Long-lasting blockade of P2-receptors of the urinary bladder in vivo following photolysis of arylazido aminopropionyl ATP, a photoaffinity label

In the present study the possibility of provoking an irreversible blockade of P2-receptors in vivo by photolyzing ANAPP3 was investigated. ANAPP3 administered as an intraarterial infusion produced a short-lived blockade of P2-receptor mediated contractions of cat urinary bladders in situ. However, irradiation of the bladders with visible light during the infusion of ANAPP3 resulted in a blockade of the response for at least 120 minutes. Thus, photoactivation of ANAPP3 in vivo produced an essentially irreversible blockade of P2-purinergic receptors.     

Effect of arylazido aminopropionyl ATP (ANAPP3), a putative ATP antagonist,on ATP responses of isolated guinea pig smooth muscle

Arylazido aminopropionyl ATP (ANAPP₃), a photoaffinity analogue of adenosine 5′-triphosphate, photoactivated with visible light (+hv), specifically and irreversibly antagonized ATP contractions of the guinea pig vas deferens. ANAPP₃ (30 μM) antagonized responses to exogenously added ATP in untreated, and in tissues pretreated with indomethacin (2.9 μM) and 6-(2-hydroxy-5-nitrobenzyl)-thio guanosine (10 μM). It was of interest to see if this pharmacological antagonist of ATP could be used to assess the validity of the purinergic nerve hypothesis by allowing a differentiation between, or proof of the identity of, responses to ATP and the non-adrenergic inhibitory transmitter in guinea pig stomach fundus. After photoactivation (+hv) in the organ bath and subsequent washout, ANAPP₃ (30 and 100 μM) failed to antagonize relaxant responses to ATP (1.0 – 1000 μM) in fundic strips. In addition ANAPP₃ failed to antagonize ATP-induced inhibition of the twitch response in electrically stimulated guinea pig ileum longitudinal muscle strips. We conclude that ANAPP₃ does not antagonize all actions of ATP, which may limit its usefulness in assessing the above hypothesis. Results with this compound suggest that ATP excitatory receptors may differ from those mediating relaxation and other ATP actions.     

Motilin acts within the CNS to inhibit urinary bladder contractions

Although peripheral actions have been shown for the brain-gut peptide, motilin, its localization in the CNS of mammals suggests some physiological role at this site. In the present experiments intracerebroventricular or intrathecal, but not peripheral, administrations of motilin produced a dose-related and naloxone reversible inhibition of the micturition reflex. Cross-tolerance was demonstrated between motilin and morphine in this respect. These data suggest a physiological role for motilin within CNS to alter urinary bladder motility, possibly through an enkephalinergic or naloxone-sensitive link.     

The cellular specificity of the effect of vasopressin on toad urinary bladder

Summary Phase and electron micrographs of toad bladders were obtained following dilution of bathing media in the presence and absence of vasopressin. Dilution of the mucosal medium alone resulted in no morphologic changes. Subsequent addition of vasopressin produced an increase in the cell volume of the granular cells, manifested by some or all of the following changes: increased area of granular cell profiles as observed in sections, rounding of the cell nucleus, displacement of the two components of the nuclear envelope, loss of nuclear heterochromatin, sacculation of the endoplasmic reticulum and the Golgi apparatus, and reduction in the electron density of the cell cytoplasm. No such morphologic changes were noted in the other cell types comprising the mucosal epithelium — the mitochondria-rich, the goblet, and the basal cells. On the other hand, dilution of the serosal bathing medium in the absence of vasopressin caused a marked increase in the cell volume of all these cell types. The results demonstrate that the action of vasopressin to enhance bulk water flow across toad bladder is exerted specifically on the apical surface of the granular cells. It is suggested that the hormonal effect on sodium transport may also be limited to the granular cells. The route of osmotic water flow and the possible role of the other mucosal epithelial cells is discussed.     

Interneuronal synapses in the local ganglia of the cat urinary bladder.

The interneuronal synapses of the urinary bladder in the cat were studied by electron microscopy. The great majority of the fibres containing vesicles are found within the ganglia occurring in the trigonum area. Morphologically differentiated synaptic contacts could be observed on the surface of the local neurons and between the different nerve processes. The presynaptic terminals can be divided into three types based on a combination of synaptic vesicles. Type I terminals, presumably cholinergic synaptic terminals, contain only small clear vesicles of 40-50 nm in diameter. Type II terminals, presumably adrenergic terminals, are characterized by small granulated vesicles of 40-60 nm in diameter. Type III terminals, probably of local origin, contain a variable number of large granulated vesicles of 80-140 nm in diameter. Occasionally, a single nerve fibre contacted several (two or four) other nerve processes forming a typical synapse. In other cases, on one nerve cell soma or on other nerve processes there are two or three different-type nerve terminals establishing synapses. It might be inferred from these observations that convergence and divergence can occur in the local ganglia and that cholinergic and adrenergic synaptic terminals can modulate the ganglionic activity. However, a local circuit also can play an important role in coordinating the function of the bladder.     

The effect of indomethacin and substance P on the guinea pig urinary bladder

The effects of indomethacin on the responses of the guinea pig urinary bladder to nerve stimulation, acetylcholine, adenosine 5′ triphosphate and Substance P have been investigated. Indomethacin alone had no significant effect on responses of the bladder to nerve stimulation but did significantly reduce the atropine-resistant contractions. Responses of the tissue to acetylcholine were unaffected by indomethacin but responses to Substance P were significantly reduced. Only the highest dose of ATP (10^?3 M) was significantly reduced by indomethacin. The possibility that Substance P is the transmitter responsible for the atropine-resistant contractions of the urinary bladder to nerve stimulation is discussed.     

H+/ATP stoichiometry of proton pump of turtle urinary bladder

Urinary acidification in the turtle urinary bladder is due to a reversible proton-translocating ATPase. To estimate the H+/ATP stoichiometry of this pump, we measured the delta G'ATP in the epithelial cells and the maximum e.m.f. generated by the pump. The latter is the maximal transepithelial electrochemical gradient for protons placed across the epithelium that is needed to nullify the rate of transport and averaged 179 +/- 7 mV. The delta G'ATP averaged 50.1 kJ/mol. The H+/ATP stoichiometry of these bladders was 2.92 +/- 0.1. In other experiments, the bladders were poisoned by iodoacetate and cyanide and a variable transepithelial electrochemical gradient for protons was placed across them. It was noted that ATP synthesis occurred at a transepithelial electrochemical gradient for protons greater than 120 mV. The delta G'ATP in other bladders treated identically averaged 40.0 kJ/mol, giving a H+/ATP stoichiometry of 3.4 +/- 0.1. We conclude that the H+/ATP stoichiometry of the proton pump of turtle urinary bladder is approximately 3.     

The effect of partial outlet obstruction on prostaglandin generation in the rabbit urinary bladder

Partial outlet obstruction of the urinary bladder has been demonstrated to induce specific dysfunctions in cellular and sub-cellular membrane structures within the bladder's smooth muscle and mucosal compartments. Recent studies have linked these membrane dysfunctions to alterations in phospholipid metabolism leading to mobilization of free arachidonic acid, the precursor for synthesis of prostaglandins (PG). The purpose of this study was to determine if partial outlet obstruction of the urinary bladder induces changes in the capacity of bladder smooth muscle and mucosa to generate PG. PG were isolated from control and partially obstructed urinary bladder smooth muscle and mucosa of male New Zealand White (NZW) rabbits. PG concentrations (PGE2, PGF2alpha and PGI2, as its stable metabolite 6-keto-PGF1alpha) were determined after 30 minute incubations using enzyme-linked immunoassay (ELISA) kits. In both control and obstructed rabbit urinary bladders, PG generation was significantly higher in isolated mucosa than muscle tissues. A significantly higher concentration of PGF2alpha, and 6-keto-PGF1alpha was measured in obstructed muscle tissue relative to controls. The concentration of 6-keto-PGF1alpha was also significantly higher than the concentrations measured for PGE2 and PGF2alpha in both control and obstructed smooth muscle samples. The generation of PGE2 was significantly higher in rabbit urinary bladder mucosa than either PGF2alpha or 6-keto-PGF1alpha in both control and obstructed samples. The capacity of obstructed mucosal tissue to generate 6-keto-PGF1alpha was significantly higher than control tissue, while no significant differences in PGE or PGF2alpha generation were noted. These data suggest obstruction of the urinary bladder induce specific elevations in PG in both smooth muscle and mucosal tissues.     

The effect of acid-base changes on vasopressin-stimulated water flow in toad urinary bladder

Water flow was measured gravimetrically in the presence and absence of vasopressin across the toad urinary bladder. Four groups of toads in different states of acid-base balance were used; a normal group, a group in NH₄Cl induced metabolic acidosis, respiratory acidosis, and a group in NaHCO₃ induced metabolic alkalosis. Vasopressin induced water flow was significantly reduced during metabolic acidosis and respiratory acidosis. Metabolic alkalosis had no effect on the hydro-osmotic response to vasopressin. Dibutyryl cyclic-AMP-stimulated water flow on the other hand was not affected by either a metabolic or respiratory acidosis. Treatment with indomethacin was able to reverse the observed reduction in the vasopressin-stimulated water flow response in the toad bladder during metabolic and respiratory acidosis. We conclude that the vasopressin stimulated water flow is altered during acidosis and evidence suggests that prostaglandins may be involved in the observed reduction in vasopressin-stimulated water flow.     

The effect of aldosterone on cyclic nucleotide phosphodiesterase activity in toad urinary bladder

Cyclic AMP phosphodiesterase activity was assayed in the 700 xg supernatant solution of homogenates of epithelial cells scraped from toad urinary bladders. The activity of the enzyme was lower in cells obtained from bladders incubated with aldosterone for 24 hours than in cells from paired tissue incubated without aldosterone. This difference may well account for the permissive effect of aldosterone on the physiologic and biochemical responses of the toad bladder to vasopressin.     

The effect of diuretics and vasopressin on the osmotic permeability of the frog urinary bladder.

1. Large concentrations (in mM) of ethacrynic acid (0.1), furosemide (1.0), theophylline (5.0) and osmotic diuretics (100.0) sharply increased the flux of water along an osmotic gradient through the frog urinary bladder wall. Spironolactone (0.1), and hydrochlorothiazide (5.0) showed only a weak action on osmotic permeability. MercusalR, clopamide and triamterene did not affect water transport. 2. The presence of 0.2--1.0 mU/ml vasopressin (ADH) after pretreatment with a diuretic did not result in summation of the effects of both drugs used. 0.01--0.1 mM ethacrynic acid and 0.01 mM MercusalR significantly decreased the reaction to ADH. 1.0 mM furosemide, 0.1 mM spironolactone, 0.01 mM clopamide and 0.8 mM acetazolamide did not change the reaction to ADH. A reduction in the cellular response to ADH and a decrease in the osmotic permeability of the tubular wall may be responsible in part for the diuretic action of ethacrynic acid and MercusalR.     

Effects of elastic loads on the contractions of cat muscles

The nerves to plantaris and soleus muscles in the cat were stimulated with maximal single shocks and with random stimulus trains which produced partially fused contractions. In order to obtain information on the mechanism of muscular contraction, the effects of allowing the muscles to shorten against various elastic loads were studied in the time domain and in the frequency domain. When springs of increasing stiffness were placed in series with the muscle, the twitch tension increased greatly. The gain of the frequency response curve was also much greater with stiffer springs. The shape of the frequency response curve for plantaris muscle could usually be described by that expected for a second-order system with two real time constants or rate constants. The rate constants changed in qualitatively similar ways in response to increased stiffness of an elastic load, increased muscle length and increased mean rate of nerve stimulation. These results are in agreement with the hypothesis that the linear responses of muscles working against elastic loads are determined by the values of two rate constants. Thus, of the many processes associated with contraction, only two are rate-limiting: one associated with the viscoelastic properties of muscle and the second associated with the reuptake of Ca into the sarcoplasmic reticulum. Non-linear aspects of muscular contraction are also discussed. These are more prominent in soleus muscle than in plantaris muscle.Graduate student of the Medical Research Council of Canada.Formerly a Post-doctoral Fellow of the Muscular Dystrophy Association of Canada.     

Transient contractions of urinary bladder smooth muscle are drivers of afferent nerve activity during filling

Activation of afferent nerves during urinary bladder (UB) filling conveys the sensation of UB fullness to the central nervous system (CNS). Although this sensory outflow is presumed to reflect graded increases in pressure associated with filling, UBs also exhibit nonvoiding, transient contractions (TCs) that cause small, rapid increases in intravesical pressure. Here, using an ex vivo mouse bladder preparation, we explored the relative contributions of filling pressure and TC-induced pressure transients to sensory nerve stimulation. Continuous UB filling caused an increase in afferent nerve activity composed of a graded increase in baseline activity and activity associated with increases in intravesical pressure produced by TCs. For each ∼4-mmHg pressure increase, filling pressure increased baseline afferent activity by ∼60 action potentials per second. In contrast, a similar pressure elevation induced by a TC evoked an ∼10-fold greater increase in afferent activity. Filling pressure did not affect TC frequency but did increase the TC rate of rise, reflecting a change in the length-tension relationship of detrusor smooth muscle. The frequency of afferent bursts depended on the TC rate of rise and peaked before maximum pressure. Inhibition of small- and large-conductance Ca²⁺-activated K⁺ (SK and BK) channels increased TC amplitude and afferent nerve activity. After inhibiting detrusor muscle contractility, simulating the waveform of a TC by gently compressing the bladder evoked similar increases in afferent activity. Notably, afferent activity elicited by simulated TCs was augmented by SK channel inhibition. Our results show that afferent nerve activity evoked by TCs represents the majority of afferent outflow conveyed to the CNS during UB filling and suggest that the maximum TC rate of rise corresponds to an optimal length-tension relationship for efficient UB contraction. Furthermore, our findings implicate SK channels in controlling the gain of sensory outflow independent of UB contractility.     

The effect of indomethacin and adenosine 5'-triphosphate on the excitatory innervation of the rate urinary bladder

Adenosine 5'-triphosphate (ATP) (20-400 microM) contracted 48% of isolated rat urinary bladder preparations but induced no response in the remainder. The response to ATP never exceeded 25% of the response to electrical stimulation in the presence of indomethacin (50 microM) plus hyoscine (25 microM) and usually developed more slowly than that to electrical stimulation. Autoinhibition could be produced to ATP by incubating the tissue with ATP (200 microM) for 20 min. Incubation of the tissue with ATP (200 microM) for 60 min in the presence of indomethacin (50 microM) and either hyoscine (25 microM) or hemicholinium-3 (500 microM) reduced but failed to abolish responses to electrical stimulation. Responses to acetylcholine were not affected by ATP (200 microM) in the presence of indomethacin and the output of acetylcholine induced by neuronal stimulation at 10 Hz was not inhibited by ATP (200 microM) or by indomethacin (50 microM). The results suggest a possible modulatory role for ATP in the excitatory innervation of the rat urinary bladder.     

Ultrastructure of the nerve cells and fibres in the urinary bladder wall of the cat.

The nerve elements in the urinary bladder of the cat were studied by electron microscopy. According to their ultrastructure, nerve cell somata can be classified into three types: the large cells with a cytoplasm rich in organelles, several processes and numerous synaptic contacts on their surface; the cytoplasm contained 80- 120-nm granulated vesicles. The second type is poor in cytoplasmic organelles and has very few processes and virtually no synaptic contacts on the soma. The third type contains numerous large 160- to 220-nm 'neurosecretory' vesicles in the cytoplasm. According to the morphology of the vesicle population, four types of nerve processes could be distinguished: Type a, with a dominant population of small (40-60 nm) agranular vesicles. These are thought to be sacral parasympathetic fibres. Type b, with small (40-60 nm) granular vesicles, which may be the noradrenergic sympathetic fibres. Type c, with 80- to 120-nm granulated vesicles, probably of local origin. Typed d, with large 160- to 220-nm 'neurosecretory' vesicles also of local origin. Different types of nerve fibres are converging on the local nerve cells. This suggests that the local circuits can play an important role in coordinating the function of the bladder. Therefore, ganglia may be considered as an elementary functional unit.