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Multidrug resistance proteins type 4 (MRP4) and 5 (MRP5) play pivotal roles in the transport of cyclic nucleotides in various tissues. However, their specific functions within the lower urinary tract remain relatively unexplored. This study aimed to investigate the effect of pharmacological inhibition of MRPs on cyclic nucleotide signaling in isolated pig bladder. The relaxation responses of the bladder were assessed in the presence of the MRP inhibitor, MK571. The temporal changes in intra- and extracellular levels of cAMP and cGMP in stimulated tissues were determined by mass spectrometry. The gene (ABCC4) and protein (MRP4) expression were also determined. MK571 administration resulted in a modest relaxation effect of approximately 26% in carbachol-precontracted bladders. The relaxation induced by phosphodiesterase inhibitors such as cilostazol, tadalafil, and sildenafil was significantly potentiated in the presence of MK571. In contrast, no significant potentiation was observed in the relaxation induced by substances elevating cAMP levels or stimulators of soluble guanylate cyclase. Following forskolin stimulation, both intracellular and extracellular cAMP concentrations increased by approximately 15.8-fold and 12-fold, respectively. Similarly, stimulation with tadalafil + BAY 41-2272 resulted in roughly 8.2-fold and 3.4-fold increases in intracellular and extracellular cGMP concentrations, respectively. The presence of MK571 reduced only the extracellular levels of cGMP. This study reveals the presence and function of MRP4 transporters within the porcine bladder and paves the way for future research exploring the role of this transporter in both underactive and overactive bladder disorders.NEW & NOTEWORTHY This study investigates the impact of pharmacological inhibition of MRP4 and MRP5 transporters on cyclic nucleotide signaling in isolated pig bladders. MK571 administration led to modest relaxation, with enhanced effects observed in the presence of phosphodiesterase inhibitors. However, substances elevating cAMP levels remained unaffected. MK571 selectively reduced extracellular cGMP levels. These findings shed light on the role of MRP4 transporters in the porcine bladder, opening avenues for further research into bladder disorders.

Assuntos

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Priapism, defined as a prolonged and often painful penile erection occurring without sexual stimulation or desire, is a common complication in sickle cell disease (SCD), affecting up to 48% of male patients. This condition presents significant clinical challenges and can lead to erectile dysfunction if not properly managed. Current pharmacological treatments for SCD-related priapism are primarily reactive rather than preventative, highlighting a gap in effective medical intervention strategies. A critical factor in developing priapism is the reduced basal bioavailability of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) in erectile tissues. New prevention strategies should ideally target the underlying pathophysiology of the disease. Compounds that stimulate and activate soluble guanylate cyclase (sGC) emerge as potential therapeutic candidates since these compounds have the property of inducing cGMP production by sGC. This review explores the potential of sGC stimulators and activators in treating priapism associated with SCD. We discuss the advantages of these agents in the face of the challenging pathophysiology of SCD. Additionally, the review underscores the impact of intravascular hemolysis and oxidative stress on priapism pathophysiology in SCD, areas in which sGC stimulators and activators may also have beneficial therapeutic effects.

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BACKGROUND: Patients with sickle cell disease (SCD) experience intravascular hemolysis, leading to elevated plasma heme levels. This phenomenon has been associated with increased priapism in men with SCD. The heme group can be metabolized by heme oxygenase (HO), generating carbon monoxide (CO), which is known to promote smooth muscle relaxation via soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP). However, the effects of heme on the relaxation responses of corpus cavernosum (CC) have not been investigated. OBJECTIVES: To evaluate the functional and biochemical effects of the heme group on mouse CC smooth muscle in vitro. MATERIALS AND METHODS: Male C57BL/6 mice were used. CC tissues were mounted in organ baths. Measurement of cGMP in mice CC was evaluated. RESULTS: The cumulative addition of heme concentrations promoted the relaxation of CC. HO inhibitor (1J, 100 µM) or sGC inhibitor (ODQ, 10 µM) blocked the relaxing effect of the heme group. Pre-incubation of CC with heme (100 µM) enhanced relaxation induced by acetylcholine, sodium nitroprusside, and nitrergic relaxation (electrical field stimulation), which was abolished by 1J or ODQ. The heme group increased the cGMP production in CC, which was abolished by 1J or ODQ. cGMP levels were significantly higher in CC treated with heme, and pre-incubation with compound 1J or ODQ abolished the effect of heme in raising cGMP levels. DISCUSSION AND CONCLUSION: The HO-CO-sGC-cGMP pathway appears to play a crucial role in promoting CC relaxation. Our study provides novel insight into the role of group heme in CC relaxation and its potential contribution to priapism in SCD. Heme may serve as a pharmacological target for new therapies to prevent priapism.

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It has already been shown that serotonin can release endocannabinoids at the spinal cord level, culminating in inhibition of the dorsal horn. At the peripheral level, cannabinoid receptors modulate primary afferent neurons by inhibiting calcium conductance and increasing potassium conductance. Studies have shown that after the activation of opioid receptors and cannabinoids, there is also the activation of the NO/cGMP/KATP pathway, inducing cellular hyperpolarization. In this study, we evaluated the participation of the cannabinoid system with subsequent activation of the NO/cGMP/KATP pathway in the peripheral antinociceptive effect of serotonin. The paw pressure test of mice was used in animals that had their sensitivity to pain increased due to an intraplantar injection of PGE2 (2 µg). Serotonin (250 ng/paw), administered locally in the right hind paw, induced antinociceptive effect. CB1 and CB2 cannabinoid receptors antagonists, AM251 (20, 40 and 80 µg) and AM630 (25, 50 and 100 µg), respectively, reversed the serotonin-induced antinociceptive effect. MAFP (0.5 µg), an inhibitor of the FAAH enzyme that degrades anandamide, and JZL184 (3.75 µg), an inhibitor of the enzyme MAGL that degrades 2-AG, as well as the VDM11 (2.5 µg) inhibitor of anandamide reuptake, potentiated the antinociceptive effect induced by a low dose (62. 5 ng) of serotonin. In the evaluation of the participation of the NO/cGMP/KATP pathway, the antinociceptive effect of serotonin was reversed by the administration of the non-selective inhibitor of NOS isoforms L-NOarg (12.5, 25 and 50 µg) and by the selective inhibitor for the neuronal isoform LNPA (24 µg), as well as by the soluble guanylate cyclase inhibitor ODQ (25, 50 and 100 µg). Among potassium channel blockers, only Glibenclamide (20, 40 and 80 µg), an ATP-sensitive potassium channel blocker, reversed the effect of serotonin. In addition, intraplantar administration of serotonin (250 ng) was shown to induce a significant increase in nitrite levels in the homogenate of the plantar surface of the paw of mice. Taken together, these data suggest that the antinociceptive effect of serotonin occurs by activation of the cannabinoid system with subsequent activation of the NO/cGMP/KATP pathway.

Assuntos

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Introduction: The local peripheral combination of analgesic drugs with herbal derivatives may have beneficial effects. Information on the action mechanism of these interactions between drugs is scarce. Therefore, the main of the present study was to determine the pharmacological interaction and action mechanism of the combination α-Bisabolol and diclofenac. Methods: Rats were injected in the dorsal surface of the right hind paw with 1% formalin. Rats received subcutaneous injections in the dorsal surface of paw of vehicles or increasing doses of α-Bisabolol, diclofenac or their combination before formalin injection into the paw. Antinociception of the α-Bisabolol + diclofenac combination was evaluated with and without the local treatment of naloxone, metformin, NG-nitro-L-arginine methyl ester (L-NAME), 1H- (1,2,4)-oxadiazolo (4,2-a) quinoxalin-1-one (ODQ), glibenclamide, glipizide, 4-aminopyridine, tetraethylammonium, apamin, or charybdotoxin. Results: α-Bisabolol, diclofenac or α-Bisabolol-diclofenac combinations produced significant antinociception in the rat (p < 0.05). The experimental effective dose (ED) value of 109.2 µg/paw was different significantly of the theoretical effective dose (ED) of 245.7 µg/paw (synergism). Blockers significantly reverted the antinociception produced by the synergistic combination of α-Bisabolol and diclofenac. Discussion: Data showed a synergism of the α-Bisabolol-diclofenac combination and the activation of the opioid receptor-Nitric Oxide-cyclic GMP-K+ channels pathway and a biguanide-dependent mechanism in order to produce the potentiation of its peripheral antinociception in the formalin test.

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Left congenital diaphragmatic hernia (CDH) can lead to pulmonary arteries abnormalities in the contralateral and ipsilateral sides of the diaphragm. Nitric oxide (NO) is the main therapy used to attenuate the vascular effects of CDH, but it is not always effective. We hypothesized that the left and right pulmonary arteries do not respond similarly to NO donors during CDH. Therefore, vasorelaxant responses of the left and right pulmonary arteries to sodium nitroprusside (SNP, a NO donor) were determined in a rabbit experimental model of left CDH. CDH was surgically induced in the fetuses of rabbits on the 25th day of pregnancy. On the 30th day of pregnancy, a midline laparotomy was performed to access the fetuses. The fetuses' left and right pulmonary arteries were isolated and mounted in myograph chambers. Vasodilation was evaluated by cumulative concentration-effect curves to SNP. Protein expression of guanylate cyclase isoforms (GCα, GCß) and the α isoform of cGMP-dependent protein kinase 1 (PKG1α), and the concentration of NO and cGMP were determined in the pulmonary arteries. The left and right pulmonary arteries of newborns with CDH exhibited increased vasorelaxant responses to SNP (i.e. the potency of SNP was increased) compared to the control group. GCα, GCß, and PKG1α expression were decreased, while NO and cGMP concentrations were increased in the pulmonary arteries of newborns with CDH compared to the control group. The increased cGMP mobilization may be responsible for the increased vasorelaxant responses to the SNP in the pulmonary arteries during left CDH.

Assuntos

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BACKGROUND: Intracellular levels of cyclic nucleotides can also be controlled by the action of multidrug resistance protein types 4 (MRP4) and 5 (MRP5). To date, no studies evaluated the role of their inhibition in an animal model of erectile dysfunction (ED). OBJECTIVES: To evaluate the effect of a 2-week treatment with MK571, an inhibitor of the efflux of cyclic nucleotides in the ED of obese mice. MATERIALS AND METHODS: Mice were divided in three groups: (i) lean, (ii) obese, and (iii) obese + MK571. The corpus cavernosum (CC) were isolated, and concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP), and tadalafil in addition to electrical field stimulation (EFS) were carried out in phenylephrine pre-contracted tissues. Expression of ABCC4 and ABCC5, intracellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), the protein levels for pVASPSer157 and pVASPSer239 , and the intracavernous pressure (ICP) were also determined. The intracellular and extracellular (supernatant) ratios in CC from obese and lean stimulated with a cGMP-increasing substance (BAY 58-2667) in the absence and presence of MK571 (20 µM, 30 min) were also assessed. RESULTS: The treatment with MK571 completely reversed the lower relaxing responses induced by EFS, ACh, SNP, and tadalafil observed in obese mice CC in comparison with untreated obese mice. Cyclic GMP and p-VASPSer239 expression were significantly reduced in CC from obese groups. MK571 promoted a sixfold increase in cGMP without interfering in the protein expression of p-VASPSer239 . Neither the cAMP levels nor p-VASPSer157 were altered in MK571-treated animals. The ICP was ∼50% lower in obese than in the lean mice; however, the treatment with MK571 fully reversed this response. Expressions of ABCC4 and ABCC5 were not different between groups. The intra/extracellular ratio of cGMP was similar in CC from obese and lean mice stimulated with BAY 58-2667. CONCLUSIONS: The MRPs inhibition by MK571 favored the accumulation of cGMP in the smooth muscle cells, thus improving the smooth muscle relaxation and the erectile function in obese mice.

Assuntos

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Pamabrom is a diuretic that is effective in treating premenstrual syndrome and primary dysmenorrhea. The aim of this study was to examine the effect of metformin and modulators of the opioid receptor-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-K+ channel pathway on the local antinociception induced by pamabrom. The rat paw 1% formalin test was used to assess the effects. Rats were treated with local administration of pamabrom (200-800 µg/paw) or indomethacin (200-800 µg/paw). The antinociception of pamabrom or indomethacin was evaluated with and without the local pretreatment of the blockers. Local administration of pamabrom and indomethacin produced dose-dependent antinociception during the second phase of the test. Local pretreatment of the paws with naloxone (50 µg/paw), l-nitro-arginine methyl ester (10-100 µg/paw), or 1H-(1,2,4)-oxadiazolo[4,2-a]quinoxalin-1-one (10-100 µg/paw) reverted the antinociception induced by local pamabrom, but not of indomethacin. Similarly, the K+ channel blockers glibenclamide, glipizide, 4-aminopyridine, tetraethylammonium, charybdotoxin, or apamin reverted the pamabrom-induced antinociception, but not of indomethacin. Metformin significantly blocked the antinociception of pamabrom and indomethacin. Our data suggest that pamabrom could activate the opioid receptor-NO-cGMP-K+ channel pathway to produce its peripheral antinociception in the formalin test. Likewise, a biguanide-dependent mechanism could be activated by pamabrom and indomethacin to generate antinociception.

Assuntos

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In the present study, we investigated whether magnesium sulphate activates the L-arginine/NO/cGMP pathway and elicits peripheral antinociception. The male Swiss mice paw pressure test was performed with hyperalgesia induced by intraplantar injection of prostaglandin E2. All drugs were administered locally into the right hind paw of animals. Magnesium sulphate (20, 40, 80 and 160 µg/paw) induced an antinociceptive effect. The dose of 80 µg/paw elicited a local antinociceptive effect that was antagonized by the non-selective NOS inhibitor, L-NOArg, and by the selective neuronal NOS inhibitor, L-NPA. The inhibitors, L-NIO and L-NIL, selectively inhibited endothelial and inducible NOS, respectively, but were ineffective regarding peripheral magnesium sulphate injection. The soluble guanylyl cyclase inhibitor, ODQ, blocked the action of magnesium sulphate, and the cGMP-phosphodiesterase inhibitor, zaprinast, enhanced the antinociceptive effects of intermediate dose of magnesium sulphate. Our results suggest that magnesium sulphate stimulates the NO/cGMP pathway via neuronal NO synthase to induce peripheral antinociceptive effects.

Assuntos

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Cyclic Nucleotides are important in regulating platelet function. Increases in 3'5'-cyclic adenosine monophosphate (cAMP) and 3'5'-cyclic guanosine monophosphate (cGMP) inhibit platelet aggregation and are pharmacological targets for antiplatelet therapy. Here we report an improved method for determining cAMP and cGMP concentrations and, for the first time, in washed platelet supernatants by combining high-performance liquid chromatography and tandem mass spectrometry (LC-MS/MS). Characteristic peaks of the substrates, cGMP or cAMP and their internal standards were identified in negative-ion electrospray ionisation using multiple reaction monitoring. Compared with previously reported methods, the method presented here shows high precision with the lowest lower limit of quantification (LLoQ) to date (10 pg/mL). The effect of a novel catecholamine, 6-nitrodopamine, on cyclic nucleotide levels was quantified. Our results showed that this new method was fast, sensitive, and highly reproducible.

Assuntos