Desensitization Of Purinoceptors Research Articles

Changed Responsiveness of the Detrusor in Rabbits With Alloxan Induced Hyperglycemia: Possible Role of 5-Hydroxytryptamine for Diabetic Bladder Dysfunction

We assessed whether the responsiveness of the detrusor is changed in rabbits with alloxan induced hyperglycemia. Hyperglycemia was induced by a bolus intravenous injection of alloxan (60 mg./kg.) in Japanese White male rabbits. At 16 weeks after alloxan detrusor muscle strips prepared from age matched normoglycemic and hyperglycemic rabbits were mounted in organ chambers. Contractile responses to KCl, carbachol, adenosine triphosphate, 5-hydroxytryptamine and electrical field stimulation were compared in the 2 groups. The effect of sarpogrelate as a selective antagonist of 5-hydroxytryptamine 2A receptor on the contractile response to 5-hydroxytryptamine was also compared. The current experiments demonstrated that hyperglycemia caused significant decreases in neurogenic and carbachol induced contractions accompanied by unchanged adenosine triphosphate and KCl induced contractions. Neurogenic bladder contraction in the hyperglycemic rabbit was significantly potentiated by exogenously applied 5-hydroxytryptamine. Potentiation was detectable even after the desensitization of purinoceptors but undetectable in the presence of atropine. Hyperglycemia resulted in enhancement of the 5-hydroxytryptamine induced bladder contraction. Sarpogrelate tended to normalize the enhanced contraction. The decrease in neurogenic bladder contraction possibly accompanied by the decreased density of muscarinic receptors, the potentiation of neurogenic bladder contraction with 5-hydroxytryptamine probably due to facilitated cholinergic transmission and the enhanced contractility to 5-hydroxytryptamine would be at least in part involved in bladder dysfunction associated with hyperglycemia.

Na+ reabsorption in cultured rat epididymal epithelium via the Na+/nucleoside cotransporter.

The effect of nucleoside on Na+ reabsorption via Na+/nucleoside cotransporter in cultured rat epididymal epithelia was studied by short-circuit current (Isc) technique. Guanosine added apically stimulated Isc in a dose-dependent manner, with a median effective concentration (EC50) of 7 +/- 2 microM (mean +/- SEM). Removal of Na+ from the apical bathing solution or pretreatment with a nonspecific Na+/nucleoside cotransporter inhibitor, phloridzin, completely blocked the Isc response to guanosine. Moreover, the guanosine response was abolished by pretreatment of the tissue with ouabain, a Na+/K+-ATPase inhibitor, suggesting the involvement of Na+/nucleoside cotransporter on the apical side and Na+/K+-ATPase on the basolateral side in Na+ reabsorption. In contrast, the Isc response to guanosine was not affected after desensitization of purinoceptors by ATP. Addition of the Na+/K+/2Cl- symport inhibitor bumetanide to the basolateral side or the nonspecific Cl- channel blocker diphenylamine-2-carboxylate to the apical side showed no effect on the Isc response to guanosine, excluding stimulation of Cl- secretion by guanosine as the cause of the guanosine-induced Isc. The Isc response to purine nucleoside (guanosine and inosine) was much higher than that to pyrimidine nucleoside (thymidine and cytidine). Consistent with substrate specificity, results of reverse transcription-polymerase chain reaction revealed mRNA for concentrative nucleoside transporter (CNT2), which is a purine nucleoside-selective Na+/nucleoside cotransporter in the epididymis, but not for CNT1. It is suggested that the Na+/nucleoside cotransporter (i.e., CNT2) may be one of the elements involved in Na+ and fluid reabsorption in the epididymis, thereby providing an optimal microenvironment for the maturation and storage of spermatozoa.

Functional antagonism between nitric oxide and ATP in the motor responses of guinea-pig ileum.

1. The interaction of nitric oxide and ATP in the non-adrenergic, non-cholinergic (NANC) motor responses and the presence of NADPH-diaphorase and quinacrine-positive myenteric neurones were studied on guinea-pig ileum using mechanographic, histochemical and quinacrine-fluorescence techniques. In the presence of phentolamine, propranolol and atropine, the non-precontracted longitudinally oriented organ bath preparations responded to sodium nitroprusside (1-100 microM) or ATP (5-50 microM) with tetrodotoxin (0.1 microM)-resistant relaxation or contraction, respectively. The effects of ATP were suramin (50 microM)- and apamin (5 microM)-sensitive. 2. The NANC motor responses elicited by electrical stimulation (0.8 ms, 1-20 Hz, 20 s) consisted of tetrodotoxin-sensitive relaxation phase followed by a phase of twitch-like and tonic contractions. 3. NG-nitro-L-arginine (L-NNA, 0.1-0.5 mM) inhibited or abolished the relaxation phase. L-arginine (0.5 mM), but not D-arginine (0.5 mM), restored the relaxation phase in L-NNA-pretreated preparations. The relaxation phase increased after ATP-induced desensitization of purinoceptors and in the presence of suramin (50 mciroM) but was abolished by apamin (5 microM). 4. The phase of contractions was enhanced by L-NNA (0.1-0.5 mM) and restored by L-arginine (0.5 mM). The twitch-like and tonic contractions were decreased during ATP-induced purinoceptor desensitization and in the presence of suramin (50 microLM). Apamin (5 microM) inhibited the tonic contractions. 5. The desensitization of purinoceptors by ATP did not change the L-NNA-induced inhibition of the relaxation phase but decreased the L-NNA-increased phase of contractions. L-NNA reduced the relaxation phase and increased the phase of contractions during purinoceptor desensitization. 6. We conclude that in the longitudinal muscle layer of the guinea-pig ileum, nitric oxide mediates the relaxation phase while ATP contributes via smooth muscle P2 purinoceptors to the phase of contractions suggesting a postjunctional functional antagonism between nitric oxide and ATP. The presence of NADPH-diaphorase- and quinacrine-positive neuronal cells and processes running to the muscle cells confirms a physiological role of nitric oxide and ATP in the ileal neurotransmission.

Activation of neurotensin receptors and purinoceptors in human colonie adenocarcinoma cells detected with the microphysiometer

Activation of endogenous neurotensin (NT) receptors and P 2-purinoceptors expressed by human colonic adenocarcinoma HT-29 cells increased extracellular acidification rates that were detected in the microphysiometer. NT (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-lle-Leu), NT[8–13] (Arg-Arg-ProTyr-Ile-Leu), NT[9–13] (Arg-Pro-Tyr-Ile-Leu), and NT1 (Nαmethyl-Arg-Lys-Pro-Trp-Tle-Leu [Tie = tert-leucine]) were full agonists, whereas XL 775 (N-[N-[2-[3-[[6-amino-l-oxo-2-[[(phenylmethoxy)carbonyl]-amino] hexyl]amirio]phenyl]-3-(4-hydroxyphenyl)-l-oxo-2-propenyl]-L-isoleucyl]-L-leucine) was a partial agonist for activating NT receptors expressed by HT-29 cells. Desensitization induced by NT was rapid and monophasic with 85% of the initial response lost by a 30-s exposure. Once initiated, the rate and extent of desensitization were similar for different concentrations of a given agonist, for agonists of different potencies, and for agonists of different efficacies, which suggests that desensitization may be independent of receptor occupancy or agonist efficacy. Resensitization was a much slower process, requiring 60 min before the full agonist response to NT was recovered. ATP, via P 2-purinoceptors, also activated cellular acidification rates in a concentration-dependent manner. ATP induced a biphasic desensitization of purinoceptors with a loss of ca. 50% of the initial stimulation detectable between 30 and 90 s of exposure to the agonist. Desensitization of NT receptors did not influence the activation of P 2-purinoceptors by ATP, suggesting there was no heterologous desensitization between the two types of receptors. Superfusion with NT receptor agonists for 15 min at concentrations that did not elicit changes in extracellular acidification rates blocked, in a concentration-dependent manner, the agonist response induced by 100 nM NT. This may reflect sequestration of the receptor. These results suggest that the high agonist affinity state of NT receptors may modulate receptor sequestration, whereas activation of the low agonist affinity state may be linked to cellular metabolism. Comparison of our results with published data found differences as well as similarities of NT responses among three lines of HT-29 cells.

Pattern of nonadrenergic, noncholinergic responses during short- or long-lasting electrical stimulation in guinea-pig ileum

1. 1. The pattern of responses of longitudinally oriented guinea pig ileum organ bath preparations was studied during short- (1–5 sec) or long-lasting (20 sec) electrical field stimulation (EFS, 0.8 msec, 40 V, 1–20 Hz). 2. 2. In the presence of phentolamine (5 μM), propranolol (5 μM), and atropine (3 μM), the EFS elicited nonadrenergic, noncholinergic (NANC), tetrodotoxin (0.3 μM)-sensitive responses. 3. 3. The 1-sec EFS evoked relaxation. The response to 5-sec EFS consisted of relaxation followed by twitch, whereas relaxation, twitch and tonic contraction characterized the NANC response to 20-sec EFS. The maximum relaxation was observed at 10-Hz short- or long-lasting EFS. 4. 4. Both N-G-nitro- l-arginine ( l-NNA, 0.1-0.5 mM) and apamin (1–5 μM) concentration dependently inhibited the relaxation of the NANC response to 10-Hz 20-sec EFS. During L-NNA treatment, the twitch and the tonic contractions were increased. The inhibitory effect of L-NNA was reversed by L-arginine (0.1-0.5 mM) but not by D-arginine. Sodium nitroprusside (1–10 μM) was without effect. 5. 5. AP 13.2 ACOH (0.1 μM), a blocker of Substance P receptors, inhibited the twitch and the tonic contractions. The contractions were decreased after desensitization of purinoceptors by ATP and in the presence of the GABA A receptor antagonist bicuculline (30 μM). 6. 6. Depending on the EFS duration, a subsequent occurrence of relaxation and contractions characterized the NANC responses. It seems that relaxation is mediated by nitric oxide whereas Substance P and ATP are involved in the maintenance of the twitch and the tonic contractions. Nitric oxide appears to exert an inhibitory effect on the excitatory transmitters, whereas purinergic mechanism(s) could modulate the nitric oxide-dependent relaxation.

Potentiation by Bradykinin and Substance P of Purinergic Neurotransmission in Urinary Bladder

Purpose The higher than normal levels of substance P (SP) and the kinins in patients suffering from interstitial cystitis suggest that they may contribute to the complex symptoms of the condition. The purpose of our experiments was to determine whether SP and bradykinin (BK) influence the excitatory motor innervation of the urinary bladder. Materials and Methods Strips of guinea pig urinary bladder were placed in isolated tissue baths, and the influence of SP and BK on contractions induced by transmural electrical stimulation and cholinergic and purinergic agonists was evaluated. Results Substance P and BK potentiated responses to the purinergic component of the neurogenic stimulation (that part of the contractile response that remains after treatment with atropine) and potentiated responses to exogenously applied adenosine triphosphate (ATP). The peptides did not potentiate the response to the cholinergic component of the nerve-induced contraction (that part of the neurogenic response that remains after desensitization of purinoceptors with alpha, beta-methylene ATP) nor responses to carbachol. The potentiating actions of SP and BK were reduced but not abolished by treatment with meclofenamic acid. Conclusions Substance P and BK potentiate the neurogenic response of the bladder by influencing the purinergic component of the excitatory motor innervation, apparently at a postjunctional site. Prostaglandins may be involved in mediating some of the actions of these peptides.

Potentiation by bradykinin and substance P of purinergic neurotransmission in urinary bladder.

The higher than normal levels of substance P (SP) and the kinins in patients suffering from interstitial cystitis suggest that they may contribute to the complex symptoms of the condition. The purpose of our experiments was to determine whether SP and bradykinin (BK) influence the excitatory motor innervation of the urinary bladder. Strips of guinea pig urinary bladder were placed in isolated tissue baths, and the influence of SP and BK on contractions induced by transmural electrical stimulation and cholinergic and purinergic agonists was evaluated. Substance P and BK potentiated responses to the purinergic component of the neurogenic stimulation (that part of the contractile response that remains after treatment with atropine) and potentiated responses to exogenously applied adenosine triphosphate (ATP). The peptides did not potentiate the response to the cholinergic component of the nerve-induced contraction (that part of the neurogenic response that remains after desensitization of purinoceptors with alpha, beta-methylene ATP) nor responses to carbachol. The potentiating actions of SP and BK were reduced but not abolished by treatment with meclofenamic acid. Substance P and BK potentiate the neurogenic response of the bladder by influencing the purinergic component of the excitatory motor innervation, apparently at a postjunctional site. Prostaglandins may be involved in mediating some of the actions of these peptides.

Potentiation of purinergic transmission by angiotensin in prostatic rat vas deferens.

1. Angiotensin II (AII) elicited only a minute, if any, direct contractile response in smooth muscle cells of prostatic rat vas deferens, but it potentiated contractile responses to field stimulation. 2. Angiotensin-potentiated contractile response to field stimulation was concentration-dependent, and the order of potency was AII > AIII approximately AI. The EC50 of AII was 8.11 +/- 2.79 nM. 3. AII did not modify the contractile response of exogenous noradrenaline (NA) on non-stimulated prostatic vas deferens. Furthermore, the concentration-response curve for AII-potentiated contractile responses to field stimulation in reserpine-treated rats did not significantly differ from the control group. 4. Desensitization of purinoceptors with 30 microM alpha, beta-methylene-ATP almost completely abolished the potentiation of the contractile response to field stimulation by AII. 5. The response to AII in the prostatic rat vas deferens was blocked by the AT1 selective antagonist losartan, but not by the AT2 selective antagonist CGP 42112. Losartan acted as a competitive antagonist with a pA2 value of 8.75. 6. In conclusion, AII potentiated purinergic transmission in the prostatic rat vas deferens via the AT1 receptor.

Potentiation of Purinergic Neurotransmission in Guinea Pig Urinary Bladder by Histamine

Patients suffering from the inflammatory condition of interstitial cystitis frequently exhibit an increased number of mast cells in the bladder. To determine whether mast cell mediators have the potential to influence the neurogenic contraction of the bladder smooth muscle and thereby possibly contribute to the symptoms of interstitial cystitis, we examined the effects of histamine, a major inflammatory mediator of mast cell origin, on nerve- and agonist-induced contractions of in vitro strips of guinea pig urinary bladder. Histamine (10μM.) potentiated by more than 50% the nerve-induced contraction of bladder strips evoked by field stimulation with 0.5msec, pulses at 4Hz. Because the neurogenic contraction of the bladder is mediated by at least two neurotransmitters, acetylcholine (ACh) and ATP, we examined the effects of histamine on each of these transmitters. Histamine potentiated responses to the purinergic component of the neurogenic response (that part of the neurogenic response that remains after treatment with atropine) and potentiated responses to exogenously applied ATP. Histamine did not potentiate the response to the cholinergic component of the neurogenic response (that part of the neurogenic response that remains after desensitization of purinoceptors with α,β-methylene ATP) nor responses to carbachol, a cholinergic agonist. These results indicate that histamine potentiates the neurogenic response of the bladder by influencing the purinergic component, apparently at postjunctional sites.

Electrical and mechanical responses of guinea-pig bladder muscle to nerve stimulation.

1 The electrical and mechanical responses to transmural stimulation of intrinsic nerves have been recorded from smooth muscle strips dissected from the dome of the guinea-pig bladder, by use of intracellular microelectrodes, and conventional tension recording techniques. 2 Stimulation of intrinsic nerves evoked action potentials in all cells studied. Hyperpolarization of the cells by extracellular current injection revealed subthreshold excitatory junction potentials (e.j.ps) in about a quarter of the cells studied. 3 Action potentials could still be evoked in the presence of atropine and neostigmine, but were abolished after desensitization of the cells to alpha, beta-methylene ATP, a stable analogue of ATP. 4 In the presence of neostigmine, the evoked action potential was followed by a slow depolarization of the membrane. The mechanical response increased in amplitude and duration. 5 The contractile response to transmural nerve stimulation was reduced but not abolished in the presence of either atropine or desensitizing doses of alpha, beta-methylene ATP. Atropine was more effective at high frequencies of stimulation (greater than or equal to 30 Hz), and alpha, beta-methylene ATP at low frequencies (less than or equal to 15 Hz). In combination the drugs abolished the response. 6 The results suggest that the mechanical response to excitatory nerve stimulation is biphasic. The early transient response is elicited by e.j.ps and evoked spikes, is resistant to atropine, but sensitive to desensitization of purinoceptors. The late response is mediated through muscarinic receptors, involves little membrane depolarization, and is unaffected by desensitization of purinoceptors. These responses are analogous to the responses seen in rabbit bladder, and in the sympathetically innervated rat tail artery and guinea-pig vas deferens.

Influence of purinoceptors' agonists and antagonists on opossum internal anal sphincter.

Studies were performed in alpha-chloralose-anesthetized and pancuronium-treated opossums. Resting internal anal sphincter pressures (IASP) were monitored using low-compliant continuously perfused catheters. P1 and P2 purinoceptor agonists, adenosine and ATP, respectively, administered close intra-arterially, caused dose-dependent decreases in the IASP. The inhibitory effect of these agonists on the IASP was tetrodotoxin resistant. Rectal balloon distension (RD) (which mimics the rectoanal inhibitory reflex) caused volume-dependent IAS relaxation. Electrical stimulation of the sacral nerve (SNS) also produced frequency-dependent decreases in IASP. The inhibitory response to adenosine (P1 purinoceptor agonist), ATP (P2 purinoceptor agonist), RD, and SNS on the internal anal sphincter (IAS) was examined before and after 8-phenyltheophylline (P1 purinoceptor antagonist) and alpha,beta-methylene ATP (P2 purinoceptor antagonist that irreversibly binds and desensitizes P2 purinoceptor). P1 and P2 purinoceptor antagonists produced selective antagonism of the inhibitory responses on the IAS of their respective agonists only. Furthermore, high doses of adenosine and ATP produced desensitization and block of their own actions only. The purinoceptors' antagonists, and the desensitization of purinoceptors by high doses of adenosine and ATP, failed to modify the fall in IASP in response to RD or SNS. From these studies we conclude that distinct inhibitory P1 and P2 purinoceptors are present on the IAS smooth muscle. However, these inhibitory purinoceptors may not be responsible for the rectoanal reflex-mediated IAS relaxation.