A non-nitrergic smooth muscle relaxant factor released from rat urinary bladder by muscarinic receptor stimulation.

PURPOSE: To study the possible release of a relaxant factor from isolated rat bladder tissue. MATERIALS AND METHODS: Thoracic aortae and urinary bladders were obtained from 55 female Sprague-Dawley rats. The bladder body was used in its original tubular shape as the donor tissue in a co-axial bioassay system, and the aorta served as acceptor tissue. RESULTS: In a co-axial bioassay system with endothelium-free, norepinephrine-contracted, rat aortic preparations mounted within urothelium-intact urinary bladder, carbachol caused a concentration-dependent relaxation, amounting to 64+/-7% (n = 10) of the induced contraction, suggesting release of a relaxing factor. The relaxant effect of carbachol was lost if the urinary bladder segment was removed. However, the relaxation was affected neither by removal of the urothelium, nor by bladder segment inversion. It was resistant to inhibition of the L-arginine/nitric oxide and cyclo-oxygenase pathways, and unaffected by beta-adrenoceptor blockade and K+ channel inhibition. The relaxation was not associated with any significant changes in the intracellular levels of cGMP or cAMP. CONCLUSION: A previously unrecognized non-adrenergic, non-nitrergic, non-prostanoid inhibitory mediator is released from the rat urinary bladder by muscarinic receptor stimulation. The physiological importance of such a factor remains to be established.

Muscarinic receptor stimulation of phosphoinositide hydrolysis in the human isolated urinary bladder.

The stimulatory action of carbachol and acetylcholine (ACh) on phosphoinositide turnover, as well as their contractile effects, were investigated in human isolated detrusor muscle. Carbachol, and ACh in combination with 10(-7) M physostigmine, induced increases in phosphoinositide turnover. However, at all the concentrations tested, carbachol was more effective than ACh (plus physostigmine), and at the highest concentration used (10(-4) M), the difference was significant (p less than 0.05). Also in a Ca(2+)-free medium containing the chelator EGTA (10(-4) M), both agonists (10(-4) M) induced small but distinct increases in phosphoinositide breakdown. Carbachol and ACh contracted the detrusor preparations concentration-dependently, and the responses were almost identical when ACh was combined with 10(-7) M physostigmine. In Ca(2+)-free medium the agonists elicited a moderate but concentration-dependent contractile response at high concentrations. The results show that muscarinic receptor agonists stimulate phosphoinositide turnover in the human bladder. Possibly, this effect is coupled to multiple muscarinic receptor subtypes. More studies are required to elucidate to what extent phosphoinositide breakdown participates in the contractile activation of this tissue.

K(+)-channel openers for relaxation of isolated penile erectile tissue from rabbit.

The effects of the K(+)-channel openers (KCOs) cromakalim (BRL 34915) and pinacidil were investigated and compared with those of papaverine on isolated corpus cavernosum from rabbit. Preparations were mounted in organ baths and isometric tension was recorded. Spontaneous contractile activity was effectively abolished by the KCOs tested, cromakalim being the most potent of them. The KCOs concentration-dependently and effectively depressed electrically induced contractions and also contractions induced by exogenously applied noradrenaline and by low (less than or equal to 20 mM) concentrations of K+. Cromakalim was three to four times more potent than pinacidil. Pinacidil and cromakalim were shown to cause increases in the efflux of 86Rb from preloaded cavernous tissue. Papaverine also effectively depressed spontaneous contractile activity, and contractions evoked by electrical stimulation and noradrenaline. It had a potency 19 to 36 times lower than that of cromakalim. However, papaverine did not increase 86Rb efflux from preloaded tissue. The results show that cromakalim and pinacidil effectively relax penile erectile tissue, probably by the opening of K(+)-channels and subsequent hyperpolarization. Further investigations on human material seems motivated in order to elucidate if the principle of K(+)-channel opening offers any therapeutic advantages to other drugs in the diagnosis and treatment of penile erectile dysfunction.

Effects of cromakalim (BRL 34915) and pinacidil on normal and hypertrophied rat detrusor in vitro.

Normal and hypertrophied rat detrusor were investigated in vitro with regard to effects of the K(+)-channel openers pinacidil and cromakalim. Both drugs abolished spontaneous contractile activity and induced a relaxation of normal and hypertrophied detrusor preparations. In both types of preparation, contractions elicited by K+, carbachol or electrical field stimulation were depressed in the presence of the K(+)-channel openers. Responses induced by K+ or electrical stimulation were more reduced in the hypertrophied than in the normal detrusor. Both K(+)-channel openers increased the efflux of 86Rb+ in a concentration-dependent way and this increase was similar in normal and hypertrophied detrusor. If applicable to man, this data suggest that K(+)-channel openers may be effective in the treatment of bladder instability secondary to outflow obstruction.

The action of pinacidil in the isolated human bladder.

The need for effective symptomatic treatment of patients with detrusor hyperactivity is widely recognized. In search of new principles of decreasing bladder contraction we have studied the effects of pinacidil on the isolated human bladder. Pinacidil is a recently developed antihypertensive agent classified as a K+ channel opener, and is believed to depress smooth muscle activity by this action. Pinacidil concentration-dependently depressed contractions elicited by carbachol, low concentrations of K+ (less than 60 mM) and electrical stimulation. In addition it caused a concentration-related increase in the efflux of 86Rb from preloaded detrusor cells. The effects on 86Rb efflux could be inhibited by tetraethylammonium chloride and procaine, but not by apamin, agents known to block K+-channels. The results support the view that part of the pinacidil effect on the human bladder is caused by an opening of K+-channels, efflux of K+ and subsequent hyperpolarization of the detrusor cells. Clinical testing of this new therapeutic principle for treatment of bladder hyperactivity seems justified.

Characterization of contraction-mediating prostanoid receptors in human penile erectile tissues.

The effects of the thromboxane-receptor antagonist L-636,499 were studied on contractions induced by the thromboxane A2 mimic U44069 and by prostaglandin F2 alpha in isolated preparations of the human corpus cavernosum and corpus spongiosum. The objectives were to characterize prostanoid receptors and, in particular, to elucidate whether more than one receptor was involved in prostanoid-induced contraction of penile erectile tissues. L-636,499 at concentrations 10(-6) M to 3 x 10(-5) M induced a parallel shift to the right of the concentration-response curve to U44069 in both corpus cavernosum and corpus spongiosum suggesting competitive antagonism. Schild plots using U44069 as the agonist and L-636,499 as the antagonist revealed slope indexes near unity in both tissues, and the pA2 values were almost identical. In contrast, L-636,499 concentration-dependently reduced the maximum response to prostaglandin F2 alpha, indicating a non-competitive action. The results suggest that the main contraction-mediating prostanoid receptor in human penile erectile tissues is a thromboxane A2 sensitive receptor. However, the presence of more than one contraction-mediating prostanoid receptor cannot be excluded.

Immunoreactive arginine vasopressin (AVP) and effects of AVP in the human vas deferens.

Immunoreactive (IR) arginine vasopressin (AVP) was found to occur in the epididymal part of the human vas deferens. Segments from nine different subjects all contained IR-AVP in concentrations ranging from 37 to 717 fmol/gm. wet weight, concentrations severalfold higher than those normally found in the circulation. IR-AVP was shown by high performance liquid chromatography to elute in the same position as synthetic AVP. AVP added to isolated preparations of the human vas deferens induced concentration-related repetitive phasic contractions without significant changes of baseline tension. These contractions seemed to be mediated via stimulation of vasopressin V1-receptors and were abolished in the presence of vasopressin antagonists. Contractions induced by electrical field stimulation were frequency-dependent and sensitive to tetrodotoxin and prazosin. They were not affected by the vasopressin antagonists used. AVP increased the response to electrical field stimulation and this effect was inhibited by vasopressin antagonists. The results suggest either that circulating AVP is taken up and accumulated by the human vas deferens, and/or that AVP is synthesized locally. They do not suggest co-release of AVP and noradrenaline from nerve endings. The physiological role of the AVP occurring in the human vas deferens remains to be established.

Tetrodotoxin-resistant contractions induced by electrical stimulation of bladder muscle from man, rabbit and rat.

Isolated detrusor preparations from man, rabbit and rat were suspended in an organ bath and isometric tension was recorded. The preparations were stimulated electrically in the presence of Bay K8644 and nifedipine before and after neuronal blockade with tetrodotoxin. Transmural electrical stimulation produced frequency-dependent contractions in all preparations. Bay K8644 significantly increased and nifedipine decreased these contractions. TTX effectively suppressed the response to electrical field stimulation in all species. When Bay K8644 was added to TTX blocked preparations, the responses to electrical stimulation were partly restored in bladder strips from man and rat. No increase in response was seen in the rabbit preparations. However, if the extracellular K+-concentration was increased to 10 mM (which per se did not affect the response) Bay K8644 significantly increased the contractions. All responses elicited by electrical stimulation in the presence of TTX were abolished by nifedipine. It is concluded that if the bladder smooth muscle is exposed to factors that can increase its sensitivity to contractile agents, this may result in uncontrolled (unstable) bladder contractions. Such contractions may use the 'normal' transmitter substances, but may be triggered at a lower stimulus intensity than normal. As a non-specific increase in membrane excitability seems to be associated with an influx of calcium through voltage-sensitive calcium channels, calcium antagonists, together with agents specifically blocking relevant transmitter substances, would offer an effective therapy against the unstable bladder.

Effects of pinacidil on bladder muscle.

Infravesical outflow obstruction and bladder hypertrophy are often associated with bladder hyperactivity causing frequency, urge and urinary incontinence. This hyperactivity may be due to a supersensitivity to depolarising stimuli. Drugs that inhibit smooth muscle activity by opening K+ channels, resulting in hyperpolarisation, would therefore seem to be an attractive therapeutic principle. Pinacidil is an effective vasodilator classified as a K+ channel opener. The drug has been shown to effectively depress spontaneous contractile activity, the contractions induced by low (less than 40 mmol/L) concentrations of K+, carbachol and by electrical stimulation of nerves in isolated normal human bladder tissue and also in normal and hypertrophied rat bladder. The effect was more pronounced in hypertrophied detrusor. Pinacidil in concentrations inhibiting muscle activity also increased the efflux of 86Rb in bladder tissue. In vivo pinacidil suppressed spontaneous contractile activity in rats with infravesical bladder obstruction and detrusor hypertrophy. The findings make K+ channel openers an interesting, potentially useful therapeutic principle in hyperactivity associated with bladder hypertrophy.

Effects of some calcium channel blockers on isolated human penile erectile tissues.

The effects of the calcium channel blockers (CCBs) verapamil, nifedipine and diltiazem on contractile activation of isolated human penile erectile tissues were investigated. Specimens of the corpus spongiosum (CS) and corpora cavernosa (CC) were obtained from men with a history of normal penile erection undergoing cystourethrectomy because of bladder malignancy. Preparations were mounted in organ baths and isometric tension was recorded. Deprivation of extracellular calcium abolished electrically induced contractions in both CS and CC preparations within 15 min.; norepinephrine (NE)-induced contractions were reduced by 90% (CS) and 83% (CC) after 30 min. All the CCBs reduced electrically induced contractions concentration-dependently, nifedipine being the most potent agent. Contractions induced by exogenous NE were depressed by about 50%, whereas high K+ (124 mM) induced responses were abolished. It is concluded that contraction in penile erectile tissues is mediated mainly by neuronally released NE stimulating postjunctional alpha-adrenoceptors. The contraction is highly dependent on extracellular calcium and can partly be inhibited by CCBs. It cannot be excluded that some CCBs injected intracavernosally may be useful for diagnosis and even treatment of erectile dysfunction. However, calcium channel blockade may not be as effective as a therapeutic principle as blockade of alpha-adrenoceptors.

Calcium channel blockade and contractile responses in the isolated human vas deferens.

The effects of removal of extracellular calcium and of the calcium channel blockers nifedipine, verapamil and diltiazem were studied on contractions induced by electrical field stimulation and high K+-solution in isolated preparations of the human vas deferens. Electrically induced contractions were blocked by tetrodotoxin and alpha-adrenoceptor blockade. They were abolished in calcium-deficient medium, and suppressed by the calcium channel blockers in the order of potency nifedipine greater than verapamil greater than diltiazem. The maximum blocking effect of nifedipine was approximately 40%. All the blockers practically abolished K+-induced contractions. It is concluded that even if the contractile response of the human vas deferens to electrical stimulation is dependent on extracellular calcium, calcium channel blockers seem to have only a limited effect on this contraction and their capability of impairing the function of the vas deferens in patients is questioned.

Source of calcium for contractions induced by depolarization and muscarinic receptor stimulation in rabbit urinary bladder.

Omission of calcium or the inclusion of lanthanum in the bathing medium resulted in an almost complete inhibition of contractile responses induced by either K+ depolarization or carbachol in strips of rabbit urinary bladder. D-600 inhibited K+-induced contractions significantly more than carbachol-induced responses. The influx of 45Ca into cells was stimulated both by K+ depolarization and carbachol. Over a 2-min period the increase in 45Ca influx induced by high K+ and carbachol was 98 and 65%, respectively. Both lanthanum and D-600 blocked 45Ca influx stimulated by either K+ depolarization or carbachol. The inhibition of 45Ca influx by these calcium-channel blocking agents, particularly by D-600, was dependent on the length of exposure. Application of carbachol during 45Ca efflux in pre-loaded muscle strips had no effect on the rate of 45Ca efflux. These results indicate that the contractile responses of the urinary bladder to depolarization and to carbachol are highly dependent on an extracellular source of calcium.

Characterization of immunoreactive arginine vasopressin (AVP) in and effects of AVP on isolated human penile erectile tissues.

Arginine vasopressin (AVP), which has been shown to have a marked contractant effect on human penile erectile tissues, particularly the corpus spongiosum (CS), was demonstrated to occur in these tissues. Out of nine CS samples, eight contained vasopressin-like immunoreactivity in concentrations ranging from 3.5 to 107.2 fmol./gm. wet weight. These amounts suggest that the hormone is taken up and/or synthesized locally. The effects of four different vasopressin antagonists were characterized on human CS strips, and they were found to effectively inhibit the contractile response to exogenous AVP. However, none of these antagonists had any effects on electrically induced contractions in penile erectile tissues. These results do not favour the view that AVP is released on electrical stimulation in amounts that contract the erectile tissues. Whether or not the peptide is involved in the mechanisms controlling penile erection remains unclear.

Effects of calcium, calcium channel blockers and Bay K 8644 on contractions induced by muscarinic receptor stimulation of isolated bladder muscle from rabbit and man.

In isolated bladder smooth muscle from both rabbit and man, carbachol-induced contractions were reduced by the calcium channel blocker nifedipine, whereas the calcium channel promotor Bay K 8644 had no effect. In nominally calcium-free medium containing 10(-4) M EGTA, carbachol-induced contractions were reduced by 69% (rabbit) and 87% (man). These contractions were abolished by nifedipine, whereas Bay K 8644 significantly increased their amplitude, in rabbit preparations almost to control level. Electrical field stimulation produced contractions which could be suppressed by scopolamine by about 50% (rabbit) and more than 90% (man). These contractions were abolished by calcium-free medium (10(-4) M EGTA), suppressed by nifedipine, but significantly enhanced by Bay K 8644. The depressant effects of nifedipine, verapamil and diltiazem were reversed by Bay K 8644. The calcium channel blockers relaxed K+-induced contractions to base line, and this action was counteracted by Bay K 8644, less effectively when relaxations were induced by diltiazem. It is concluded that contractions produced by muscarinic receptor stimulation are primarily dependent on calcium bound to the outside of the membrane of the smooth muscle, and/or coming from the extracellular medium. Electrically evoked, scopolamine sensitive contractions seem to be mediated by a mechanism different from that of contractions produced by exogenously added muscarinic receptor agonist. The present data support the view that combined blockade of muscarinic receptors and calcium channels is an effective way of inhibiting bladder contractions in both rabbit and man.