Characterization of the retina-induced relaxation in mice.

PURPOSE: The retinal relaxing factor (RRF) is a continuously released factor from the retina that causes vasorelaxation, the identity and potential role in physiology of which remain largely unknown. Experiments were performed to find out whether the RRF-induced relaxation is influenced by serotonin, glutamate, L-cysteine, the cytochrome P450 pathway, the cyclooxygenase pathway, or oxidative stress. In addition, the sensitivity of retinal and non-retinal arteries towards the RRF was compared. METHODS: In vitro tension measurements were performed on isolated mouse femoral or bovine retinal arteries to study the vasorelaxing effect of the RRF, induced by mouse or bovine retinas. RESULTS: The presence of serotonin, glutamate, or L-cysteine did not alter the RRF-induced relaxation. Increasing oxidative stress by hydroquinone and diethyldithiocarbamic acid sodium salt enhanced the RRF response. Inhibition of the cytochrome P450 or the cyclooxygenase pathway did not cause any alteration. Surprisingly, the RRF-induced relaxation was enhanced by the presence of flufenamic acid or carbenoxolone. Furthermore, bringing retinal tissue in close contact with retinal or non-retinal arteries induced comparable relaxations. CONCLUSIONS: Serotonin, glutamate, L-cysteine, the cytochrome P450, and the cyclooxygenase pathway do not influence the RRF-induced relaxation and the RRF-induced relaxation seems to be resistant to oxidative stress. The mechanism responsible for the enhanced RRF-induced relaxation in the presence of flufenamic acid or carbenoxolone remains elusive and the RRF does not show more effectivity on retinal arteries.

Inhibition of Cyclic GMP Export by Multidrug Resistance Protein 4: A New Strategy to Treat Erectile Dysfunction?

BACKGROUND: Intracellular cyclic guanosine monophosphate (cGMP) concentrations are regulated by degradation enzymes (phosphodiesterases) and by active transport across the plasma membrane by multidrug resistance proteins (MRPs) 4 and 5. AIM: To evaluate the functional effect of MRP-4 inhibition and the role of MRP-4-mediated cGMP export in mouse corpora cavernosa. METHODS: Isometric tension of mouse corpora cavernosa was measured after cumulative addition of MK-571, an inhibitor of MRP-4, or sildenafil, a phosphodiesterase type 5 inhibitor. In addition, the effect of MRP-4 inhibition on cGMP-independent and cGMP-dependent relaxations was studied. In vivo intracavernosal pressure and mean arterial pressure measurements were performed after intracavernosal injection of MK-571. The effect of MRP-4 inhibition on cGMP content was determined using an enzyme immunoassay kit. OUTCOMES: Measurement of the effect of MK-571 on cGMP content, relaxant responses of mouse corpora cavernosa to cGMP-independent and cGMP-dependent vasodilating substances, and determination of the ratio of intracavernosal pressure to mean arterial pressure after intracavernosal injection of MK-571. RESULTS: MK-571 and sildenafil relaxed the corpora cavernosa concentration dependently, with sildenafil being the more potent relaxing compound. Furthermore, MK-571 enhanced relaxing responses to cGMP-dependent substances, such as sodium nitroprusside, sildenafil, acetylcholine, and electrical field stimulation, with the latter even under in vitro diabetic conditions. In contrast, cGMP-independent relaxations were not altered by MRP-4 inhibition. Intracavernosal administration of MK-571 significantly increased intracavernosal pressure, with minimal effect on mean arterial pressure. The cGMP analysis showed that MRP-4 inhibition was accompanied by increased cGMP levels. CLINICAL TRANSLATION: MRP-4, at least when targeted locally in the penis or when combined with a phosphodiesterase type 5 inhibitor, might be a valuable alternative strategy for the treatment of (diabetic) erectile dysfunction. STRENGTHS AND LIMITATIONS: This study is the first to demonstrate an in vitro direct relaxant and an in vivo pro-erectile effect of the MRP-4 inhibitor, MK-571, on mouse corpora cavernosa. However, the functional effect of MRP-5-mediated export in mouse corpora cavernosa was not explored, which has been suggested to play the predominant role in cGMP export. CONCLUSION: Inhibition of MRP-4 increases basal and stimulated levels of cGMP, leading to corpora cavernosa relaxation and penile erection. Therefore, in addition to degradation of cGMP, export of cGMP by MRP-4 could contribute substantially to regulating cGMP levels in mouse corpora cavernosa. Boydens C, Pauwels B, Vanden Daele L, Van de Voorde J. Inhibition of Cyclic GMP Export by Multidrug Resistance Protein 4: A New Strategy to Treat Erectile Dysfunction? J Sex Med 2017;14:502-509.

Erectile Dysfunction in Heme-Deficient Nitric Oxide-Unresponsive Soluble Guanylate Cyclase Knock-In Mice.

INTRODUCTION: The nitric oxide (NO), soluble guanylate cyclase (sGC), and cyclic guanosine monophosphate (cGMP) pathway is the leading pathway in penile erection. AIM: To assess erectile function in a mouse model in which sGC is deficient in heme (apo-sGC) and unresponsive to NO. METHODS: Mutant mice (sGCß1ki/ki) that express an sGC enzyme that retains basal activity but fails to respond to NO because of heme deficiency (apo-sGC) were used. Isolated corpora cavernosa from sGCß1ki/ki and wild-type mice were mounted in vitro for isometric tension recordings in response to sGC-dependent and -independent vasorelaxant agents. In addition, the erectile effects of some of these agents were tested in vivo at intracavernosal injection. MAIN OUTCOME MEASURES: In vitro and in vivo recordings of erectile responses in sGCß1ki/ki and wild-type mice after stimulation with sGC-dependent and -independent vasorelaxant agents. RESULTS: NO-induced responses were abolished in sGCß1ki/ki mice in vitro and in vivo. The ability of the heme-dependent, NO-independent sGC stimulator BAY 41-2272 to relax the corpora cavernosa was markedly attenuated in sGCß1ki/ki mice. In contrast, the relaxation response to the heme- and NO-independent sGC activator BAY 58-2667 was significantly enhanced in sGCß1ki/ki mice. The relaxing effect of sGC-independent vasorelaxant agents was similar in wild-type and sGCß1ki/ki mice, illustrating that the observed alterations in vasorelaxation are limited to NO-sGC-cGMP-mediated processes. CONCLUSION: Our results suggest that sGC is the sole target of NO in erectile physiology. Furthermore, this study provides indirect evidence that, in addition to sGCα1ß1, sGCα2ß1 is important for erectile function. In addition, the significant relaxation observed in sGCß1ki/ki mice with the cumulative addition of the sGC activator BAY 58-2667 indicates that sGC activators might offer value in treating erectile dysfunction.

Protective effect of resveratrol and quercetin on in vitro-induced diabetic mouse corpus cavernosum.

BACKGROUND: Hyperglycemia and increased levels of methylglyoxal (MGO) can trigger the development of vascular complications in diabetes. Resveratrol and quercetin are red wine polyphenols with known beneficial cardiovascular properties, including an antioxidant capacity. This study evaluated whether resveratrol and/or quercetin could prevent in vitro-induced diabetic changes in neurogenic and vascular relaxant responses of mouse arteries and corpora cavernosa. METHODS: Isometric tension of isolated aorta, mesenteric arteries and corpora cavernosa was measured using organ bath systems. Diabetic conditions were mimicked in vitro by co-incubating the tissues for 2 h with high glucose (HG, 30 mM) and MGO (120 µM). RESULTS: The presence of HG and MGO significantly blunted acetylcholine (Ach)-induced relaxations in corpora cavernosa and mesenteric arteries but not in aorta. Electrical field stimulated (EFS) responses of corpora cavernosa were also significantly inhibited by these diabetic conditions. In corpora cavernosa 2 h co-incubation with resveratrol (30 µM) or quercetin (30 µM) significantly attenuated HG and MGO-induced deficits in Ach- and EFS-responses. CONCLUSIONS: Our study demonstrates that in mouse arteries, HG and MGO rather affect endothelium derived hyperpolarizing factor-mediated than nitric oxide (NO)-mediated relaxations. In corpora cavernosa HG and MGO interfere with NO release. Resveratrol and quercetin protect mouse corpora cavernosa from diabetic-induced damage to NO-mediated relaxant responses. This might rely on their antioxidant capacity.

Effect of resveratrol and orchidectomy on the vasorelaxing influence of perivascular adipose tissue.

Perivascular adipose tissue (PVAT) releases several adipo(cyto)kines. Some are vasoactive substances that elicit a net beneficial anticontractile effect. Resveratrol and testosterone are known to modulate adipo(cyto)kine release from adipose tissue and could therefore influence the anticontractile effect of PVAT. In vitro tension measurements were performed using thoracic aorta segments with and without adipose tissue from sham-operated or orchidectomized male Swiss mice. Concentration-response curves to norepinephrine (NOR) were constructed in the presence and absence of resveratrol (10 µM, 15 min) or the relaxant effect of resveratrol (10-100 µM) was investigated after inducing tone with NOR (5 µM). Aortas with PVAT displayed significantly attenuated contractions to NOR compared with aortas without PVAT. In aortas without PVAT, resveratrol (10 µM) significantly decreased NOR responses and elicited concentration-dependent (10-100 µM) relaxations. However, in aortas with adherent PVAT, resveratrol (10 µM) neither decreased NOR responses, nor did resveratrol (10-100 µM) induce arterial relaxations. The anticontractile effect of PVAT was less pronounced in the presence of resveratrol and unaltered by orchidectomy. Orchidectomy did not influence contractions induced by NOR. Orchidectomy does not modulate the anticontractile capacity of PVAT, while resveratrol decreases the vasorelaxing influence of PVAT. The positive effects associated with resveratrol addition are neutralized by the presence of PVAT. This is thought to result from a dual effect of resveratrol: (1) inhibition of the influence of vasodilatory adipo(cyto)kines and (2) a direct relaxant effect on the vascular smooth muscle. Overall, the beneficial relaxing effect of resveratrol is lost in mice thoracic aorta surrounded by PVAT.

Oximes induce erection and are resistant to oxidative stress.

INTRODUCTION: Because of their nitric oxide (NO)-donating capacities, oxime derivatives have shown to offer some therapeutic perspective for the treatment of erectile dysfunction (ED) as well as cardiovascular diseases. However, to date the in vivo effect of these oximes on erectile function remains unknown. In many disease states oxidative stress occurs, impairing NO-mediated relaxations. Hence the influence of oxidative stress on oxime-induced effects is also of interest. AIMS: This study aimed to evaluate the in vivo effect of formaldoxime (FAL) and formamidoxime (FAM) on blood pressure and intracavernosal pressure (ICP); and to examine the role of soluble guanylyl cyclase (sGC) and the influence of oxidative stress on the FAL and FAM responses. METHODS: Blood pressure and ICP were monitored in vivo after resp. intravenous or intracavernosal injection of FAL and FAM. Moreover isometric tension was measured in vitro on isolated mice corpora cavernosa (CC), thoracic aorta, and femoral artery in organ baths. The role of sGC was investigated using transgenic mice lacking the alpha 1 subunit of sGC. MAIN OUTCOME MEASURES: Mean arterial pressure (MAP) and ICP were measured after FAL/FAM injection. In vitro relaxation of CC strips was evaluated in response to addition of FAL/FAM. RESULTS: In vivo both FAL and FAM elicit a dose-dependent lowering of blood pressure (maximal ΔMAP: 33.66 ± 4.07 mm Hg [FAL] and 20.43 ± 2.06 mm Hg [FAM] ) as well as an increase of ICP (maximal increase of ICP/MAP: 70.29 ± 2.88% [FAL] and 52.91 ± 8.61% [FAM] ). The FAL/FAM effect is significantly lower in knockout vs. wild-type mice. Oxidative stress has an inhibitory effect on corporal NO-mediated relaxations induced by electrical field stimulation, acetylcholine, and sodium nitroprusside whereas the responses to 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate sodium salt, FAL and FAM were not influenced. CONCLUSIONS: Oximes induce erection which is mediated by sGC. The oxime-induced relaxations are resistant to oxidative stress, which increases their therapeutic potential for the treatment of ED.

Relaxant and antioxidant capacity of the red wine polyphenols, resveratrol and quercetin, on isolated mice corpora cavernosa.

INTRODUCTION: The red wine polyphenols resveratrol and quercetin are known for their vasorelaxant and antioxidant capacity, which is assumed to rely on the activation of the nitric oxide (NO)/soluble guanylyl cyclase (sGC) pathway. Vasodilators as well as antioxidants can regulate penile erection and be beneficial for the treatment of erectile dysfunction (ED). AIMS: The goal of this study was to evaluate the NO/sGC dependency of the relaxant effect of resveratrol and quercetin on mice aorta and corpora cavernosa (CC), as well as to explore their influence on oxidative stress-induced ED. METHODS: Isolated mice aorta and CC were mounted for isometric tension recordings into organ baths. Cumulative concentration-response curves were constructed for resveratrol and quercetin in the absence/presence of inhibitors of the NO/sGC pathway. In addition, in CC the effect of resveratrol and quercetin was studied on NO-mediated relaxations using acetylcholine (Ach), sodium nitroprusside (SNP), and electrical field stimulation (EFS). In certain experiments, corporal tissues were exposed to oxidative stress using palmitic acid (PA, 0.5 mM). MAIN OUTCOME MEASURES: Corporal responses to resveratrol and quercetin were measured in the presence/absence of inhibitors of different molecular pathways. The effect of resveratrol and quercetin incubation on Ach-, SNP-, or EFS-mediated responses was explored in the presence/absence of PA. RESULTS: While both polyphenols are potent vasodilators of mice aorta, only resveratrol relaxes mice CC. The relaxation response to resveratrol on aorta was diminished in sGCα1 (-/-) mice, but not on CC. The polyphenols did not influence Ach-, SNP-, or EFS-mediated relaxations as such. Resveratrol, but not quercetin, was able to significantly reverse PA-induced decrease of EFS relaxations. CONCLUSION: The red wine compound resveratrol, but not quercetin, relaxes isolated mice CC concentration-dependently through mechanisms independent of the NO/sGC pathway. Resveratrol is a more potent antioxidant than quercetin, being able to restore decreased neuronal NO responses in mice CC.

NO-donating oximes relax corpora cavernosa through mechanisms other than those involved in arterial relaxation.

INTRODUCTION: Erectile dysfunction (ED), as well as many cardiovascular diseases, is associated with impaired nitric oxide (NO) bioavailability. Recently, oxime derivatives have emerged as vasodilators due to their NO-donating capacities. However, whether these oximes offer therapeutic perspectives as an alternative NO delivery strategy for the treatment of ED is unexplored. AIMS: This study aims to analyze the influence of formaldoxime (FAL), formamidoxime (FAM), and cinnamaldoxime (CAOx) on corporal tension and to elucidate the underlying molecular mechanisms. METHODS: Organ bath studies were carried out measuring isometric tension on isolated mice corpora cavernosa (CC), thoracic aorta, and femoral artery. After contraction with norepinephrine (NOR), cumulative concentration-response curves of FAL, FAM, and CAOx (100 nmol/L-1 mmol/L) were performed. MAIN OUTCOME MEASURES: FAL-/FAM-induced relaxations were evaluated in the absence/presence of various inhibitors of different molecular pathways. RESULTS: FAL, FAM, and CAOx relax isolated CC as well as aorta and femoral artery from mice. ODQ (soluble guanylyl cyclase-inhibitor), diphenyliodonium chloride (nonselective flavoprotein inhibitor), and 7-ethoxyresorufin (inhibitor of CYP450 1A1 and NADPH-dependent reductases) substantially blocked the FAL-/FAM-induced relaxation in the arteries but not in CC. Only a small inhibition of the FAM response in CC was observed with ODQ. CONCLUSIONS: This study shows for the first time that NO-donating oximes relax mice CC. Therefore, oximes are a new group of molecules with potential for the treatment of ED. However, the underlying mechanism(s) of the FAL-/FAM-induced corporal relaxation clearly differ(s) from the one(s) involved in arterial vasorelaxation.

Protein-bound uremic toxins stimulate crosstalk between leukocytes and vessel wall.

Leukocyte activation and endothelial damage both contribute to cardiovascular disease, a major cause of morbidity and mortality in CKD. Experimental in vitro data link several protein-bound uremic retention solutes to the modulation of inflammatory stimuli, including endothelium and leukocyte responses and cardiovascular damage, corroborating observational in vivo data. However, the impact of these uremic toxins on the crosstalk between endothelium and leukocytes has not been assessed. This study evaluated the effects of acute and continuous exposure to uremic levels of indoxylsulfate (IS), p-cresylsulfate (pCS), and p-cresylglucuronide (pCG) on the recruitment of circulating leukocytes in the rat peritoneal vascular bed using intravital microscopy. Superfusion with IS induced strong leukocyte adhesion, enhanced extravasation, and interrupted blood flow, whereas pCS caused a rapid increase in leukocyte rolling. Superfusion with pCS and pCG combined caused impaired blood flow and vascular leakage but did not further enhance leukocyte rolling over pCS alone. Intravenous infusion with IS confirmed the superfusion results and caused shedding of heparan sulfate, pointing to disruption of the glycocalyx as the mechanism likely mediating IS-induced flow stagnation. These results provide the first clear in vivo evidence that IS, pCS, and pCG exert proinflammatory effects that contribute to vascular damage by stimulating crosstalk between leukocytes and vessels.

Catecholamine and insulin control of lipolysis in subcutaneous adipose tissue during long-term diet-induced weight loss in obese women.

The aim of this study was to investigate the evolution of the adrenergic and insulin-mediated regulation of lipolysis during different phases of a 6-mo dietary intervention. Eight obese women underwent a 6-mo dietary intervention consisting of a 1-mo very low-calorie diet (VLCD) followed by a 2-mo low-calorie diet (LCD) and 3-mo weight maintenance (WM) diet. At each phase of the dietary intervention, microdialysis of subcutaneous adipose tissue (SCAT) was performed at rest and during a 3-h hyperinsulinemic euglycemic clamp. Responses of dialysate glycerol concentration (DGC) were determined at baseline and during local perfusions with adrenaline or adrenaline and phentolamine before and during the last 30 min of the clamp. Dietary intervention induced a body weight reduction and an improved insulin sensitivity. DGC progressively decreased during the clamp, and this decrease was similar during the different phases of the diet. The adrenaline-induced increase in DGC was higher at VLCD and LCD compared with baseline condition and returned to prediet levels at WM. In the probe with adrenaline and phentolamine, the increase in DGC was higher than that in the adrenaline probe at baseline and WM, but it was not different at VLCD and LCD. The results suggest that the responsiveness of SCAT to adrenaline-stimulated lipolysis increases during the calorie-restricted phases due to a reduction of the α(2)-adrenoceptor-mediated antilipolytic action of adrenaline. At WM, adrenaline-stimulated lipolysis returned to the prediet levels. Furthermore, no direct relationship between insulin sensitivity and the diet-induced changes in the regulation of lipolysis was found.

Divergent molecular mechanisms underlay CO- and CORM-2-induced relaxation of corpora cavernosa.

INTRODUCTION: Similar to nitric oxide (NO), the principal mediator of penile erection, carbon monoxide (CO) possesses vasodilator capacities. However, whether CO could be a therapeutic target for treating erectile dysfunction (ED) is unexplored. The danger associated with systemic administration of CO has led to the development of CO-releasing molecules (CORMs), releasing CO in a local, safe and controlled way. These CORMs have shown positive outcomes in cardiovascular studies. More knowledge on the (patho)physiological functions of CO in erectile function and the potential therapeutic role of CORMs is required. AIM: The present study aims the assessment of the effect of CO and CO donor CORM-2 on the corporal tension and the underlying molecular mechanisms. METHODS: Organ bath studies were performed measuring isometric tension on isolated mice corpora cavernosa (CC) strips. Responses to CO (10-300 µmol/L) and CORM-2 (10-100 µmol/L) were measured in the presence/absence of activators/inhibitors of different molecular pathways. MAIN OUTCOME MEASURES: CO and CORM-2 relax corporal strips concentration dependently, although the molecular mechanisms behind the corporal relaxation seem to differ completely. RESULTS: CO induces corporal relaxation by activating soluble guanylyl cyclase (sGC), increasing cyclic guanosine monophosphate (cGMP) concentrations. The molecular mechanism involved in CORM-2-induced corporal relaxation is not related to sGC activation and remains obscure. CONCLUSIONS: Both CO and CORM-2 induce corporal relaxation, although the underlying molecular mechanisms show no resemblance. That CO induces corporal relaxation through a mechanism similar to that of NO could be of importance as it indirectly offers the possibility that endogenous CO might serve as a backup system for insufficient NO availability in cases of ED. Whether CORM-2 possesses the same capacity remains questionable and requires further research.

Adipose tissue as regulator of vascular tone.

Adipokines secreted by visceral, subcutaneous, and perivascular adipocytes are involved in the regulation of vascular tone by acting as circulatory hormones (leptin, adiponectin, omentin, visfatin, angiotensin II, resistin, tumor necrosis factor-α, interleukin-6, apelin) and/or via local paracrine factors (perivascular adipocyte-derived relaxing and contractile factors). Vascular tone regulation by adipokines is compromised in obesitas and obesity-related disorders. Hypoxia created in growing adipose tissue dysregulates synthesis of vasoactive adipokines in favor of harmful proinflammatory adipokines, while the levels of the cardioprotective adipokines adiponectin and omentin decrease. Considering the potential of the role of adipokines in obesity-related vascular diseases, strategies to counter these diseases by targeting the adipokines are discussed.

Regulation of vascular tone by adipocytes.

Recent studies have shown that adipose tissue is an active endocrine and paracrine organ secreting several mediators called adipokines. Adipokines include hormones, inflammatory cytokines and other proteins. In obesity, adipose tissue becomes dysfunctional, resulting in an overproduction of proinflammatory adipokines and a lower production of anti-inflammatory adipokines. The pathological accumulation of dysfunctional adipose tissue that characterizes obesity is a major risk factor for many other diseases, including type 2 diabetes, cardiovascular disease and hypertension. Multiple physiological roles have been assigned to adipokines, including the regulation of vascular tone. For example, the unidentified adipocyte-derived relaxing factor (ADRF) released from adipose tissue has been shown to relax arteries. Besides ADRF, other adipokines such as adiponectin, omentin and visfatin are vasorelaxants. On the other hand, angiotensin II and resistin are vasoconstrictors released by adipocytes. Reactive oxygen species, leptin, tumour necrosis factor α, interleukin-6 and apelin share both vasorelaxing and constricting properties. Dysregulated synthesis of the vasoactive and proinflammatory adipokines may underlie the compromised vascular reactivity in obesity and obesity-related disorders.

New therapeutic targets for the treatment of erectile dysfunction.

INTRODUCTION: Despite the high efficacy and safety rates of the currently available treatments for erectile dysfunction, basic research reveals numerous new targets that are explored for therapeutic use. AIM: To overview potential new targets and to review available animal and human studies focusing on the potential of these targets for effective therapy for treating erectile dysfunction. METHODS: A comprehensive literature search was conducted using the PubMed and Medline database, and citations were selected based on relevance. MAIN OUTCOME MEASURES: Data are presented based on the analysis of the selected scientific information and published clinical trials. RESULTS: Fundamental research has, in the past decade, increased the understanding in both the physiological and the pathophysiological pathways that play a role in erectile function. As this information increases each day, new targets to treat erectile dysfunction are frequently presented. Currently a number of new therapeutic targets have been published. Some of them target the nitric oxide/cyclic guanosine monophosphate relaxation pathway as the phosphodiesterase type 5 inhibitors do, others primarily target pathways involved in contraction. Also, targets within the central nervous system currently receive much attention. Some of these targets have already been used in clinical trials to test their efficacy and safety, with either disappointing or promising results. CONCLUSIONS: This review overviews potential therapeutic targets and summarizes animal as well as human studies evaluating their perspectives for the treatment of erectile dysfunction.

Combined glucocorticoid resistance and hyperlactatemia contributes to lethal shock in sepsis.

Sepsis is a potentially lethal syndrome resulting from a maladaptive response to infection. Upon infection, glucocorticoids are produced as a part of the compensatory response to tolerate sepsis. This tolerance is, however, mitigated in sepsis due to a quickly induced glucocorticoid resistance at the level of the glucocorticoid receptor. Here, we show that defects in the glucocorticoid receptor signaling pathway aggravate sepsis pathophysiology by lowering lactate clearance and sensitizing mice to lactate-induced toxicity. The latter is exerted via an uncontrolled production of vascular endothelial growth factor, resulting in vascular leakage and collapse with severe hypotension, organ damage, and death, all being typical features of a lethal form of sepsis. In conclusion, sepsis leads to glucocorticoid receptor failure and hyperlactatemia, which collectively leads to a lethal vascular collapse.

Lipid mobilization in subcutaneous adipose tissue during exercise in lean and obese humans. Roles of insulin and natriuretic peptides.

The aim of this study was to evaluate the relative contributions of various hormones involved in the regulation of lipid mobilization in subcutaneous adipose tissue (SCAT) during exercise and to assess the impact of obesity on this regulation. Eight lean and eight obese men performed a 60-min cycle exercise bout at 50% of their peak oxygen uptake on two occasions: during intravenous infusion of octreotide (a somatostatin analog) or physiological saline (control condition). Lipolysis in SCAT was evaluated using in situ microdialysis. One microdialysis probe was perfused with the adrenergic blockers phentolamine and propranolol while another probe was perfused with the phosphodiesterase and adenosine receptor inhibitor aminophylline. Compared with the control condition, infusion of octreotide reduced plasma insulin levels in lean (from approximately 3.5 to 0.5 microU/ml) and in obese (from approximately 9 to 2 microU/ml), blunted the exercise-induced rise in plasma GH and epinephrine levels in both groups, and enhanced the exercise-induced natriuretic peptide (NP) levels in lean but not in obese subjects. In both groups, octreotide infusion resulted in higher exercise-induced increases in dialysate glycerol concentrations in the phentolamine-containing probe while no difference in lipolytic response was found in the aminophylline-containing probe. The results suggest that insulin antilipolytic action plays a role in the regulation of lipolysis during exercise in lean as well as in obese subjects. The octreotide-induced enhancement of exercise lipolysis in lean subjects was associated with an increased exercise-induced plasma NP response. Adenosine may contribute to the inhibition of basal lipolysis in both subject groups.

In vitro and in vivo studies on the importance of the soluble guanylyl cyclase α1 subunit in penile erection.

PURPOSE: Soluble guanylyl cyclase (sGC), which plays a pivotal role in penile erection, is a heterodimer build up by an α and a ß subunit. For both subunits two isoforms have been characterized, but only the sGCα(1)ß(1) and sGCα(2)ß(1) isoforms seem to be functionally active. To elucidate the functional role of the sGCα(1)ß(1) heterodimer in the mechanism of erection, experiments were performed in vivo and on isolated corpora cavernosa (CC) using sGCα(1)(-/-) mice. MATERIALS AND METHODS: For the in vivo study sGC-dependent and -independent vasorelaxing agents were injected intracavernosally in sGCα(1)(-/-) and sGCα(1)(+/+) mice and the rise in intracavernosal pressure was recorded. For the in vitro study, isolated CC tissues from sGCα(1)(-/-) and sGCα(1)(+/+) mice were mounted in organ baths for isometric tension recording and concentration-dependent curves were obtained for sGC-dependent and -independent vasorelaxing agents. These experiments were performed on 2 different mice strains (129SvEvS7 and C57BL6/J) to determine potential strain differences. RESULTS: The responses in sGCα(1)(-/-) after administration of the NO-donors, sodium nitroprusside (SNP) and spermine-NO, and to electrical stimulation are significantly reduced although not completely abolished. Responses to sGC-independent vasorelaxing agents are similar in sGCα(1)(-/-) and sGCα(1)(+/+) mice from both strains suggesting that the decreased potential of smooth muscle relaxation is not related to structural changes or changes in the pathway downstream sGC. CONCLUSION: This study illustrates the strain-independent importance of the sGCα(1)ß(1) heterodimer, although remaining vasorelaxing responses in the sGCα(1)(-/-) mice suggest a complementary role for the sGCα(2)ß(1) isoform or (an) sGC-independent mechanism(s).

Adenosine enhances the relaxing influence of retinal tissue.

Retinal tissue from different species continuously releases an as yet unidentified retinal relaxing factor (RRF) lowering tone of isolated arteries. The potential influence of adenosine on this relaxing influence was investigated using isometric tension recording of different isolated arteries. The presence of bovine retinal tissue or rat retinal tissue enhanced the vasorelaxing effect of adenosine on isolated bovine retinal artery. In isolated rat carotid artery adenosine elicited no relaxation. However, a small relaxation is observed in the presence of rat retinal tissue, but not in the presence of porcine retina. The fact that adenosine potentiates the effect of rat retinal tissue but not that of a similar piece of porcine retinal tissue indicates species differences. Neither a NO-synthase inhibitor (nitro-L-arginine, 0.1mM), a cyclooxygenase inhibitor (indomethacin, 10 microM) or an epoxygenase inhibitor (miconazole, 10 microM) influenced the enhanced vasodilating effect of adenosine on bovine retinal arteries in the presence of bovine retinal tissue. On the other hand, when the retinal arteries were contracted with 120 mM K(+), adenosine no longer induced relaxation of the preparation with bovine retinal tissue. This is in line with the concept that adenosine enhances the influence of RRF. Also, the fact that rat carotid artery is less sensitive to RRF than bovine retinal artery - corresponding with a less enhanced adenosine response in rat carotid artery - is in line with the potential involvement of the RRF in the enhanced adenosine response. However, experiments using a bioassay setup for RRF gave no evidence for an increased RRF-release from the retina, nor for an increased RRF-sensitivity of the retinal artery in the presence of adenosine. In conclusion, our findings indicate that adenosine potentiates the relaxing influence of bovine and rat retinal tissue. This effect is species dependent as it is not seen with porcine retinal tissue. Neither NO, cyclooxygenase metabolites or epoxyeicosatrienoic acids seem to be involved in this enhanced vasorelaxing response. The involvement of the RRF cannot be excluded.

Functional role of the soluble guanylyl cyclase alpha(1) subunit in vascular smooth muscle relaxation.

OBJECTIVE: Soluble guanylyl cyclase (sGC), the predominant receptor for nitric oxide (NO), exists in 2 active isoforms (alpha(2)beta(1) and alpha(1)beta(1)). In vascular tissue sGCalpha(1)beta(1) is believed to be the most important. The aim of our study was to investigate the functional importance of the sGCalpha(1)-subunit in vasorelaxation. METHODS: Aortic and femoral artery segments from male and/or female sGCalpha(1)(-/-) mice and wild-type littermates were mounted in a small-vessel myograph for isometric tension recording. This was supplemented with biochemical measurements of the cGMP concentration and sGC enzyme activity. RESULTS: The functional importance of sGCalpha(1)beta(1) was demonstrated by the significantly decreased relaxing effects of acetylcholine (ACh), sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), NO gas, YC-1, BAY 41-2272 and T-1032 in the sGCalpha(1)(-/-) mice of both genders. Moreover, the basal and SNP-stimulated cGMP levels and basal sGC activity were significantly lower in the sGCalpha(1)(-/-) mice. However, the relaxing effects of NO, BAY 41-2272 and YC-1 seen in blood vessels from sGCalpha(1)(-/-) mice indicate a role for an sGCalpha(1)beta(1)-independent mechanism. The increase in sGC activity after addition of BAY 41-2272 and the inhibition of the ACh-, SNP-, SNAP- and NO gas-induced response by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) in the sGCalpha(1)(-/-) mice are observations suggesting that the sGCalpha(2)beta(1) isoform is also functionally active. However, the insignificant increase in cGMP in response to SNP and the non-upregulated sGCalpha(2) expression level in the sGCalpha(1)(-/-) mice suggest rather the involvement of (an) sGC-independent mechanism(s). CONCLUSIONS: We conclude that sGCalpha(1)beta(1) is involved in the vasorelaxation induced by NO-dependent and NO-independent sGC activators in both genders. However, the remaining relaxation seen in the sGCalpha(1)(-/-) mice suggests that besides sGCalpha(1)beta(1) also the minor isoform sGCalpha(2)beta(1) and/or (an) sGC-independent mechanism(s) play(s) a substantial role.

Search for the Source of the Retinal Relaxing Factor.

Purpose/Aim of the study: the retinal relaxing factor (RRF) is an unidentified paracrine factor, which is continuously released from retinal tissue and causes smooth muscle cell relaxation. This study tried to identify the cellular source of the RRF. Furthermore, the possible RRF release by voltage-dependent sodium channel activation and the calcium-dependency of the RRF release were investigated. MATERIALS AND METHODS: mouse femoral arteries were mounted in myograph baths for in vitro isometric tension measurements. The vasorelaxing effect of chicken retinas, which contain no vascular cells, and of solutions incubated with MIO-M1 or primary Müller cell cultures were evaluated. The RRF release of other retinal cells was investigated by using cell type inhibitors. Concentration-response curves of veratridine, a voltage-dependent sodium channel activator, were constructed in the presence or absence of mouse retinal tissue to evaluate the RRF release. The calcium-dependency of the RRF release was investigated by evaluating the vasorelaxing effect of RRF-containing solutions made out of chicken retinas in the absence or presence of calcium. RESULTS: Chicken retinas induced vasorelaxation, whereas solutions incubated with Müller cell cultures did not. Moreover, the gliotoxin DL-α-aminoadipic acid, the microglia inhibitor minocycline, and the tetrodotoxin-resistant voltage-dependent sodium channel 1.8 inhibitor A-803467 could not reduce the RRF-induced relaxation. Concentration-response curves of veratridine were not enlarged in the presence of retinal tissue, and RRF-containing solutions made in the absence of calcium induced a substantial, but reduced vasorelaxation. CONCLUSIONS: the RRF is not released from vascular cells and probably neither from glial cells. The retinal cell type that does release the RRF remains unclear. Veratridine does not stimulate the RRF release in mice, and the RRF release in chickens is calcium-dependent as well as calcium-independent.