Does diabetes mellitus affect the progress of tolerance to isosorbide dinitrate (ISDN) in corporal tissue?

For erection to take place, the penile arteries and sinusoids have to dilate, thereby increasing the blood flow into the penis. There is increasing evidence that release of l-arginine derived nitric oxide (NO) from nonadrenergic-noncholinergic (NANC) nerves and from the sinusoidal endothelium is a major event in penile smooth muscle relaxation and promotes the endogenous formation of cyclic guanosine monophosphate (cGMP). Nitrovasodilators can be attributed to the activation of soluble guanylate cyclase, resulting in an increase in intracellular level of cyclic guanosine monophosphate, but prolonged exposure to high levels of nitroglycerine and other organic nitroesters induces tolerance against the cardiovascular effect. In this study, the aim was to determine the effect of diabetes on the corporal smooth muscle relaxant effect of ISDN and the effect of diabetes on the process of tolerance to the drug. For this purpose, alloxan-induced diabetic rabbits were used to form diabetes group. The responses of the corpus cavernous strips obtained from control and alloxan-induced diabetic rabbit were studied in organ chamber. In conclusion, prolonged in vitro exposure of corpus cavernosum strips obtained from control and diabetic groups to high concentrations of ISDN caused significant desensitization to the relaxant effect the drug. So, prolonged exposure of corporal tissue to the agents like nitroglycerine, used for treatment of impotence, may render ineffective the therapy in diabetic erectile impotence. However, intolerance to nitric oxide provides a rationale for the concept of using nitro oxide agents (like SNP) in the treatment of diabetic erectile dysfunction.     

Evidence of relaxant effect of omeprazole in rabbit corpus cavernosum in vitro

The present experiments were designed to investigate the effects of omeprazole, a H(+)-K+ ATPase inhibitor, on corporal smooth muscle tone in vitro. All spontaneous contractile activity in the corpus cavernosum was blocked following omeprazole (0.1 mM-1 mM) administration. However atropine (1 microM), Nw-nitro L-arginine methyl ester (L-NAME, 30 microM) or indomethacin (10 microM) did not affect the spontaneous contraction. Omeprazole (10 microM-1 mM) concentration-dependently induced relaxation in corporal smooth muscle precontracted with 10 microM phenylephrine or 80 mM KCl. Pretreatment of corporal tissue with L-NAME (30 microM), indomethacin (10 microM), ammonium chloride (7.5 mM), sodium acetate (7.5 mM), tetraethyl ammonium chloride (0.5 mM) or glibenclamide (1 microM) had no effect on the omeprazole induced relaxant responses. Nimodipine, an L-type Ca++ channel blocker, relaxed corporal strips precontracted with 80 mM KCl. Collectively, these results indicate that the inhibition of spontaneous contraction and the relaxation of precontracted corporal smooth muscle by omeprazole is probably mediated by the blockade of calcium channels. Further work is needed to determine the cellular mechanism(s) of action by which omeprazole acts on corpus cavernosum smooth muscle.     

Time-dependent alteration in cromakalim-induced relaxation of corpus cavernosum from streptozocin-induced diabetic rats

The purpose of the present study was to investigate the relaxant responses to the ATP-sensitive potassium (K(ATP)) channel opener cromakalim in corpus cavernosum strips from 1-, 2-, 4-, 6-, and 8-week streptozocin-induced diabetic rats. Cromakalim (1 nM-0.1 mM) produced concentration-dependent relaxation in phenylephrine (7.5 microM)-precontracted isolated rat corporal strips. Compared with age-matched control animals, a significant enhancement in cromakalim-induced relaxation of corpus cavernosum was observed in 2-week diabetic animals, whereas the relaxant responses to cromakalim were decreased in 6-and 8-week diabetic animals. However, the cromakalim-induced relaxation was not altered in either 1-week or 4-week rat corporal strips in comparison with corresponding age-matched non-diabetic groups. Preincubation with the K(ATP) channel blocker glibenclamide (10 microM) significantly inhibited the cromakalim-induced relaxation in both non-diabetic and diabetic rat corpus cavernosum, but neither the voltage-dependent K(+) channel (K(V)) antagonist 4-aminopyridine (1 mM) nor the calcium-activated K(+) channel (K(Ca)) antagonist charybdotoxin (0.1 microM) had significant effect on cromakalim-induced relaxation in both control and diabetic rat corporal strips. Relaxation responses to the nitric oxide donor sodium nitroprusside (1 nM-0.1 mM) in diabetic rat corpus cavernosum were similar to that of age-matched controls. These data demonstrated that the relaxant responses to cromakalim were altered in diabetic cavernosal strips in a time dependent manner, suggesting that the period of diabetes mellitus may play a key role in the K(ATP) channels function in rat corpus cavernosum.     

Influence of relaxin on the neurally induced relaxant responses of the mouse gastric fundus

The peptide hormone relaxin has been reported to depress the amplitude of contractile responses in the mouse gastric fundus by upregulating nitric oxide (NO) biosynthesis at the neural level. In the present study, we investigated whether relaxin also influenced nonadrenergic, noncholinergic (NANC) gastric relaxant responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. In carbachol precontracted strips from control mice and in the presence of guanethidine, electrical field stimulation (EFS) elicited fast relaxant responses that may be followed by a sustained relaxation. All relaxant responses were abolished by tetrodotoxin. Relaxin increased the amplitude of the EFS-induced fast relaxation without affecting either the sustained one or the direct smooth muscle response to papaverine. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), that abolished the EFS-induced fast relaxation without influencing the sustained one, relaxin was ineffective. In strips from relaxin-pretreated mice, EFS-induced fast relaxations were enhanced in amplitude with respect to the controls, while sustained ones as well as direct smooth muscle responses to papaverine were not changed. Further addition of relaxin to the bath medium did not influence neurally induced fast relaxant responses, whereas L-NNA did. In conclusion, in the mouse gastric fundus, relaxin enhances the neurally induced nitrergic relaxant responses acting at the neural level.     

Erectile dysfunction in spontaneously hypertensive rats: pathophysiological mechanisms

Hypertensive men have a higher prevalence of erectile dysfunction (ED) than the general population. Experimental evidence of ED in hypertensive animals is scarce. This study evaluates the erectile function of spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY) in vivo by the increase in intracavernosal pressure after electrical stimulation of the cavernous nerve (CN) and by isometric tension studies on corporal strips. Frequency-dependent erectile responses to CN stimulations were reduced in SHR. Phenylephrine induced lower corporal contractions in SHR although pD2 values were similar to WKY. Endothelium-dependent relaxations to ACh were impaired significantly in SHR, and indomethacin improved these relaxations in both WKY and SHR, the latter thus reaching values similar to WKY. Corporal relaxations to sodium nitroprusside were enhanced in SHR. Thus a dysfunctional alpha-adrenergic contraction of the corporal smooth muscle, an increased cyclooxygenase-dependent constrictor tone, and/or a defect in endothelium-dependent reactivity are associated with the altered erectile mechanisms in SHR. Drugs targeting endothelial dysfunction may delay the occurrence of ED as a complication of hypertension.     

Cardiovascular risk factors, erection disorders and endothelium dysfunction

Upon sexual stimulation, penile erection, occurring in response to the activation of pro-erectile autonomic pathways, is greatly dependent on adequate inflow of blood to the erectile tissue and requires coordinated arterial endothelium-dependent vasodilatation and sinusoidal endothelium-dependent corporal smooth muscle relaxation. Nitric oxide (NO) is the principal peripheral pro-erectile neurotransmitter which is released by both non-adrenergic, non-cholinergic neurons and the sinusoidal endothelium to relax corporal smooth muscle through the cGMP pathway. Any factors modifying the basal corporal tone, the arterial inflow of blood to the corpora, the synthesis/release of neurogenic or endothelial NO are prime suspects for being involved in the pathophysiology of erectile dysfunction (ED). In fact, conditions associated with altered endothelial function, such as ageing, hypertension, hypercholesterolemia and diabetes, may, by changing the balance between contractant and relaxant factors, cause circulatory and structural changes in penile tissues, resulting in arterial insufficiency and defect in smooth muscle relaxation and thus, ED. There is increasing evidence to suggest that ED is predominantly a vascular disease and may even be a marker for occult cardiovascular disease. Recent results illustrating the importance of endothelial dysfunction in the pathophysiology of different forms of experimental ED are discussed. These pathways may represent new potential treatment targets.     

Synthetic nitrovasodilators are effective, in vitro, in relaxing penile tissue from impotent men: the findings and their implications

Normal penile erectile function is dependent on arterial adequacy, appropriate venous occlusion, neurohumoral factors, and finally the relaxation of penile cavernous trabecular smooth muscle. The present experiments were designed to test whether compounds related to endothelium-derived relaxing factor have a role in penile smooth muscle relaxation and whether this role is preserved in clinically impotent tissue. Isometric tension experiments were conducted using strips of human tissue (appropriately obtained) from patients found to be impotent by clinical criteria. Glyceryl trinitrate and isosorbide dinitrate produced maximal relaxations of 66 and 63%, respectively, in tissues contracted with norepinephrine: 50% relaxation was observed at 6 x 10(-7) and 8 x 10(-5) M, respectively. The finding of a relaxant response to synthetic nitrovasodilators in "impotent" tissue implies that (i) complete end organ (smooth muscle) failure is not always, if ever, seen, (ii) endothelium-derived factors probably play a role in erectile tissue parallel with their role in other vascular tissues, (iii) more proximal factors may be responsible for clinical impotence, and (iv) synthetic nitrovasodilators may have a role in the therapy of clinical impotence.     

Effects of hypercholesterolemia on the electrical and contractile properties of the blood vessel wall

The investigations were performed on the ring strips of rabbit aorta. The electrical activity of the vessels smooth muscle cells was registered by the "sucrose gap" method; the contractile activity of the strips was determined simultaneously. Experimental atherosclerosis was induced by keeping rabbits on a special diet enriched by cholesterol for 2 and 4 months. Strips were prepared with intact or mechanically removed endothelium. Hypercholesterolemia was shown to inhibit the reactivity of the vessel's wall to the weakening action of acetylcholine due to endothelial stimulation. The cause of these changes was the inhibition of the endothelial functional activity and inactivation of mechanisms by which endothelium influences smooth muscle cells.     

Modulation of nitrergic relaxant responses by peptides in the mouse gastric fundus

The effects of pituitary adenylate cyclase-activating peptide (PACAP-38) and vasoactive intestinal polypeptide (VIP) were investigated in the gastric fundus strips of the mouse. In carbachol (CCh) precontracted strips, in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor L-N(G)-nitro arginine (L-NNA) or by the guanylate cyclase inhibitor (ODQ), the sustained one by alpha-chymotrypsin. alpha-Chymotrypsin also increased the amplitude of the EFS-induced fast relaxation. PACAP-38 and VIP caused tetrodotoxin-insensitive sustained relaxant responses that were both abolished by alpha-chymotrypsin. Apamin did not influence relaxant responses to EFS nor relaxation to both peptides. PACAP 6-38 abolished EFS-induced sustained relaxations, increased the amplitude of the fast ones and antagonized the smooth muscle relaxation to both PACAP-38 and VIP. VIP 10-28 and [D-p-Cl-Phe6,Leu17]-VIP did not influence the amplitude of both the fast or the sustained response to EFS nor influenced the relaxation to VIP and PACAP-38. The results indicate that in strips from mouse gastric fundus peptides, other than being responsible for EFS-induced sustained relaxation, also exerts a modulatory action on the release of the neurotransmitter responsible for the fast relaxant response, that appears to be NO.     

Beta-adrenergic agonists and cyclic AMP decrease intracellular resting free-calcium concentration in ileum smooth muscle

Intracellular free-calcium levels were measured in strips of longitudinal smooth muscle from guinea-pig ileum; fura-2 was used as a calcium monitor. At rest the calcium concentration was about 180 nM, and this rose to 300-400 nM following electrical stimulation and during spontaneous calcium transients (all measurements at 23-25 degrees C). Isoprenaline suppressed the spontaneous calcium transients, and reduced the resting calcium level to about 130 nM. This fall in resting calcium concentration was seen even in muscle strips which did not have spontaneous activity. Elevation of intracellular cyclic AMP levels, produced by forskolin or dibutyryl cyclic AMP, mimicked the actions of isoprenaline. We conclude that the relaxant effects of beta-adrenergic agonists of visceral smooth muscle may be explained partly by a fall in intracellular resting free-calcium level, mediated via an increase in cyclic AMP.     

Effect of aging on the response of biochemical markers in the rabbit subjected to short-term partial bladder obstruction

Purpose Partial bladder outlet obstruction (PBOO) results in marked biochemical alterations in the bladder. In this study, we focused on comparison of thapsigargin sensitive sarco/endoplasmic reticulum Ca²⁺ ATPase activity (SERCA) and Citrate Synthase after short term PBOO in young versus old rabbits. Materials and methods A total of 20 young and 20 mature male rabbits were divided into 4 sub-groups of 5 rabbits each (4 obstructed and 1 sham-control rabbit). The rabbits in the groups were evaluated after 1, 3, 7, and 14 days of obstruction, respectively. The activities of SERCA and citrate synthase were examined as markers for sarcoplasmic reticular calcium storage and release and mitochondrial function, respectively. Results The SERCA activity of bladder body smooth muscle in the young animals increased at 7 and 14 days. For the old rabbits, the SERCA activity decreased significantly by 1 day and remained this level throughout the course of obstruction, and was significantly lower than young at all time periods. The citrate synthase activity in the young animals decreased over the 1–7 days, and then returned toward control level by 14 days following obstruction. In the old animals, citrate synthase activity of bladder body smooth muscle progressively decreased over the course of the study, and was significantly lower in the old than the young animals after 14 days obstructed. Conclusion The urinary bladders of the young rabbits have a considerable greater ability to adapt to PBOO than do those of the old rabbits. The deterioration of mitochondrial and SR function may be important mechanisms underlying geriatric voiding dysfunction.     

The role of prostaglandins in penile erection

The balance of penile smooth muscle tone is finely controlled, with contractile factors acting in opposition to relaxant factors. The principal agents in this process are undoubtedly noradrenaline and nitric oxide. Prostaglandins probably have a crucial role in the 'fine tuning' of corporal smooth muscle tone. Their effects on control mechanisms in the healthy penis are more likely to be modulatory rather than direct.     

Intercellular communication through gap junctions: a potential role in pharmacomechanical coupling and syncytial tissue contraction in vascular smooth muscle isolated from the human corpus cavernosum

Kinetic and steady-state protocols were used to examine the effects of disruption of intercellular communication with heptanol, on contractile responses elicited by activation of the alpha 1-adrenergic receptor in human corporal vascular smooth muscle. For the steady-state studies, strips of corporal tissue from 19 patients were submaximally precontracted with phenylephrine (PE) and then relaxed by the cumulative addition of heptanol. Heptanol completely and reversibly relaxed all tissues studied in a concentration-dependent manner. The heptanol concentration response data were then computer fit to the general logistic equation to obtain pEC50 (negative logarithm of the concentration that elicits one-half of the maximal effect) and slope factor values, with Emax (maximal relaxation) set to 100%. The mean pEC50 and slope factor values, respectively, were 2.86 +/- 0.04 and 1.86 +/- 0.17. Furthermore, kinetic studies on corporal tissues from a subset of the patient population (11 patients) revealed that preincubation of tissues with 2 mM heptanol caused a significant decrease in both the rate and magnitude of PE-induced contractions in all tissues studied, without affecting the rate constant for onset of contraction (k(obs)). The present results on intact tissue extend our previous observations on cultured corporal cells, and support the supposition that intercellular communication through gap junctions may play an important role in the initiation, maintenance and modulation of alpha 1-adrenergic contractions in human vascular smooth muscle.     

Possible role of nitric oxide in transmitting information from vasodilator nerve to cerebroarterial muscle

Treatment with L-NG-monomethyl arginine (L-NMMA), an inhibitor of nitric oxide (NO) synthesis from L-arginine, suppressed the relaxant response of dog cerebral artery strips to transmural electrical stimulation and nicotine, as did oxyhemoglobin. The inhibition by L-NMMA was reversed or prevented by L-, but not D-, arginine. It is concluded that NO or an NO-related compound may play a crucial role in transmitting information from excited vasodilator nerves to cerebroarterial smooth muscle.     

Effect of nedocromil sodium on non-adrenergic, non-cholinergic neural responses in guinea pig bronchi in vitro.

OBJECTIVE: Nedocromil sodium (nedocromil) improves the clinical condition of asthmatic subjects but its mechanism of action is not fully understood. This study aimed to determine whether nedocromil alters the ability of contractile and relaxant non-adrenergic, non-cholinergic neural (NANC) responses to stabilise tone by inhibiting or potentiating these responses in bronchial smooth muscle and, if so, whether the action is on a pre- or postjunctional level. RESULTS: Nedocromil attenuated contractile but not relaxant NANC responses (elicited by electric field stimulation) significantly (P < 0.05) in guinea pig main bronchi in vitro. However, the ability of NANC responses to stabilise tone (convergence effect) was not significantly impaired by nedocromil. Furthermore, nedocromil did not significantly shift the concentration response curve (-log EC50) to neurokinin A (NKA), the dominating contractile NANC transmitter, or alter the maximum response to NKA (P > 0.05). Submaximum or maximum contractile responses to histamine were not markedly affected by nedocromil (P > 0.05). CONCLUSIONS: Nedocromil exerts selective neural inhibition of the contractile but not of the relaxant NANC responses on a pre-junctional level in bronchial smooth muscle. Nedocromil does not, however, markedly impair the ability of NANC response to stabilise bronchial smooth muscle tone.     

Effects of Met-enkephalin on the mechanical activity and distribution of Met-enkephalin-like immunoreactivity in the cat large intestine.

The effects of Met-enkephalin on the spontaneous and electrically evoked activity were investigated in longitudinal and circular strips isolated from different regions of the large intestine, i.e., proximal colon, distal colon and rectum. Met-enkephalin induced dose-dependent contractile responses which were reversibly blocked by naloxone (10(-6) M). In all longitudinal strips and in the circular strips of the rectum, the effects of Met-enkephalin were prevented by TTX (10(-7) M), demonstrating their neurogenic nature. In the circular strips from the colon, Met-enkephalin induced contractile responses after TTX, proving the existence of smooth muscle opioid receptors. The comparison between the EC50 values of Met-enkephalin showed that the opioid receptors in the different regions have different sensitivity to Met-enkephalin, while the opioid receptors in the longitudinal and circular layers of the same region have equal affinity. Atropine (10(-6) M) and guanethidine (10(-6) M) did not alter significantly the EC50 values, showing that the neurogenic effects of Met-enkephalin on the spontaneous activity involve mainly nonadrenergic, noncholinergic (NANC) neurotransmitter mechanisms. When the preparations were stimulated electrically, Met-enkephalin (10(-9) M) suppressed the cholinergic components of the responses. Met-enkephalin-containing nerve fibers were found in the myenteric plexus of the three intestinal regions. In the colon, where direct smooth muscle effects were observed, fibers containing Met-enkephalin-like immunoreactivity were found to go deep into the circular layer, suggesting that they could supply Met-enkephalin input to the smooth muscle cells.     

Cooling is a potent vasodilator of deep vessels in the rat.

The objectives of this study were to determine the effect of cooling on smooth muscle tone of the pulmonary artery and aorta and to clarify the basic mechanism of these responses. We recorded isometric tension in smooth muscle strips of rat pulmonary artery and aorta in organ baths during stepwise cooling. Cooling responses were tested before and after the addition of various standard agents that interfere with known neurogenic (autonomic blockers, tetrodotoxin) and myogenic mechanisms (calcium channel blockers) of relaxation. We also examined the hypothesis of the presence of a cooling-released substance. Stepwise cooling (37degrees C to 4 degrees C) of aortic smooth muscle induced reproducible graded relaxations that were inversely proportional to temperature. Cooling-induced relaxation was not dependent on a neural mechanism nor the release of neurotransmitters or a cooling-released substance such as NO or CO. Cooling of pulmonary arterial and aortic smooth muscle preparations induced a graded myogenic relaxation inversely proportional to the cooling temperature. The mechanism is not dependent on local nervous or known mediators but related to a direct physico-chemical effect of cooling.     

Deletion of Dicer in smooth muscle affects voiding pattern and reduces detrusor contractility and neuroeffector transmission

MicroRNAs have emerged as important regulators of smooth muscle phenotype and may play important roles in pathogenesis of various smooth muscle related disease states. The aim of this study was to investigate the role of miRNAs for urinary bladder function. We used an inducible and smooth muscle specific Dicer knockout (KO) mouse which resulted in significantly reduced levels of miRNAs, including miR-145, miR-143, miR-22, miR125b-5p and miR-27a, from detrusor preparations without mucosa. Deletion of Dicer resulted in a disturbed micturition pattern in vivo and reduced depolarization-induced pressure development in the isolated detrusor. Furthermore, electrical field stimulation revealed a decreased cholinergic but maintained purinergic component of neurogenic activation in Dicer KO bladder strips. The ultrastructure of detrusor smooth muscle cells was well maintained, and the density of nerve terminals was similar. Western blotting demonstrated reduced contents of calponin and desmin. Smooth muscle α-actin, SM22α and myocardin were unchanged. Activation of strips with exogenous agonists showed that depolarization-induced contraction was preferentially reduced; ATP- and calyculin A-induced contractions were unchanged. Quantitative real time PCR and western blotting demonstrated reduced expression of Cav1.2 (Cacna1c). It is concluded that smooth muscle miRNAs play an important role for detrusor contractility and voiding pattern of unrestrained mice. This is mediated in part via effects on expression of smooth muscle differentiation markers and L-type Ca(2+) channels in the detrusor.