adrenergic, non-cholinergic (NANC) neurotransmitters. The neurotransmitters responsible for excitatory and inhibitory NANC neurotransmission (e-NANC and i-NANC respectively) have not been conclusively identified, but ATP, nitric oxide (NO) and peptides such as VIP and substance P are candidates for these roles. In this review, we discuss the possible role of ATP and NO as e- or i-NANC neurotransmitter in the digestive, respiratory and urinary tracts. Much of the work on NANC innervation in the digestive tract has been carried out on the circular muscle layers of the ileum. This receives inhibitory NANC innervation with ATP responsible for fast relaxation and VIP, and possibly NO, for the slow response. Early and late excitatory junction potentials can be recorded in the presence of atropine. The second is due to substance P since it is blocked in the presence of spantide and by desensitization of the tissue with high doses of substance P. The transmitter responsible for the early NANC contraction has not been identified. Electrical field stimulation (EFS) applied to the tracheal smooth muscle during contraction induced by 5-HT in the presence of atropine and guanethidine elicited monophasic NANC relaxation. By contrast, NANC relaxation elicited in the smaller airways was biphasic, comprising an initial fast component followed by a second slow one. L-NAME selectively abolished the first component without affecting the second. VIP-antagonists or alpha-chymotrypsin considerably attenuated the amplitude of the L-NAME insensitive relaxation. These results indicate that at least two neurotransmitters, possibly NO or NO-containing compounds and VIP, are involved in i-NANC neurotransmission in the airway. In the urinary bladder a large, transient atropine resistant contraction occurs in response to pelvic nerve stimulation. This is blocked by alpha, beta methylene ATP suggesting that it is due to ATP. There is no evidence of inhibitory innervation. In the urethra contraction is completely blocked by atropine and guanethidine; a rapid NANC relaxation is abolished by drugs that block NO synthesis. Nerves containing peptides supply both urethra and bladder and may also be involved. These results suggest that all visceral smooth muscles may receive inhibitory NANC innervation involving NO. ATP produces contraction of the urinary bladder but relaxation of the digestive tract. The role of peptides is not yet clear but there is evidence that substance P may be an excitatory transmitter and VIP an inhibitory transmitter in many organs.

Innervation of the smooth muscle of the lower urinary tract.

The lower urinary tract in mammals consists of the bladder, a storage organ for urine, and the urethra through which urine is expelled to the outside. In the male the urethra extends outside the body as the penis. The lower urinary tract is innervated by pelvic (parasympathetic) nerves and hypogastric nerves which combine on each side at the pelvic plexus. Fibres pass to the bladder, urethra, genital organs, rectum and blood vessels. In addition to the pelvic and hypogastric nerves there is sacral outflow to skeletal muscle, such as the external sphincters of the urethra and anus, in the pudendal nerve and the sympathetic chain extends caudally with branches lying in the sacral outflow that supply the penis and other organs though not the bladder. Bladder evacuation and penile erection are produced by activity of the pelvic nerves whereas sympathetic activity usually dominates during urine storage and penile flaccidity, though it may not be essential for either.

Excitatory neurotransmission in the mammalian bladder and the effects of suramin.

OBJECTIVE: To compare the electrical and mechanical activity, and assess the effect of suramin on strips of detrusor from various species. MATERIALS AND METHODS: Bladder muscle strips from guinea-pigs, rabbits, monkeys and sheep and detrusor strips from humans (obtained at operation) were studied. The mechanical responses to nerve stimulation were recorded with a force transducer and electrical activity was recorded with the double sucrose gap. RESULTS: In all species acetylcholine was released from the nerves and a prolonged contraction was produced. A second transmitter, possibly adenosine triphosphate, produced a rapid transient contraction, the amplitude of which varied with the species. In the rabbit and guinea-pig the phasic contraction and accompanying depolarization were large, whereas in primates they were small and in sheep were intermediate. At high concentrations, suramin reduced the contraction and accompanying depolarization in rabbit and guinea-pig muscle but not in sheep. Suramin enhanced the late cholinergic responses and increased spontaneous mechanical activity in all species. These latter effects were not seen after desensitization of the receptors with the ATP analogue alpha, beta- methylene ATP. CONCLUSION: Although suramin reduces the excitatory effect of nerve activity in some species, it would produce little beneficial effect in the human hyperexcitable bladder as any inhibitory effect might be offset by the increase in spontaneous activity.

The effect of cromakalim on the electrical properties of and [86Rb+] efflux from normal and hypertrophied rat bladder.

1. The activity of smooth muscle strips from normal and hypertrophied rat bladders was compared. The hypertrophied bladders were produced by partially obstructing the urethra 8-13 weeks previously. 2. Spontaneous mechanical activity was more frequent and smaller in amplitude in strips from normal than hypertrophied bladders and was less sensitive to cromakalim, being reversibly abolished by cromakalim at 10(-6) mol/L compared with 10(-7) mol/lL for hypertrophied bladder. 3. The mean resting membrane potentials of smooth muscle cells from normal and hypertrophied rat bladders were -47.2 and -47.6 mV, respectively. Bursts of spontaneous action potentials, corresponding to the mechanical activity, were seen in some cells. 4. Nifedipine at 10(-6) mol/L had no significant effect on the resting membrane potential. Occasional single spikes occurred with increased duration and the after hyperpolarization was abolished. Cromakalim at 10(-5) mol/L produced hyperpolarization of 3-9 mV and, in the continued presence of the drug, occasional singe spikes could be recorded from both normal and hypertrophied bladders. 5. Nifedipine at 10(-6) mol/L abolished movement but did not significantly alter [86Rb+] efflux from strips from either normal or hypertrophied bladders. Addition of cromakalim at 5 x 10(-6) or 5 x 10(-5) mol/L in the presence or absence of nifedipine increased efflux from the normal bladder by 30-40%. In the hypertrophied bladder the efflux increased by about 14% and 28% in the presence of 5 x 10(-6) and 5 x 10(-5) mol/L cromakalim, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotransmission in the urinary bladder of rabbits and guinea pigs.

Alpha, beta-Methyleneadenosine 5'-triphosphate (alpha, beta-mATP) produced transient contraction of strips of bladder taken from rabbits or guinea pigs, and mechanical responses to field stimulation at 5-100 Hz were reduced by this drug by 5-20%. Atropine reduced responses by approximately 50%, and both drugs together by 80-95%. In double sucrose gap experiments on the rabbit bladder, alpha, beta-mATP selectively reduced but did not abolish an initial excitatory junction potential (ejp), and atropine selectively abolished a late depolarization. In the guinea pig, a single ejp was partially inhibited by either alpha,beta-mATP or atropine. Residual responses were further reduced by tetrodotoxin in both species. The initial ejp and late depolarization in the rabbit were reduced in parallel by hemicholinium over 2 h, suggesting that release of acetylcholine (ACh) and the second transmitter by nerves may be coupled. ACh but not ATP produced an increase in intracellular concentration of inositol trisphosphate in dispersed smooth muscle cells from the rabbit bladder; ATP but not carbachol produced a small transient current across the cell membrane in this species. It is concluded that ACh mobilizes intracellular Ca2+ for contraction, whereas the effect of ATP is dependent on extracellular Ca2+.

The physiology of penile erection.

Erection of the penis results from increase in blood flow into the corpora. The blood flows through the corpus spongiosum and glans which increase in volume, whereas the blood becomes trapped in the corpus cavernosum which becomes rigid as the pressure increases. The protrusion of the penis may be aided by relaxation of the retractor penis muscle. The major erectile fibres lie in the pelvic nerve and anti-erectile fibres in the sacral sympathetic outflow. The hypogastric nerves may contain both nerve types but there is considerable species and individual variation. The neurotransmitters mediating erection have yet to be determined. There is some evidence that acetylcholine is involved in the increase in blood flow through the corpus spongiosum but not in the corpus cavernosum. Vasoactive intestinal peptide may also have a role. It is possible that these and other substances interact to control the complete process. Erection is inhibited by noradrenaline released from sympathetic nerves, and this acts mainly on alpha-1 adrenoceptors within the penis and on the retractor penis muscle. During tumescence blood flows into the sinusoids from the helicine arterioles which supply them. The sinusoids become dilated due to relaxation of smooth muscle within the trabeculae. Blood may also be redirected from anastomoses between the dorsal arteries and corpus spongiosum through other helicine arterioles supplying the sinusoids of the corpus cavernosum. The significance of polsters (smooth muscle projections into the blood vessel lumen) remains controversial. Occlusion of venous drainage from the corpora cavernosa is both passive (due to increased corpus cavernosum pressure) and active. Relaxation of trabecular smooth muscle may also modify blood flow through the corpora cavernosa.

Effects of acute alcohol on penile tumescence in normal young men and dogs.

To determine the cause of the inhibition of sexual function observed in normal subjects with elevated blood alcohol concentrations (BAC), nocturnal penile tumescence (NPT) was monitored in 11 subjects over three consecutive nights. On the third night alcohol was administered (BAC = 0.154 g/100 mL). We observed no effect of alcohol on the size, duration, or number of erections. These results were confirmed in dogs. The latent period, magnitude, and duration of corpus cavernosal pressure changes produced by pelvic nerve stimulation were not affected by mean blood alcohol levels of 0.327 g/100 mL in three dogs. These results suggest that the inhibition of sexual response caused in conscious subjects by the ingestion of alcohol is not due to a suppression of the underlying spinal reflex but may be the result of its effect on perceptual or cognitive sexual mechanisms.

Responses of erectile tissue from impotent men to pharmacological agents.

Erectile tissue was removed from the corpora cavernosa of 25 impotent men undergoing surgery for insertion of penile prostheses. Strips, set up in an organ bath, were contracted by the alpha-adrenergic agonist phenylephrine. There was no significant difference between tissue taken from men with diabetes, alcoholism, Peyronie's disease or men with no obvious condition causing the impotence. The sensitivity of tissues from hypertensive patients was significantly reduced but this was probably due to drugs being taken for hypertension. Precontracted tissues could be relaxed by acetylcholine or isoprenaline. The responses, however, were inconsistent, so that no difference between the different groups of patients was apparent.

Prostaglandins and neurotransmission at the guinea pig and rabbit urinary bladder.

High concentrations of prostaglandins (PGE1, PGE2, or PGE2 alpha) (2 x 10(-6) M) produced a slow contraction of longitudinal strips of detrusor muscle taken from the bladders of guinea pigs and rabbits. At a lower concentration (10(-6) M) prostaglandins enhanced contractions produced by field stimulation of nerves in guinea pig but not rabbit strips. The contractions were not affected by indomethacin. Contractions of guinea pig strips in response to acetylcholine at 10(-4) M were enhanced by prostaglandins and unaffected by indomethacin. Membrane potentials of smooth muscle cells recorded with micro electrodes, were unchanged up to 10(-6) M PGE2. Above this the cells were depolarized with an increase in frequency of spontaneous action potentials. Synchronous recording of electrical and mechanical activity with the double sucrose gap indicated a decrease in amplitude of the evoked excitatory junction potential and action potential even when the contraction was enhanced in the presence of PGE2. Responses to repeated stimulation at 10 Hz for 1 min were progressively depressed. This trend was slightly reduced by PGE2 but unaffected by indomethacin. It is concluded that prostaglandins are not normally released by the nerves to the urinary bladder but are able to facilitate contraction in the guinea pig. This effect is probably on the excitatory-contraction coupling, possibly by mobilizing Ca2+. Some modification of transmitter release by the nerves may also occur.